RESUMO
Equine insulin dysregulation (ID) is a significant metabolic problem because the hyperinsulinaemia that develops increases the animal's risk of developing laminitis, a debilitating foot condition. The role of gastrointestinal factors, such as incretin hormones, in the pathogenesis of ID and hyperinsulinaemia in horses is poorly understood, particularly in comparison to other species. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic peptide released from L cells in the gastrointestinal tract and is implicated in metabolic dysfunction in other species. The aim of this study in vitro was to establish basic physiological understanding about intestinal secretion of GLP-2 in horses. Basal and glucose-stimulated GLP-2 secretion was measured in post-mortem tissue samples from the duodenum, jejunum, and ileum. We observed that GLP-2 secretion was minimal in samples from the duodenum compared to the jejunum and ileum (5-9-fold higher; P < 0.05). Furthermore, GLP-2 secretion was not responsive to glucose stimulation in the ileum or duodenum but was responsive to glucose in the jejunum. This effect in the jejunum was inhibited by 30 % (P = 0.02) using phlorizin, a selective sodium-glucose cotransporter-1 (SGLT-1) inhibitor, and by 38 % (P = 0.04) using phloretin, a non-selective SGLT-1/GLUT-2 inhibitor. The localisation of glucose-responsive GLP-2 secretion in the jejunum might be relevant to the development of post-prandial hyperinsulinaemia. This study has provided data on GLP-2 secretion from the equine small intestine that will enable more complex and dynamic studies on the pathogenesis of ID.
RESUMO
Blood glucose concentration is often measured during an oral glucose test (OGT), but is not thought to aid in diagnosing insulin dysregulation (ID) or pituitary pars intermedia dysfunction (PPID). The aim of this retrospective study was to investigate whether the change in blood glucose concentration during an OGT aligned with indicators of equine metabolic syndrome or PPID, including serum insulin and plasma ACTH concentrations, clinical observations, age, sex, breed type and the test dose. The cohort included 149 horses, miniature horses, and ponies that had undergone an in-feed OGT and clinical examination between 2015 and 2021. The animals were diagnosed as either metabolically healthy, insulin-dysregulated, having PPID or both endocrinopathies. The mean ± standard error increase in blood glucose during the OGT was 3.41 ± 0.21 mM, and this change showed a weak positive correlation with the increase in serum insulin concentration (r = 0.36; P 0.001), body condition score (BCS; r = 0.26; P = 0.002) and cresty neck score (CNS; r = 0.38; P 0.001). The median [interquartile range] increase in blood glucose for miniature horses (5.25 [2.98-6.5] mM), was more than twice that seen in full-sized horses (2.4 [1.33-3.45] mM; P = 0.03). In metabolically healthy animals the increase in blood glucose during an OGT (+2.2 [1-3.5] mM) was smaller (P 0.001) than in animals with ID (+3.8 [2.73-5.33] mM), or both endocrine diseases (+6.1 [3.6-6.85] mM). There was an effect of the dose of dextrose on the blood glucose response, with higher doses yielding larger responses (P 0.001). The variability in these data support that basal and post-prandial blood glucose responses to an OGT are not appropriate as stand-alone diagnostic markers of ID or PPID. However, the association between blood glucose and CNS supports the use of CNS when evaluating animals for ID.
Assuntos
Doenças do Sistema Endócrino , Doenças dos Cavalos , Síndrome Metabólica , Doenças da Hipófise , Humanos , Cavalos , Animais , Glicemia , Estudos Retrospectivos , Doenças do Sistema Endócrino/veterinária , Síndrome Metabólica/veterinária , Insulina , Doenças da Hipófise/diagnóstico , Doenças da Hipófise/veterináriaRESUMO
Although it is understood that equine endocrinopathic laminitis can be triggered by high concentrations of insulin, it is unclear whether this represents a direct action on lamellar tissue via insulin receptors (InsR), an interaction with IGF-1 receptors (IGF-1R), or some other, indirect action. This uncertainty is because of the reported scarcity of InsR in lamellar tissue and the low affinity of insulin for equine IGF-1R. In the present study, the effects of insulin and IGF-1 (as a positive control) were examined using lamellar explants isolated from the hooves of healthy horses and incubated in cell culture medium for between 2 min and 48 h. In this system, a low physiological concentration of IGF-1 (10 nM; 1.31 ng/mL) caused a marked increase in the appearance of phosphorylated IGF-1R after 5 min (P < 0.05), and this effect was blocked by a human anti-IGF-1R monoclonal antibody (mAb). However, a high concentration of insulin (10 nM; 1,430 µIU/mL) appeared to cause dephosphorylation of the IGF-1R after 5 min (P < 0.01), 15 min, and 30 min (P < 0.001). Using 3H-thymidine as a marker, it was also demonstrated that insulin and IGF-1-stimulated cell proliferation in lamellar explants over the same concentration range as each other (1-100 nM), implying that each peptide acts via its own receptor (P < 0.001). Conversely, the effect of both peptides could be blocked using a selective anti-IGF-1R mAb (P < 0.001), implying that insulin acts via IGF1-R (either directly or indirectly). Notwithstanding this conundrum, the results demonstrate that insulin acts directly on lamellar tissue and suggest that a therapeutic anti-IGF-1R mAb could be useful in treating or preventing endocrinopathic laminitis.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Casco e Garras/metabolismo , Cavalos/metabolismo , Insulina/farmacologia , Receptor IGF Tipo 1/metabolismo , Técnicas de Cultura de Tecidos/veterinária , Animais , Anticorpos Monoclonais , Western Blotting , Proliferação de Células , Receptor IGF Tipo 1/genéticaRESUMO
Prolonged hyperinsulinemia is thought to be the cause of equine endocrinopathic laminitis, a common and crippling disease of the foot, for which there are no pharmacologic treatments other than pain relief. It has been suggested that insulin causes its effects on the lamellae by activating IGF-1 receptors (IGF-1R), as insulin receptors (InsR) are scarce in this tissue, whereas IGF-1R are abundant and become downregulated after prolonged insulin infusion. As a first step toward confirming this mechanism and beginning to develop a therapeutic anti-IGF-1R monoclonal antibody (mAb) for horses, it was necessary to identify available human IGF-1R mAbs that would recognize equine receptors. Four IGF-1R mAbs were tested using soluble equine IGF-1R, with ELISA and flow cytometry. Frozen equine lamellar and liver tissue was also used in radioligand binding assays. The results demonstrated that only one of the mAbs tested (mAb1) was able to compete effectively with IGF-1 for binding to its receptors in equine lamellar tissue, with an IC50 of 5 to 159 ng/mL. None of the 4 mAbs were able to bind to equine hepatic InsR. This study has generated valuable structure-activity information and has identified a prototype anti-IGF-1R mAb suitable for further development.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Doenças dos Cavalos/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/imunologia , Animais , Anticorpos Monoclonais/química , Doenças do Pé/tratamento farmacológico , Doenças do Pé/etiologia , Doenças do Pé/veterinária , Cavalos , Humanos , Hiperinsulinismo/complicações , Hiperinsulinismo/veterinária , Fígado/química , Receptor de Insulina/antagonistas & inibidores , Receptor de Insulina/imunologia , Relação Estrutura-AtividadeRESUMO
Endocrinopathic laminitis, related to equine metabolic syndrome and insulin dysregulation, causes marked pain and suffering in horses and represents a substantial cost to the horse industry. This study investigated the effect of feeding a diet high in nonstructural carbohydrates on concentrations of active glucagon-like peptide-1 (aGLP-1), total insulin-like growth factor-1 (IGF-1), and high-molecular-weight (HMW) adiponectin, in insulin-dysregulated ponies. Thirty-seven ponies were challenged with this diet for up to 18 d to induce hyperinsulinemia. Hormone concentrations were measured in selected samples on day 2 of the diet challenge period, over 4 h after feeding. Fourteen of the ponies developed mild laminitis induced by the diet challenge. Insulin and glucose responses to the diet have been reported previously. Feeding increased the concentrations of aGLP-1 (P < 0.05) and HMW adiponectin (P < 0.001), but there was no difference between the laminitic and nonlaminitic groups for either hormone. Concentrations of IGF-1 and insulin were inversely related, with IGF-1 being 32% lower in hyperinsulinemic/laminitic ponies compared with nonlaminitic ponies (P = < 0.05). These results indicate that unlike insulin and possibly IGF-1, concentrations of aGLP-1 and HMW adiponectin do not have a strong association with, or play a major role in, the pathogenesis of equine laminitis.
Assuntos
Adiponectina/metabolismo , Doenças do Pé/veterinária , Peptídeo 1 Semelhante ao Glucagon/sangue , Casco e Garras/patologia , Inflamação/veterinária , Fator de Crescimento Insulin-Like I/metabolismo , Adiponectina/genética , Animais , Carboidratos da Dieta/efeitos adversos , Doenças do Pé/induzido quimicamente , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Doenças dos Cavalos/induzido quimicamente , Doenças dos Cavalos/metabolismo , Cavalos , Inflamação/induzido quimicamente , Insulina/sangue , Fator de Crescimento Insulin-Like I/genéticaRESUMO
Although it is well established that equine laminitis can be triggered by extreme hyperinsulinemia, the mechanism of insulin action is not known. High concentrations of insulin lead to separation of the weight-bearing apparatus from the hoof wall and are associated with an increased cycle of cell death and proliferation in the lamellae. Gene expression and immunohistochemistry studies have indicated that the lamellae are sparsely populated with insulin receptors, whereas IGF-1 receptors (IGF-1R) are abundant, suggesting that the action of insulin may be mediated by insulin binding to the IGF-1R. To investigate this possibility, cell membrane fragments containing IGF-1R were extracted from the livers of 6 horses and the lamellae of >50 horses euthanized for nonresearch purposes at an abattoir. Radioligand-binding studies using 125I-IGF-1 and 125I-insulin confirmed an abundance of high-affinity IGF-1R in the liver (KD 0.11 nM, Bmax 223 fmol/mg protein) and lamellae (KD 0.16 nM, Bmax 243 fmol/mg protein). However, the affinity of insulin for binding to the lamellar IGF-1R (Ki 934 nM) was >5,800 fold less than that of IGF-1, suggesting that insulin is unlikely to bind to equine IGF-1R at physiological concentrations. Although insulin receptors could be detected in the liver (KD 0.48 nM, Bmax 123 fmol/mg protein), they were barely detectable in lamellae (estimated Bmax 14 fmol/mg protein). There was no evidence to support the presence of insulin/IGF-1 hybrid receptors in either tissue. These findings suggest that insulin does not act directly through IGF-1 receptors and that an alternative theory is required to explain the mechanism of insulin action in laminitis.
Assuntos
Casco e Garras/metabolismo , Doenças dos Cavalos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Doenças do Pé/veterinária , Cavalos , Hiperinsulinismo/complicações , Hiperinsulinismo/veterinária , Radioisótopos do IodoRESUMO
BACKGROUND: Equine insulin dysregulation (ID) is a common and poorly understood disorder that increases the risk of laminitis. Recent data show that the condition may be associated with alteration of the enteroinsular axis and enhanced glucose bioavailability. Upregulation of glucagon-like peptide-2 (GLP-2), an intestinotrophic peptide, leads to enhanced nutrient uptake and metabolic dysfunction in other species. OBJECTIVES: The study aimed to 1) determine whether GLP-2 is differentially expressed in insulin-dysregulated ponies, compared with healthy ponies, and 2) confirm intestinal expression of the GLP-2 receptor in horses (eGLP-2R). STUDY DESIGN: Cohort study. METHODS: Fasting and post-prandial GLP-2 concentrations were measured in archived plasma samples obtained from 25 mixed-breed ponies during two feeding studies. Measurements were undertaken with an ELISA that was validated for equine use as part of the current study. Ponies were designated as healthy or insulin-dysregulated based on an oral glucose test, and the results were compared between groups. The gene expression of the eGLP-2R was determined with polymerase chain reaction. RESULTS: Basal, fasted plasma GLP-2 concentrations were higher in ponies with ID, compared with healthy ponies. Grazing increased GLP-2 in healthy, but not in insulin-dysregulated, ponies. The eGLP-2R gene was expressed in the small intestine and pancreas. MAIN LIMITATIONS: The study was performed with a relatively small sample size. The specificity of the GLP-2 assay could not be determined due to the lack of equine-specific assay standards. CONCLUSIONS: This study has demonstrated that GLP-2 may be important in the pathogenesis of equine ID and suggests that the eGLP-2R may be a novel therapeutic target for the treatment of equine ID.
Assuntos
Peptídeo 2 Semelhante ao Glucagon/fisiologia , Receptor do Peptídeo Semelhante ao Glucagon 2/metabolismo , Cavalos/metabolismo , Insulina/metabolismo , Intestino Delgado/metabolismo , Animais , Estudos de Coortes , Ingestão de Alimentos/fisiologia , Ensaio de Imunoadsorção Enzimática/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Jejum/metabolismo , Feminino , Peptídeo 2 Semelhante ao Glucagon/sangue , Peptídeo 2 Semelhante ao Glucagon/imunologia , Teste de Tolerância a Glucose/veterinária , Cavalos/sangue , Masculino , Regulação para CimaRESUMO
The aim of this study was to investigate the relationship between laminitis development in ponies and insulin/glucose concentrations in response to the oral glucose test (OGT) and a dietary challenge high in nonstructural carbohydrates (NSCs). After undergoing an OGT (1 g dextrose/kg BW in feed), 37 ponies with 2-h serum insulin concentrations ranging from 22 to 1,133 µIU/mL were subjected to a diet challenge period (DCP), consuming 12 g NSC/kg BW/d for up to 18 d. Insulin and glucose responses were measured on day 2 of the DCP. Clinical laminitis was diagnosed by blinded experts and confirmed radiographically. Basal ACTH levels and clinical signs were assessed to investigate concurrent putative pituitary pars intermedia dysfunction (PPID). The diet induced Obel grade 1 or 2 laminitis in 14 ponies (38%). The ponies that developed laminitis had higher maximum concentrations of blood glucose (P = 0.04) and serum insulin (P = 0.02) in response to the diet. The geometric mean (95% CI) blood glucose concentration for laminitis cases was 14.9 (12.9-17.2) mM, compared to 10.7 (9.2-12.5) mM for ponies who did not develop laminitis. Similarly, the geometric mean (95% CI) for serum insulin was 396 (301-520) µIU/mL for laminitis cases, compared to 216 (148-316) µIU/mL for ponies who did not develop laminitis. Laminitis incidence was likewise associated with insulin concentrations measured during the OGT. Laminitis occurred at frequencies of 0% (0/7) if postdextrose insulin (µIU/mL) was <50; 35% (8/23) if insulin was 50 to 195; and 86% (6/7) if insulin was >195 µIU/mL. Basal ACTH concentrations were above seasonally accepted reference ranges in 16/37 ponies, and 8 of these animals (50%) developed laminitis. This included all 5 ponies in the study that had clinical signs of PPID (100%). In contrast, hyperinsulinemia and laminitis occurred in only 3/11 ponies (27%) with elevated ACTH concentrations and no clinical signs of PPID (P = 0.009). Thus, laminitis occurrence was associated with higher glucose and insulin responses to both the OGT and challenge diet, and the frequency of laminitis can be predicted based on insulin and glucose hyperresponsiveness to these oral carbohydrate challenges.
Assuntos
Carboidratos da Dieta/efeitos adversos , Doenças do Pé/veterinária , Teste de Tolerância a Glucose , Doenças dos Cavalos/induzido quimicamente , Inflamação/veterinária , Hormônio Adrenocorticotrópico/sangue , Animais , Glicemia , Carboidratos da Dieta/administração & dosagem , Feminino , Doenças do Pé/induzido quimicamente , Casco e Garras/patologia , Cavalos , Inflamação/patologia , Insulina/sangue , Masculino , Doenças da Hipófise/veterináriaRESUMO
Metabolic disease is a significant problem that causes a range of species-specific comorbidities. Recently, a better understanding of glucose-dependent insulinotropic polypeptide (GIP) biology has led to the suggestion that inhibiting its action may attenuate obesity in several species. In horses, antagonism of GIP may also reduce hyperinsulinemia, which leads to insulin-associated laminitis, a painful comorbidity unique to this species. However, little is known about GIP in horses. The aims of this study were to examine the tissue distribution of equine GIP receptors (eGIPR), to determine whether eGIPR can be blocked using a GIP antagonist not tested previously in horses, and to establish whether there is any association between GIP concentrations and body mass in this species. Archived tissues from healthy horses were used to establish that eGIPR gene expression was strong in pancreas, heart, liver, kidney, and duodenum and absent in gluteal muscle. Pancreatic islets were isolated from fresh horse pancreas using collagenase digestion and layering through a density gradient. Islet viability was confirmed microscopically and by demonstrating that insulin production was stimulated by glucose in a concentration-dependent manner. Insulin release was also shown to be concentration-dependent with GIP up to 0.1µM, and the response to GIP was decreased ( = 0.037) by the antagonist (Pro3)GIP. As for the relationship between body mass and GIP in vivo postprandial GIP concentrations in archived plasma samples were positively correlated with body condition and cresty neck scores ( < 0.05). Thus, the eGIPR is a potential therapeutic target for insulin dysregulation and obesity in horses.
Assuntos
Polipeptídeo Inibidor Gástrico/metabolismo , Cavalos/fisiologia , Insulina/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Duodeno/metabolismo , Feminino , Glucose/metabolismo , Ilhotas Pancreáticas/metabolismo , Masculino , Obesidade/metabolismo , Pâncreas/metabolismo , Período Pós-PrandialRESUMO
REASONS FOR PERFORMING STUDY: Insulin dysregulation can be difficult to diagnose from basal insulin and glucose concentrations, so a field-based oral glucose test (OGT) is preferred. However, the repeatability of this test has not been reported. OBJECTIVES: To determine the repeatability of an in-feed OGT in ponies and examine some factors affecting the palatability of the test meal. STUDY DESIGN: A repeated measures, longitudinal study. METHODS: An in-feed OGT was performed at 08.00 h on 3 consecutive occasions under controlled conditions in 8 mixed breed ponies. d-glucose (0.75 g/kg bwt) was dissolved in water and combined with wheat bran and lucerne chaff. Blood samples were taken before and 90, 120, 180 min and 24 h after d-glucose. The repeatability of the test was analysed with repeated measures ANOVA. Insulin and glucose responses to d-glucose were also compared to an equivalent dose of dietary carbohydrate provided with a commercial grain mixture. RESULTS: The overall insulin responses to the OGTs did not differ between tests. Individual insulin responses were more variable (P<0.05) than glucose responses. There was no difference in insulin concentration in post d-glucose samples over time. Insulin and glucose responses to grain and d-Glucose were not different. CONCLUSIONS: An OGT is reasonably repeatable in ponies. The currently recommended post-glucose sampling time point of 2 h is acceptable, with sampling at 90 min also likely to produce a consistent result. The use of an alternative carbohydrate source to d-glucose, such as a commercial grain-based product, may be a viable and more palatable option for the test.
Assuntos
Glicemia/fisiologia , Teste de Tolerância a Glucose/veterinária , Cavalos/fisiologia , Ração Animal , Animais , Ingestão de Alimentos , Cavalos/sangue , Insulina/sangue , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Obesity in horses is increasing in prevalence and can be associated with insulin insensitivity and laminitis. Current treatment strategies for obesity include dietary restriction and exercise. However, whether exercise alone is effective for decreasing body fat is uncertain. HYPOTHESIS: Our hypothesis was that twice daily use of a dynamic feeding system for 3 months would induce sustained, low-intensity exercise thereby decreasing adiposity and improving insulin sensitivity (SI). ANIMALS: Eight, university-owned, mixed-breed, adult ponies with body condition scores (BCS) ≥5/9 were used. METHODS: Two treatments ("feeder on" or "feeder off") were administered for a 3-month period by a randomized, crossover design (n = 4/treatment). An interim equilibration period of 6 weeks at pasture separated the 2 study phases. Measurements of body mass (body weight, BCS, cresty neck score [CrNS], and morphometry), body fat (determined before and after the "feeder on" treatment only), triglycerides, and insulin sensitivity (SI; combined glucose-insulin test) were undertaken before and after treatments. RESULTS: The dynamic feeding system induced a 3.7-fold increase in the daily distance travelled (n = 6), compared to with a stationary feeder, which significantly decreased mean BCS (6.53 ± 0.94 to 5.38 ± 1.71), CrNS (2.56 ± 1.12 to 1.63 ± 1.06) and body fat (by 4.95%). An improvement in SI did not occur in all ponies. CONCLUSIONS AND CLINICAL IMPORTANCE: A dynamic feeding system can be used to induce sustained (daily), low-intensity exercise that promotes weight loss in ponies. However, this exercise may not be sufficient to substantially improve SI.
Assuntos
Tecido Adiposo/fisiologia , Doenças dos Cavalos/terapia , Obesidade/veterinária , Condicionamento Físico Animal , Criação de Animais Domésticos/métodos , Animais , Estudos Cross-Over , Cavalos , Obesidade/terapia , Fatores de TempoRESUMO
Managing equine hyperinsulinemia is crucial for preventing laminitis, but our understanding of the mechanisms involved in insulin dysregulation in this species is incomplete. C-peptide is co-secreted with insulin but is resistant to hepatic metabolism and can be used to study insulin dysregulation. This study examined C-peptide secretion in serial blood samples collected after oral and i.v. dextrose (0.75 g/kg) administration to 9 ponies (BCS, 7.1 ± 0.5). The ponies were designated as hyperinsulinemic (HI) or normoinsulinemic (NI) responders before the study, using oral glucose tests and fasted glucose-to-insulin ratios, and responses were compared between the 2 groups. C-peptide concentrations increased ( < 0.01) rapidly from fasted levels after both oral and i.v. dextrose, with similar area under the concentration-time curve (AUC) for both tests and a significant correlation with AUC. The AUC was similar in HI and NI ponies after i.v. dextrose, indicating similar pancreatic capacity for both groups. However, for oral dextrose, the AUC and the AUC were markedly higher ( < 0.05) in the HI ponies, indicating a greater secretion rate of these peptides. Slower insulin clearance might have also contributed to the larger AUC in HI ponies, but this hypothesis requires further investigation with specific measures of hepatic insulin clearance.
Assuntos
Glicemia/análise , Peptídeo C/efeitos dos fármacos , Glucose/administração & dosagem , Cavalos/fisiologia , Administração Intravenosa/veterinária , Administração Oral , Animais , Peptídeo C/metabolismo , Feminino , Hiperinsulinismo/sangue , Hiperinsulinismo/veterinária , Insulina/sangue , Masculino , Pâncreas/metabolismoRESUMO
Compared with some other species, insulin dysregulation in equids is poorly understood. However, hyperinsulinemia causes laminitis, a significant and often lethal disease affecting the pedal bone/hoof wall attachment site. Until recently, hyperinsulinemia has been considered a counterregulatory response to insulin resistance (IR), but there is growing evidence to support a gastrointestinal etiology. Incretin hormones released from the proximal intestine, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide, augment insulin secretion in several species but require investigation in horses. This study investigated peripheral and gut-derived factors impacting insulin secretion by comparing the response to intravenous (iv) and oral d-glucose. Oral and iv tests were performed in 22 ponies previously shown to be insulin dysregulated, of which only 15 were classified as IR (iv test). In a more detailed study, nine different ponies received four treatments: d-glucose orally, d-glucose iv, oats, and commercial grain mix. Insulin, glucose, and incretin concentrations were measured before and after each treatment. All nine ponies showed similar iv responses, but five were markedly hyperresponsive to oral d-glucose and four were not. Insulin responsiveness to oral d-glucose was strongly associated with blood glucose concentrations and oral glucose bioavailability, presumably driven by glucose absorption/distribution, as there was no difference in glucose clearance rates. Insulin was also positively associated with the active amide of GLP-1 following d-glucose and grain. This study has confirmed a functional enteroinsular axis in ponies that likely contributes to insulin dysregulation that may predispose them to laminitis. Moreover, iv tests for IR are not reliable predictors of the oral response to dietary nonstructural carbohydrate.
Assuntos
Trato Gastrointestinal/metabolismo , Doenças dos Cavalos/metabolismo , Cavalos/metabolismo , Hiperinsulinismo/metabolismo , Resistência à Insulina , Insulina/metabolismo , Ração Animal , Animais , Dieta , Carboidratos da Dieta/farmacologia , Células Enteroendócrinas/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Teste de Tolerância a Glucose/métodos , Teste de Tolerância a Glucose/veterinária , Hiperinsulinismo/veterinária , Incretinas/metabolismoRESUMO
REASONS FOR PERFORMING STUDY: An increased incidence of metabolic disease in horses has led to heightened recognition of the pathological consequences of insulin resistance. Laminitis, failure of the weightbearing digital lamellae, is an important consequence. Altered trafficking of specialised glucose transporters (GLUTs), responsible for glucose uptake, is central to the dysregulation of glucose metabolism and may play a role in the pathophysiology of laminitis. OBJECTIVES: We hypothesised that prolonged hyperinsulinaemia alters the regulation of glucose transport in insulin-sensitive tissue and digital lamellae. Our objectives were to compare the relative protein expression of major GLUT isoforms in striated muscle and digital lamellae in healthy horses and during marked and moderate hyperinsulinaemia. STUDY DESIGN: Randomised, controlled study. METHODS: Prolonged hyperinsulinaemia and lamellar damage were induced by a prolonged euglycaemic-hyperinsulinaemic clamp or a prolonged glucose infusion, and results were compared with those of electrolyte-treated control animals. Protein expression of GLUTs was examined with immunoblotting. RESULTS: Lamellar tissue contained more GLUT1 protein than skeletal muscle (P = 0.002) and less GLUT4 than the heart (P = 0.037). During marked hyperinsulinaemia and acute laminitis (induced by the prolonged euglycaemic-hyperinsulinaemic clamp), GLUT1 protein expression was decreased in skeletal muscle (P = 0.029) but unchanged in the lamellae, while novel GLUTs (8 and 12) were increased in the lamellae (P = 0.03) but not in skeletal muscle. However, moderate hyperinsulinaemia and subclinical laminitis (induced by the prolonged glucose infusion) did not cause differential GLUT protein expression in the lamellae compared with control horses. CONCLUSIONS: The results suggest that lamellar tissue functions independently of insulin and that insulin resistance may not be an essential component of the aetiology of laminitis. Marked differences in GLUT expression exist between insulin-sensitive and insulin-independent tissues during metabolic dysfunction in horses. The different expression profiles of novel GLUTs during acute and subclinical laminitis may be important to disease pathophysiology and require further investigation.
Assuntos
Glucose/metabolismo , Cavalos/metabolismo , Hiperinsulinismo/veterinária , Músculo Esquelético/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Técnica Clamp de Glucose/veterinária , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismoRESUMO
Equine laminitis, a disease of the lamellar structure of the horse's hoof, can be incited by numerous factors that include inflammatory and metabolic aetiologies. However, the role of inflammation in hyperinsulinaemic laminitis has not been adequately defined. Toll-like receptor (TLR) activation results in up-regulation of inflammatory pathways and the release of pro-inflammatory cytokines, including interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), and may be a pathogenic factor in laminitis. The aim of this study was to determine whether TLR4 expression and subsequent pro-inflammatory cytokine production is increased in lamellae and skeletal muscle during equine hyperinsulinaemia. Standardbred horses were treated with either a prolonged, euglycaemic hyperinsulinaemic clamp (p-EHC) or a prolonged, glucose infusion (p-GI), which induced marked and moderate hyperinsulinaemia, respectively. Age-matched control horses were treated simultaneously with a balanced electrolyte solution. Treated horses developed clinical (p-EHC) or subclinical (p-GI) laminitis, whereas controls did not. Skeletal muscle and lamellar protein extracts were analysed by Western blotting for TLR4, IL-6, TNF-α and suppressor of cytokine signalling 3 (SOCS3) expression. Lamellar protein expression of TLR4 and TNF-α, but not IL-6, was increased by the p-EHC, compared to control horses. A significant positive correlation was found between lamellar TLR4 and SOCS3. Skeletal muscle protein expression of TLR4 signalling parameters did not differ between control and p-EHC-treated horses. Similarly, the p-GI did not result in up-regulation of lamellar protein expression of any parameter. The results suggest that insulin-sensitive tissues may not accurately reflect lamellar pathology during hyperinsulinaemia. While TLR4 is present in the lamellae, its activation appears unlikely to contribute significantly to the developmental pathogenesis of hyperinsulinaemic laminitis. However, inflammation may have a role to play in the later stages (e.g., repair or remodelling) of the disease.
Assuntos
Doenças do Pé/veterinária , Regulação da Expressão Gênica/imunologia , Casco e Garras/imunologia , Doenças dos Cavalos/imunologia , Hiperinsulinismo/veterinária , Receptor 4 Toll-Like/imunologia , Animais , Western Blotting/veterinária , Citocinas/análise , Citocinas/imunologia , Doenças do Pé/etiologia , Doenças do Pé/genética , Doenças do Pé/imunologia , Doenças dos Cavalos/etiologia , Doenças dos Cavalos/genética , Cavalos , Hiperinsulinismo/imunologia , Músculo Esquelético/imunologia , Distribuição Aleatória , Estatísticas não Paramétricas , Receptor 4 Toll-Like/genéticaRESUMO
REASONS FOR PERFORMING THE STUDY: As growth hormone increases lean body mass, it could be a therapy for obese horses. However, growth hormone use induces hyperinsulinaemia in some species, so further investigation is warranted. OBJECTIVES: To investigate the effects of feeding, exercise and growth hormone therapy on basal insulin concentrations in healthy horses. STUDY DESIGN: In vivo experimental study. METHODS: Blood samples were obtained every 30 min from 12 geldings over 24 h, to establish basal serum insulin concentrations, before they underwent a 3-week exercise programme. Horses were allocated into 2 groups and exercised for another 4 weeks. Group A received daily i.m. injections of recombinant equine growth hormone; 5 mg/day for 5 days, then 12.5 mg/day for 16 days. Blood samples were taken daily before feeding. Insulin vs. time area under curve of Groups A and B were compared using a Student's unpaired t test. RESULTS: Horses demonstrated insulin peaks within 2 h of feeding of 577 ± 108.3 pmol/l at 09.30 h and 342.4 ± 75.7 pmol/l at 17.30 h, despite receiving the same meal. The nadir was between midnight and 07.30 h. Exercise had no effect on basal insulin concentrations prior to equine growth hormone administrations. The equine growth hormone injections increased serum insulin concentrations (P = 0.01) within Group A, from 44.4 ± 15.3 pmol/l initially to 320.9 ± 238.2 pmol/l by Day 12. Exogenous growth hormone caused variable hyperinsulinaemia, which was alleviated once equine growth hormone administration ceased. CONCLUSIONS: Single serum samples taken prior to the morning meal provide basal insulin concentrations. Exercise did not change basal insulin concentrations. However, equine growth hormone injections increased basal insulin concentrations, which were not ameliorated by exercise. POTENTIAL RELEVANCE: This therapy is not recommended to address obesity in insulin-resistant equids.
Assuntos
Criação de Animais Domésticos/métodos , Ritmo Circadiano/fisiologia , Hormônio do Crescimento/farmacologia , Cavalos/metabolismo , Insulina/sangue , Condicionamento Físico Animal/fisiologia , Animais , Relação Dose-Resposta a Droga , Hormônio do Crescimento/administração & dosagem , MasculinoRESUMO
Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6h, 12h and 24h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.
Assuntos
Doenças do Pé/veterinária , Produtos Finais de Glicação Avançada/análise , Casco e Garras/química , Doenças dos Cavalos/imunologia , Animais , Sequência de Bases , Clonagem Molecular , Transportador 2 de Aminoácido Excitatório/análise , Doenças do Pé/imunologia , Doenças do Pé/metabolismo , Regulação da Expressão Gênica/imunologia , Transportador de Glucose Tipo 4/análise , Casco e Garras/imunologia , Doenças dos Cavalos/metabolismo , Cavalos/genética , Cavalos/imunologia , Cavalos/metabolismo , Peroxidação de Lipídeos/imunologia , Dados de Sequência Molecular , Estresse Oxidativo/imunologia , Reação em Cadeia da Polimerase/veterinária , Espécies Reativas de Oxigênio/análise , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/análise , Receptores Imunológicos/genéticaRESUMO
Determining circulating equine insulin concentrations is becoming increasingly important in equine clinical practice and research. Most available assays are optimized for human medicine, but there is strong equine cross-reactivity because of the highly conserved nature of insulin. To identify an accurate and reliable assay for equine insulin, 6 commercial immunoassays were evaluated for precision, accuracy, and specificity. Only 1 assay initially reached the requisite standard: Mercodia Equine Insulin Enzyme-linked Immunosorbent assay (ELISA). Plasma matrix interferences were identified when the provided assay buffer was used with the Siemens Count-a-Coat Insulin radioimmunoassay (RIA) but not when charcoal-stripped equine plasma was used as the diluent. This modified RIA and the Mercodia Equine Insulin ELISA were evaluated further by directly examining accuracy by comparing their results for 18 equine plasma samples with values obtained using liquid chromatography and high-resolution/high-accuracy mass spectrometry (LC-MS). Compared with LC-MS measurements, the modified Siemens Insulin RIA rendered a moderate Lin's concordance coefficient (ρ(c)) of 0.41, whereas the Mercodia Equine Insulin ELISA rendered a very poor ρ(c) of 0.06. This suggests that the Siemens Insulin RIA is appropriate to use for routine evaluations when LC-MS is not available.
Assuntos
Cavalos/sangue , Insulina/sangue , Radioimunoensaio/veterinária , Animais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
REASONS FOR PERFORMING STUDY: Several conditions associated with laminitis in horses are also associated with insulin resistance, which represents the failure of glucose uptake via the insulin-responsive glucose transport proteins in certain tissues. Glucose starvation is a possible mechanism of laminitis, but glucose uptake mechanisms in the hoof are not well understood. OBJECTIVES: To determine whether glucose uptake in equine lamellae is dependent on insulin, to characterise the glucose transport mechanism in lamellae from healthy horses and ponies, and to compare this with ponies with laminitis. METHODS: Study 1 investigated the effects of insulin (300 µU/ml; acute and 24 h) and various concentrations of glucose up to 24 mmol/l, on 2-deoxy-D-[2,6-(3)H] glucose uptake in hoof lamellar explants in vitro. Study 2 measured the mRNA expression of GLUT1 and GLUT4 transport proteins by PCR analysis in coronary band and lamellar tissue from healthy horses and ponies, ponies with insulin-induced laminitis, and ponies suffering from chronic laminitis as a result of equine Cushing's syndrome. RESULTS: Glucose uptake was not affected by insulin. Furthermore, the relationship between glucose concentration and glucose uptake was consistent with an insulin-independent glucose transport system. GLUT1 mRNA expression was strong in brain, coronary band and lamellar tissue, but was weak in skeletal muscle. Expression of GLUT4 mRNA was strong in skeletal muscle, but was either absent or barely detectable in coronary band and lamellar tissue. CONCLUSIONS: The results do not support a glucose deprivation model for laminitis, in which glucose uptake in the hoof is impaired by reduced insulin sensitivity. Hoof lamellae rely on a GLUT1-mediated glucose transport system, and it is unlikely that GLUT4 proteins play a substantial role in this tissue. POTENTIAL RELEVANCE: Laminitis associated with insulin resistance is unlikely to be due to impaired glucose uptake and subsequent glucose deprivation in lamellae.