RESUMO
BACKGROUND: Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM. METHODS: Using rat models of diet-induced obesity (DIO), T1DM and T2DM (n = 14 per group), obestatin, its precursor protein preproghrelin, and GPR39 expression was investigated in tissues involved in glucose and lipid homeostasis regulation. Furthermore, serum obestatin and ghrelin concentrations were determined. RESULTS: Serum obestatin concentrations were positively correlated with glucagon (r = 0.6456; P < 0.001) and visfatin (r = 0.5560; P < 0.001), and negatively correlated with insulin (r = -0.4362; P < 0.05), adiponectin (r = -0.3998; P < 0.05), and leptin (r = -0.4180; P < 0.05). There were differences in GPR39 and preproghrelin expression in the three animal models. Hepatic GPR39 and preproghrelin mRNA expression was greater in T1DM, T2DM, and obese rats than in lean controls, whereas pancreatic GPR39 mRNA and protein and preproghrelin mRNA expression was decreased in T1DM, T2DM, and DIO rats. Higher GPR39 and preproghrelin protein and mRNA levels were found in adipose tissues of T1DM compared with control. In adipose tissues of T2DM and DIO rats, GPR39 protein levels were lower than in lean or T1DM rats. Preproghrelin mRNA was higher in adipose tissues of T1DM, T2DM, and DIO than lean rats. CONCLUSION: We hypothesize that changes in obestatin, GPR39, and ghrelin may contribute to metabolic abnormalities in T1DM, T2DM, and obesity.
Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Expressão Gênica , Obesidade/fisiopatologia , Hormônios Peptídicos/genética , Receptores Acoplados a Proteínas G/genética , Análise de Variância , Animais , Glicemia/metabolismo , Western Blotting , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Grelina/sangue , Grelina/genética , Grelina/metabolismo , Glucagon/sangue , Insulina/sangue , Masculino , Obesidade/sangue , Hormônios Peptídicos/sangue , Hormônios Peptídicos/metabolismo , Ratos Wistar , Receptores Acoplados a Proteínas G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/sangueRESUMO
Resistin participates in the regulation of energy homeostasis, insulin resistance, and inflammation. The potential expression in pancreas, and modulation of the endocrine pancreas secretion by resistin is not well characterized, therefore, we examined it on several levels. We examined the localization of resistin in rat pancreatic islets by immunohistochemistry and immunofluorescence, and the potential presence of resistin mRNA by RT-PCR and protein by Western Blot in these structures. In addition, we studied the regulation of insulin and glucagon secretion by resistin in pancreatic INS-1E ß- and InR-G9 α-cell lines as well as isolated rat pancreatic islets. We identified resistin immunoreactivity in the periphery of rat pancreatic islets and confirmed the expression of resistin at mRNA and protein level. Obtained data indicated that resistin is co-localized with glucagon in pancreatic α-cells. In addition, we found that in vitro resistin decreased insulin secretion from INS-1E cells and pancreatic islets at normal (6 mM) and high (24 mM) glucose concentrations, and also decreased glucagon secretion from G9 cells and pancreatic islets at 1 mM, whereas a stimulation of glucagon secretion was observed at 6 mM glucose. Our results suggest that resistin can modulate the secretion of insulin and glucagon from clonal ß or α cells, and from pancreatic islets.
Assuntos
Glucagon/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Resistina/metabolismo , Animais , Linhagem Celular , Células Secretoras de Glucagon/metabolismo , Glucose/farmacologia , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Resistina/genética , Resistina/farmacologiaRESUMO
Obestatin is a 23-amino acid peptide encoded by the ghrelin gene, which regulates food intake, body weight and insulin sensitivity. Obestatin influences glucose and lipid metabolism in mature adipocytes in rodents. However, the role of this peptide in rat preadipocytes remains to be fully understood. The current study characterized the effects of obestatin on lipid accumulation, preadipocyte differentiation, lipolysis and leptin secretion in rat primary preadipocytes. Obestatin enhanced lipid accumulation in rat preadipocytes and increased the expression of surrogate markers of preadipocyte differentiation. At the early stage of differentiation, obestatin suppressed lipolysis. By contrast, lipolysis was stimulated at the late stage of adipogenesis. Furthermore, obestatin stimulated the release of leptin, a key satiety hormone. Overall, the results indicated that obestatin promotes preadipocyte differentiation. Obestatin increased leptin release in preadipocytes, while the modulation of lipolysis appears to depend upon the stage of differentiation.
Assuntos
Adipócitos/metabolismo , Diferenciação Celular/genética , Leptina/metabolismo , Lipólise/genética , Hormônios Peptídicos/fisiologia , Animais , Metabolismo dos Lipídeos/genética , Masculino , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Ratos , Ratos Wistar , Triglicerídeos/metabolismoRESUMO
AIMS/HYPOTHESIS: Orexin A (OXA) modulates food intake, energy expenditure, and lipid and glucose metabolism. OXA regulates the secretion of insulin and glucagon, while glucose regulates OXA release. Here, we evaluate the role of glucagon in regulating OXA release both in vivo and in vitro. METHODS: In a double-blind crossover study, healthy volunteers and type 1 diabetic patients received either intramuscular glucagon or placebo. Patients newly diagnosed with type 2 diabetes underwent hyperinsulinaemic-euglycaemic clamp experiments, and insulin-hypoglycaemia tests were performed on healthy volunteers. The primary endpoint was a change in OXA levels after intramuscular glucagon or placebo administration in healthy participants and patients with type 1 diabetes. Secondary endpoints included changes in OXA in healthy participants during insulin tolerance tests and in patients with type 2 diabetes under hyperinsulinaemic-euglycaemic conditions. Participants and staff conducting examinations and taking measurements were blinded to group assignment. OXA secretion in response to glucagon treatment was assessed in healthy and obese mice, the streptozotocin-induced mouse model of type 1 diabetes, and isolated rat pancreatic islets. RESULTS: Plasma OXA levels declined in lean volunteers and in type 1 diabetic patients injected with glucagon. OXA levels increased during hyperinsulinaemic hypoglycaemia testing in healthy volunteers and during hyperinsulinaemic euglycaemic conditions in type 2 diabetic patients. Plasma OXA concentrations in healthy lean and obese mice and in a mouse model of type 1 diabetes were lower after glucagon treatment, compared with vehicle control. Glucagon decreased OXA secretion from isolated rat pancreatic islets at both low and high glucose levels. OXA secretion declined in pancreatic islets exposed to diazoxide at high and low glucose levels, and after exposure to an anti-insulin antibody. Glucagon further reduced OXA secretion in islets pretreated with diazoxide or an anti-insulin antibody. CONCLUSIONS/INTERPRETATION: Glucagon inhibits OXA secretion in humans and animals, irrespective of changes in glucose or insulin levels. Through modifying OXA secretion, glucagon may influence energy expenditure, body weight, food intake and glucose metabolism.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Glucagon/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neuropeptídeos/metabolismo , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adulto , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Método Duplo-Cego , Feminino , Glucose/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Orexinas , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismoRESUMO
Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 2/sangue , Glucagon/antagonistas & inibidores , RNA/metabolismo , Animais , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/farmacocinética , Aptâmeros de Nucleotídeos/uso terapêutico , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biossíntese , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Modelos Animais de Doenças , Jejum/sangue , Glucagon/metabolismo , Teste de Tolerância a Glucose , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A case of a 49-year-old male with exacerbation of eosinophilic granulomatosis with polyangiitis (EGPA) with heart involvement mimicking acute coronary syndrome is presented. Institution of intensive immunosupresive treatment resulted in the improvement of clinical condition and systolic left ventricular function. Coronary angiography excluded atherosclerosis as a primary cause of heart damage.
Assuntos
Síndrome de Churg-Strauss/diagnóstico , Disfunção Ventricular Esquerda/diagnóstico , Síndrome Coronariana Aguda/diagnóstico , Angiografia Coronária , Diagnóstico Diferencial , Eletrocardiografia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Neuropeptide B (NPB) and W (NPW) regulate food intake and energy homeostasis in humans via two G-protein-coupled receptor subtypes, termed as GPR7 and GPR8. Rodents express GPR7 only. In animals, NPW decreases insulin and leptin levels, whereas the deletion of either NPB or GPR7 leads to obesity and hyperphagia. Metabolic and endocrine in vitro activities of NPW/NPB in adipocytes are unknown. We therefore characterize the effects of NPB and NPW on the secretion and expression of leptin and resistin, and on lipolysis, using rat adipocytes. Isolated rat adipocytes express GPR7 mRNA. NPB and NPW are expressed in macrophages and preadipocytes but are absent in mature adipocytes. Both, NPB and NPW reduce the secretion and expression of leptin from isolated rat adipocytes. NPB stimulates the secretion and expression of resistin, whereas both, NPB and NPW increase lipolysis. Our study demonstrates for the first time that NPB and NPW regulate the expression and secretion of leptin and resistin, and increase lipolysis in isolated rat adipocytes. These effects are presumably mediated via GPR7. The increase of resistin secretion, stimulation of lipolysis and the decrease of leptin secretion may represent mechanisms, through which NPB and NPW can affect glucose and lipid homeostasis, and food intake in rodents.
Assuntos
Adipócitos/metabolismo , Leptina/metabolismo , Lipólise , Neuropeptídeos/metabolismo , Resistina/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Lipólise/efeitos dos fármacos , Masculino , Neuropeptídeos/farmacologia , Ratos , Ratos WistarRESUMO
BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition. METHODS: Factors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2 ± 10.1 years; a mean forced expiratory volume in 1 second [FEV(1)], 55.6 ± 15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally. RESULTS: COPD patients had higher fII (115 ± 16 vs 102 ± 10%, p < 0.0001), fV (114 ± 19 vs 102 ± 12%, p = 0.0002), fVII (111 ± 15 vs 102 ± 17%, p = 0.002), fVIII (170 ± 34 vs 115 ± 27%, p < 0.0001), and fIX (119 ± 21 vs 107 ± 17%, p = 0.003), and lower fTFPI (17.7 ± 3.2 vs 18.9 ± 3.2 ng/ml, p = 0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+28.3%, p < 0.0001), rates of thrombin generation (+46.1%, p < 0.0001) and total thrombin formation (+14.4%, p < 0.001), together with shorter initiation phase of thrombin generation (p < 0.0001) and the time to maximum thrombin levels (p < 0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII , fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles. CONCLUSIONS: Prothrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.
Assuntos
Inibidores dos Fatores de Coagulação Sanguínea/sangue , Fatores de Coagulação Sanguínea/metabolismo , Coagulação Sanguínea , Doença Pulmonar Obstrutiva Crônica/sangue , Trombina/metabolismo , Idoso , Antitrombinas/sangue , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Feminino , Volume Expiratório Forçado , Humanos , Cinética , Lipoproteínas/sangue , Pulmão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Polônia , Proteína C/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Capacidade VitalRESUMO
INTRODUCTION: Increased cardiovascular mortality and risk of venous thromboembolism are serious extra-pulmonary complications of chronic obstructive pulmonary disease (COPD). Previously, circulating active tissue factor (TF) and factor XIa (FXIa) have been reported to be associated with acute coronary syndromes. OBJECTIVE: To measure plasma FXIa and active TF, prothrombin fragment 1.2 (F1.2), and markers of systemic inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], tumor necrosis factor α [TNFα] and matrix metalloproteinase 9 [MMP-9]) in 60 patients with documented stable COPD free of previous thromboembolic events. METHODS: In-house clotting assays using inhibitory monoclonal antibodies against FXIa and TF. RESULTS: FXIa was detected in 9 (15%) and TF activity in 7 (11.7%) COPD patients. Subjects positive for FXIa and/or TF (n=10; 16.7%) had higher F1.2 (median [interquartile range], 398 [216] vs 192 [42] pM, p<0.000001), fibrinogen (5.58 [2.01] vs 3.97 [2.47] g/L, p=0.0007), CRP (14.75 [1.20] vs 1.88 [2.95] mg/L, p<0.000001), IL-6 (8.14 [4.74] vs 2.45 [2.24] pg/mL, p=0.00002), and right ventricular systolic pressure (47 [15] vs 38 [12] mmHg, p=0.023), and lower vital capacity (66 [15] vs 80 [17] % predicted, p=0.04) than COPD patients without detectable FXIa and TF. COPD severity was not associated with the presence of circulating FXIa and active TF. CONCLUSIONS: This is the first study to show that active FXIa and TF are present in stable COPD patients, who exhibit enhanced systemic inflammation and thrombin generation. Our findings suggest a new prothrombotic mechanism which might contribute to elevated risk of thromboembolic complications in COPD.
Assuntos
Fator XIa/metabolismo , Inflamação/complicações , Doença Pulmonar Obstrutiva Crônica/complicações , Trombina/metabolismo , Tromboplastina/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
INTRODUCTION: There is growing evidence that chronic obstructive pulmonary disease (COPD) is a risk factor for coronary heart disease. Simvastatin is a hypolipemic drug with proven efficacy in the prevention of cardiovascular diseases. Observational studies showed that statins may be useful in the reduction of mortality from COPD. Experimental studies on animals showed anti-inflammatory effects of statins on the lung tissue. OBJECTIVES: The aim of this study was to evaluate the influence of simvastatin on inflammatory markers in patients with COPD. PATIENTS AND METHODS: Fifty-six patients (aged 44-80 years) with stable COPD (a mean forced expiratory volume in 1 second [FEV1] 55%), were randomly assigned (1:1) to receive simvastatin 40 mg/day or to receive no statin treatment. Blood samples were collected before, 2 weeks, and 3 months after statin administration. The levels of fibrinogen, C-reactive protein (CRP), tumor necrosis factor-alpha, interleukin 6 (IL-6), and matrix metalloproteinase-9 were measured. RESULTS: The groups did not differ significantly in terms of demographic data, clinical symptoms, pharmacological treatment, spirometry, and lipid profile at baseline. Among comorbidities only arterial hypertension was more frequent in the statin group (32.1% vs. 17.9%, P = 0.03). After 2 weeks as well as 3 months of simvastatin treatment, no significant reduction of any measured inflammatory markers was observed. There was a nonsignificant reduction of CRP and IL-6 in the subgroup with FEV1 >50% during simvastatin treatment. There was a decrease in total cholesterol (from 5.7 to 4.7 mmol/l, P = 0.0018) and low-density lipoprotein cholesterol (from 3.46 to 2.47 mmol/l, P = 0.000037) in the statin group. CONCLUSIONS: In COPD patients, a 3-month treatment with simvastatin does not reduce circulating inflammatory markers.
Assuntos
Proteína C-Reativa/efeitos dos fármacos , Interleucina-6/metabolismo , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Sinvastatina/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Biomarcadores/metabolismo , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Masculino , Pessoa de Meia-IdadeRESUMO
In the present study, the influence of chromium(III) complexes (acetate, chloride, glycinate, histidinate, lactate and propionate) on insulin binding and signal transduction [phosphorylation of tyrosine and serine in the insulin receptor substrate (IRS)-1] was investigated in vitro using three experimental models: isolated rat liver membranes and cultured mouse C2C12 myoblasts or 3T3-L1 preadipocytes. The examined complexes did not elevate the binding of insulin to the liver membranes. Moreover, chromium histidinate, lactate, acetate and propionate complexes diminished the specific binding of insulin. Simultaneously, chromium chloride, which did not significantly elevate insulin binding, increased the number of membrane accessible particles of the insulin receptors. However, it was accompanied by slightly diminished affinity of the receptor to the hormone. Chromium acetate and propionate significantly diminished the binding capacity of the low-affinity insulin receptor class. Investigations with the myoblast cell line C2C12 and preadipocyte cell line 3T3-L1 did not allow differentiation of the influence of the examined complexes on insulin binding. Immunodetection of phosphorylated forms of IRS-1 showed that the chromium compounds modulated the transduction of the insulin signal. Chromium glycinate, acetate and propionate decreased the amount of IRS-1 phosphorylated at serine. Since it is generally thought that phosphorylation of serine in IRS-1 may moderate insulin action, the above mentioned chromium complexes may, in this way, enhance insulin effects inside target cells. Phosphorylation of tyrosine in IRS-1, which acts as a stimulatory signal for further steps of insulin action, was elevated after the incubation of 3T3-L1 cells with insulin. Chromium supplementation did not additionally intensify this process. However, in the absence of insulin, chromium glycinate and acetate slightly elevated the level of IRS-1 phosphorylated at tyrosine. This fact may be important in vivo at low levels of insulin in blood. The results indicate that the action of chromium(III) complexes involves a direct effect on the number of receptors accessible to insulin, their affinity to the hormone and the modulation of the signal multiplying proteins by their phosphorylation.
RESUMO
Increased risk of thrombotic events occurs in chronic obstructive pulmonary disease (COPD). Elevated fibrinogen and C-reactive protein (CRP), being common in COPD, are associated with formation of dense fibrin clots resistant to lysis. Statins have been found to display anti-inflammatory and antithrombotic effects. We investigated fibrin clot properties in COPD patients prior to and following statin therapy. Ex vivo plasma fibrin clot permeability, compaction, and fibrinolysis were assessed in 56 patients with stable COPD, aged 64.9 +/- 9.2 years (mean FEV(1), 54.7 +/- 15.9% predicted), versus 56 controls matched for age, sex and cardiovascular risk factors. Patients were then randomly assigned to receive simvastatin 40 mg/day (n = 28) or to remain without statins for three months (n = 28). Patients with COPD had lower clot permeability (6.1+/- 1.07 versus 9.2 +/- 0.9 10(-9) cm(2), p < 0.0001), decreased compaction (44.9 +/- 4.5 versus 63.9 +/- 6.1%, p < 0.0001), higher maximum D-dimer levels released from clots (4.23 +/- 0.55 versus 3.53 +/- 0.31 mg/l, p < 0.0001) with a decreased rate of this release (75.0 +/- 8.3 versus 80.9 +/- 8.0 microg/l/min, p = 0.03) and prolonged lysis time (9.84 +/- 1.33 versus 8.02 +/- 0.84 min, p < 0.0001) compared with controls. Scanning electron microscopy confirmed denser clot structure in COPD. Multiple linear regression analysis after adjustment for age and fibrinogen showed that in the COPD patients, CRP was the only independent predictor of permeability (R(2) = 0.47, p < 0.001) and lysis time (R(2) = 0.43, p < 0.001). Simvastatin improved clot properties (p < 0.05) despite unaltered CRP and irrespective of cholesterol reduction. Our study shows that fibrin clots in COPD patients are composed of much denser networks that are more resistant to lysis, and these properties can be improved by statin administration.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Fibrina/fisiologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Sinvastatina/uso terapêutico , Idoso , Estudos de Casos e Controles , Feminino , Fibrina/efeitos dos fármacos , Fibrina/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/complicações , Trombose/sangue , Trombose/etiologia , Trombose/prevenção & controleRESUMO
Cerebellin (CER) is a neuromodulatory hexadecapeptide that originates from the precursor protein precerebellin (Cbln1). Four highly homologous isoforms of Cbln are known (Cbln1-Cbln4), which are expressed in the central nervous system (CNS) and in peripheral tissues. CER modulates synaptic structure formation in the CNS, whereas in the peripheral tissues CER regulates catecholamine secretion. Cbln is also expressed in the pancreas; however, its function in the pancreas is unknown. Here, we demonstrate the role of CER in regulating insulin secretion in vivo and in vitro. We identified Cbln1 and Cbln3 transcripts in rat pancreatic islets and detected Cbln-immunoreactivity, predominantly located in the periphery of the rat endocrine pancreas. In vivo, CER reduced plasma insulin levels in rats after 1 and 2 h. CER decreased insulin secretion from isolated rat pancreatic islets at high (11 mM), but not at low (3.33 mM) glucose concentration. CER inhibited stimulated insulin secretion from clonal rat insulinoma (INS-1) cells, reduced forskolin-induced production of cAMP and intracellular calcium concentration. Our study demonstrates for the first time that Cbln1 and Cbln3 are expressed in the rat endocrine pancreas. Furthermore, we identify CER as an insulinostatic factor, which decreases intracellular cAMP production and calcium in INS-1 cells.
Assuntos
Insulina/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Animais , Cálcio/análise , Linhagem Celular , AMP Cíclico/análise , Feminino , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Pâncreas Exócrino/efeitos dos fármacos , Pâncreas Exócrino/metabolismo , Precursores de Proteínas/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Agouti-related protein (AGRP) is a homolog of the agouti protein and acts as an antagonist of peptides derived from propiomelanocortin through melanocortin receptors. This peptide is produced mainly in the hypothalamus, particularly during negative energy balance and influences increased food intake. In the hypothalamus, this peptide is co-expressed in arcuate nuclei with neuropeptide Y, another important peptide that regulates energy metabolisms. In our study, we analyzed changes in the Agrp mRNA level in the hypothalamus as well as mRNA and protein levels in placenta during different stages of rat pregnancy. We also investigated the AGRP level in the blood serum. In this study, we found the AGRP level in serum increased, while its gene expression in the hypothalamus increased only up to the 13th day of pregnancy, and decreased on the 18th day. This study demonstrates that AGRP is expressed during late pregnancy in placenta. Moreover, we found that AGRP expression is higher on the 18th than on the 13th day of pregnancy. Our results indicate that AGRP may play an important role during pregnancy in the mother's and, possibly, also in the fetus's energy balance.
Assuntos
Proteína Relacionada com Agouti/sangue , Proteína Relacionada com Agouti/metabolismo , Hipotálamo/metabolismo , Placenta/metabolismo , Gravidez/sangue , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/fisiologia , Animais , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Feminino , Regulação da Expressão Gênica , Idade Gestacional , Troca Materno-Fetal/genética , Troca Materno-Fetal/fisiologia , Gravidez/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
OBJECTIVES: Neuromedin U (NmU) is a neuropeptide with anorexigenic activity. Two receptor subtypes (NmUR1 and NmUR2) confer the effects of NmU on target cells. We have recently demonstrated that NmU reduces insulin secretion from isolated pancreatic islets. Aim of our current study is to investigate the role of somatostatin at mediating the effects of NmU on insulin secretion. METHODS: Expression of NmU in the pancreas was detected by immunohistochemistry. Insulin and somatostatin secretion from in situ perfused rat pancreas and isolated pancreatic islets was measured by radioimmunoassay. The paracrine effects of somatostatin within pancreatic islets were blocked by cyclosomatostatin, a somatostatin receptor antagonist. RESULTS: Receptor subtype NmUR1, but not NmUR2, was expressed in the endocrine pancreas, predominantly in the periphery. Neuromedin U reduced insulin secretion from in situ perfused rat pancreas and stimulated somatostatin secretion from isolated pancreatic islets. Neuromedin U stimulated somatostatin secretion at both physiological and supraphysiological glucose concentrations. Cyclosomatostatin increased insulin secretion and reduced NmU-induced inhibition of insulin secretion. CONCLUSIONS: Neuromedin U reduces insulin and increases somatostatin secretion. Blockade of somatostatin action abolishes the inhibition of insulin secretion by NmU. The results of the study suggest that somatostatin mediates the inhibitory action of NmU on insulin secretion.
Assuntos
Insulina/metabolismo , Neuropeptídeos/farmacologia , Pâncreas/efeitos dos fármacos , Somatostatina/fisiologia , Animais , Secreção de Insulina , Pâncreas/metabolismo , Ratos , Ratos Wistar , Receptores de Neurotransmissores/análise , Receptores de Neurotransmissores/fisiologiaRESUMO
Somatostatin (SST) inhibits Ca(2+) entry into pancreatic B-cells via voltage-operated Ca(2+) channels (VOCCs) of L-type, leading to the suppression of insulin secretion. Activation of R-type channels increases insulin secretion. However, the role of R-type Ca(2+) channels (Ca(V)2.3) in mediating the effects of SST on insulin secretion has not been so far investigated. Here, we identify the SST-receptor subtypes (SSTR) expressed on insulin-producing INS-1 cells by RT-PCR and by functional assays. The role of R-type channels in regulating [Ca(2+)](i) in response to SST-treatment was detected by cell fluorescence imaging and patch-clamp technique. INS-1 expressed SSTR2 and SSTR3 and agonists (ag.) selective for these receptors reduced 10 nM exendin-4/20 mM glucose-stimulated insulin secretion. Surprisingly, SST and SST2-ag. transiently increased [Ca(2+)](i). Subsequently, these agonists led to a decrease in [Ca(2+)](i) below the basal levels. In contrast, SST3-ag. failed to induce a transient peak of [Ca(2+)](i). Instead, a persistent minor suppression of [Ca(2+)](i) was detected from 25 min. R-type channel blocker SNX-482 altered [Ca(2+)](i) in SST- and SST2-ag.-treated cells. Notably, the inhibition of insulin secretion by SST and SST2-ag., but not SST3-ag. was attenuated by SNX-482. Taken together, SST and SSTR2 regulate [Ca(2+)](i) and insulin secretion in INS-1 cells via R-type channels. In contrast, the R-type calcium channel does not mediate the effects of SST3-ag. on insulin secretion. We conclude that R-type channels play a major role in the inhibition of insulin secretion by somatostatin in INS-1 cells.
Assuntos
Canais de Cálcio Tipo R/fisiologia , Insulina/metabolismo , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , Canais de Cálcio Tipo R/efeitos dos fármacos , Fenômenos Eletrofisiológicos , RNA Mensageiro/metabolismo , Ratos , Receptores de Somatostatina/agonistas , Espectrometria de Fluorescência , Venenos de Aranha/farmacologia , Fatores de TempoRESUMO
Neuromedin U (NmU) is a regulatory peptide found in significant concentrations in both the brain and gut of the rat and is named according to its ability to powerfully contract the uterus. Two types of NmU receptors were recently identified and subsequent studies evidenced NmU involvement in the regulation of energy homeostasis. Such a role of neuromedin U suggests that a polypeptide may also be involved in the regulation of adipoinsular axis function. Therefore in the present study we examined the expression of NmU receptors in pancreatic islets using RT-PCR and Western blotting analysis. We also investigated the role of NmU in regulation of insulin secretion in vitro using isolated pancreatic islets. We have confirmed that NmUR1 but not NmUR2 is specifically expressed in isolated rat pancreatic islets. In all tested doses (1, 10, 100 nmol/l) NmU dose- dependently decreased insulin output by isolated pancreatic islets. These inhibitory effects of NmU on insulin secretion may suggest the involvement of NmU in regulating the pancreatic branch of adipoinsular axis function. Thus, NmU can be included in that group of anorectic peptides, which are also involved in the regulation of insulin secretion.
Assuntos
Depressores do Apetite/farmacologia , Antagonistas da Insulina/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Neuropeptídeos/farmacologia , Receptores de Neurotransmissores/metabolismo , Animais , Western Blotting , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Neuropeptídeos/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Receptores de Neurotransmissores/análise , Receptores de Neurotransmissores/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To increase our knowledge concerning the central and peripheral regulation of reproduction in mammals a series of studies were performed. In the first experiment, we found that exogenous leptin altered the activity of the hypothalmo-pituitary-gonadotropic axis in sheep during insufficient feeding. The action of leptin appears to be mediated by changes in GnRH and LH secretion as well as NPY immunoreactivity. The aim of the second experiment was to investigate the role of the adipoinsular axis hormones during pregnancy in rats. The elevated levels of plasma leptin as wells as the increased mRNAs expression of the leptin receptors in placenta indicate the significant role of the hormone in fetal growth and development. On the other hand, a decrease in leptin receptors mRNA content within hypothalamus and pituitary together with unchanged plasma insulin level may suggest that during rat pregnancy leptin resistance was developed in the hypothalamus, pituitary and pancreatic islets. The third experiment was carried out to establish the role of opioids and glucocorticoids in the regulation of the hypothalmo-pituitary-gonadal axis in ewes during natural or synchronized estrous cycle. Prolonged treatment with progesterone resulted in significant changes in plasma levels of Met-enkephalin, cortisol and steroids and altered the expression of proenkephalin mRNA in the hypothalamus, pituitary, ovary and adrenals. Injections of Met-enkephalin or naltrexone (blocker of opioid receptors) modulated the progesterone influence in tested tissues. The data clearly suggest that opioids are involved in the regulation of the estrous cycle at the hypothalamo-pituitary-gonadal/adrenal axes.
Assuntos
Gonadotropinas Hipofisárias/fisiologia , Insulina/fisiologia , Leptina/fisiologia , Mamíferos/fisiologia , Animais , Encefalina Metionina/fisiologia , Hormônios Esteroides Gonadais/fisiologia , Receptores Opioides/fisiologiaRESUMO
Control of processes responsible for food intake and regulation of energy homeostasis during pregnancy is crucial for mother as well as for fetus development. Leptin is one of the main hormonal factors involved in regulation these processes in organisms. During pregnancy leptin regulates mother's energy balance and may also affect fetus growth and development, particularly via receptors in hypothalamus arcuate nuclei (ARC), pituitary and placenta. In the present study, serum leptin levels and expression of both short (ObRs) and long (ObRb) form of leptin receptor in the hypothalamus, pituitary and placenta were measured in the course of pregnancy. The results of these studies indicate that leptin concentration in serum increases during pregnancy and decreases 24 h after the delivery. The expression of both short and long forms of the leptin receptor in the hypothalamus decreases in the course of pregnancy and increases after the delivery. In the pituitary, however, a decrease of leptin receptor mRNA during pregnancy was observed only for ObRb. Analysis of placental leptin receptor expression demonstrated an increase of ObRb and constant high levels of ObRs mRNA. Our results suggest that changes in leptin level and its receptor expression may influence the energy homeostasis during pregnancy. In addition, changes in ObR expression are suggestive for: i) leptin resistance in the hypothalamus and pituitary; and ii) an increased leptin-dependent signaling in the placenta.
Assuntos
Leptina/sangue , Prenhez , Isoformas de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Ingestão de Alimentos , Metabolismo Energético , Feminino , Homeostase , Hipotálamo/metabolismo , Leptina/genética , Hipófise/metabolismo , Placenta/metabolismo , Gravidez , Isoformas de Proteínas/genética , Ratos , Ratos Wistar , Receptores de Superfície Celular/genética , Receptores para LeptinaRESUMO
The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.