RESUMO
Pancreatic amyloid formation by islet amyloid polypeptide (IAPP) is a hallmark pathological feature of type 2 diabetes. IAPP is stored in the secretory granules of pancreatic beta-cells and co-secreted with insulin to maintain glucose homeostasis. IAPP is innocuous under homeostatic conditions but imbalances in production or processing of IAPP may result in homodimer formation leading to the rapid production of cytotoxic oligomers and amyloid fibrils. The consequence is beta-cell dysfunction and the accumulation of proteinaceous plaques in and around pancreatic islets. Beta-site APP-cleaving enzyme 2, BACE2, is an aspartyl protease commonly associated with BACE1, a related homolog responsible for amyloid processing in the brain and strongly implicated in Alzheimer's disease. Herein, we identify two distinct sites of the mature human IAPP sequence that are susceptible to BACE2-mediated proteolytic activity. The result of proteolysis is modulation of human IAPP fibrillation and human IAPP protein degradation. These results suggest a potential therapeutic role for BACE2 in type 2 diabetes-associated hyperamylinaemia.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Insulina/metabolismo , Insulina/farmacologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mutação , Placa Amiloide/metabolismo , Proteólise/efeitos dos fármacos , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
Trefoil factor 3 (TFF3), also called intestinal trefoil factor or Itf, is a 59 amino acid peptide found as a homodimer predominantly along the gastrointestinal tract and in serum. TFF3 expression is elevated during gastrointestinal adenoma progression and has been shown to promote mucosal wound healing. Here we show that in contrast to other trefoil factor family members, TFF1 and TFF2, TFF3 is highly expressed in mouse duodenum, jejunum and ileum and that its expression is regulated by food intake. Overexpression of TFF3 using a recombinant adeno-associated virus (AAV) vector, or daily administration of recombinant TFF3 protein in vivo improved glucose tolerance in a diet-induced obesity mouse model. Body weight, fasting insulin, triglyceride, cholesterol and leptin levels were not affected by TFF3 treatment. Induction of mucinous metaplasia was observed in mice with AAV-mediated TFF3 overexpression, however, no such adverse histological effect was seen after the administration of recombinant TFF3 protein. Altogether these results suggest that the therapeutic potential of targeting TFF3 to treat T2D may be limited.
Assuntos
Glicemia/metabolismo , Ingestão de Alimentos/genética , Vetores Genéticos/efeitos adversos , Metaplasia/genética , Mucinas/genética , Obesidade/genética , Animais , Colesterol/sangue , Dependovirus/genética , Dieta Hiperlipídica , Duodeno/metabolismo , Duodeno/patologia , Expressão Gênica , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Teste de Tolerância a Glucose , Humanos , Íleo/metabolismo , Íleo/patologia , Insulina/sangue , Jejuno/metabolismo , Jejuno/patologia , Leptina/sangue , Masculino , Metaplasia/etiologia , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Mucinas/administração & dosagem , Mucinas/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fator Trefoil-2 , Fator Trefoil-3 , Triglicerídeos/sangueRESUMO
Free fatty acids (FFAs) play important physiological roles in many tissues as an energy source and as signaling molecules in various cellular processes. Elevated levels of circulating FFAs are associated with obesity, dyslipidemia, and diabetes. Here we show that GPR84, a previously orphan G protein-coupled receptor, functions as a receptor for medium-chain FFAs with carbon chain lengths of 9-14. Medium-chain FFAs elicit calcium mobilization, inhibit 3',5'-cyclic AMP production, and stimulate [35S]guanosine 5'-O-(3-thiotriphosphate) binding in a GPR84-dependent manner. The activation of GPR84 by medium-chain FFAs couples primarily to a pertussis toxin-sensitive G(i/o) pathway. In addition, we show that GPR84 is selectively expressed in leukocytes and markedly induced in monocytes/macrophages upon activation by lipopolysaccharide. Furthermore, we demonstrate that medium-chain FFAs amplify lipopolysaccharide-stimulated production of the proinflammatory cytokine interleukin-12 p40 through GPR84. Our results indicate a role for GPR84 in directly linking fatty acid metabolism to immunological regulation.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Equorina/metabolismo , Animais , Medula Óssea , Células CHO , Cálcio/metabolismo , Sinalização do Cálcio , Cricetinae , AMP Cíclico/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Citometria de Fluxo , Imunofluorescência , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Leucócitos/metabolismo , Ligantes , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Camundongos , Monócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Local catabolism of the essential amino acid tryptophan is considered an important mechanism in regulating immunological and neurological responses. The kynurenine pathway is the main route for the non-protein metabolism of tryptophan. The intermediates of the kynurenine pathway are present at micromolar concentrations in blood and are regulated by inflammatory stimuli. Here we show that GPR35, a previously orphan G protein-coupled receptor, functions as a receptor for the kynurenine pathway intermediate kynurenic acid. Kynurenic acid elicits calcium mobilization and inositol phosphate production in a GPR35-dependent manner in the presence of G(qi/o) chimeric G proteins. Kynurenic acid stimulates [35S]guanosine 5'-O-(3-thiotriphosphate) binding in GPR35-expressing cells, an effect abolished by pertussis toxin treatment. Kynurenic acid also induces the internalization of GPR35. Expression analysis indicates that GPR35 is predominantly detected in immune cells and the gastrointestinal tract. Furthermore, we show that kynurenic acid inhibits lipopolysaccharide-induced tumor necrosis factor-alpha secretion in peripheral blood mononuclear cells. Our results suggest unexpected signaling functions for kynurenic acid through GPR35 activation.