RESUMO
The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.
Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Diabetes Mellitus Tipo 2/enzimologia , Células Secretoras de Insulina/enzimologia , Inibidores de Lipoxigenase/farmacologia , Estado Pré-Diabético/enzimologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/patologia , Ativação Enzimática/efeitos dos fármacos , Células Secretoras de Insulina/patologia , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Obesos , Estado Pré-Diabético/tratamento farmacológico , Estado Pré-Diabético/patologiaRESUMO
OBJECTIVE: In type 2 diabetes (T2D), pancreatic ß cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. METHODS: Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. RESULTS: A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was Elovl2, encoding Elongase of very long chain fatty acids 2. Elovl2 silencing decreased glucose-stimulated insulin secretion in mouse and human ß cell lines. CONCLUSION: Our results suggest a role for Elovl2 in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of ß cell failure under metabolic stress.
Assuntos
Acetiltransferases/genética , Diabetes Mellitus Tipo 2/genética , Insulina/metabolismo , Acetiltransferases/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Elongases de Ácidos Graxos , Redes Reguladoras de Genes , Glucose/metabolismo , Humanos , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , FenótipoRESUMO
AIMS/HYPOTHESIS: Per-Arnt-Sim kinase (PASK) is a nutrient-regulated domain-containing protein kinase previously implicated in the control of insulin gene expression and glucagon secretion. Here, we explore the roles of PASK in the control of islet hormone release, by generating mice with selective deletion of the Pask gene in pancreatic beta or alpha cells. METHODS: Floxed alleles of Pask were produced by homologous recombination and animals bred with mice bearing beta (Ins1 (Cre); PaskBKO) or alpha (Ppg (Cre) [also known as Gcg]; PaskAKO) cell-selective Cre recombinase alleles. Glucose homeostasis and hormone secretion in vivo and in vitro, gene expression and islet cell mass were measured using standard techniques. RESULTS: Ins1 (Cre)-based recombination led to efficient beta cell-targeted deletion of Pask. Beta cell mass was reduced by 36.5% (p < 0.05) compared with controls in PaskBKO mice, as well as in global Pask-null mice (38%, p < 0.05). PaskBKO mice displayed normal body weight and fasting glycaemia, but slightly impaired glucose tolerance, and beta cell proliferation, after maintenance on a high-fat diet. Whilst glucose tolerance was unaffected in PaskAKO mice, glucose infusion rates were increased, and glucagon secretion tended to be lower, during hypoglycaemic clamps. Although alpha cell mass was increased (21.9%, p < 0.05), glucagon release at low glucose was impaired (p < 0.05) in PaskAKO islets. CONCLUSIONS/INTERPRETATION: The findings demonstrate cell-autonomous roles for PASK in the control of pancreatic endocrine hormone secretion. Differences between the glycaemic phenotype of global vs cell type-specific null mice suggest important roles for tissue interactions in the control of glycaemia by PASK.
Assuntos
Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Alelos , Animais , Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Homeostase/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genéticaRESUMO
AIMS/HYPOTHESIS: Glucagon release from pancreatic alpha cells is required for normal glucose homoeostasis and is dysregulated in both Type 1 and Type 2 diabetes. The tumour suppressor LKB1 (STK11) and the downstream kinase AMP-activated protein kinase (AMPK), modulate cellular metabolism and growth, and AMPK is an important target of the anti-hyperglycaemic agent metformin. While LKB1 and AMPK have emerged recently as regulators of beta cell mass and insulin secretion, the role of these enzymes in the control of glucagon production in vivo is unclear. METHODS: Here, we ablated LKB1 (αLKB1KO), or the catalytic alpha subunits of AMPK (αAMPKdKO, -α1KO, -α2KO), selectively in â¼45% of alpha cells in mice by deleting the corresponding flox'd alleles with a preproglucagon promoter (PPG) Cre. RESULTS: Blood glucose levels in male αLKB1KO mice were lower during intraperitoneal glucose, aminoimidazole carboxamide ribonucleotide (AICAR) or arginine tolerance tests, and glucose infusion rates were increased in hypoglycemic clamps (p < 0.01). αLKB1KO mice also displayed impaired hypoglycemia-induced glucagon release. Glucose infusion rates were also elevated (p < 0.001) in αAMPKα1 null mice, and hypoglycemia-induced plasma glucagon increases tended to be lower (p = 0.06). Glucagon secretion from isolated islets was sensitized to the inhibitory action of glucose in αLKB1KO, αAMPKdKO, and -α1KO, but not -α2KO islets. CONCLUSIONS/INTERPRETATION: An LKB1-dependent signalling cassette, involving but not restricted to AMPKα1, is required in pancreatic alpha cells for the control of glucagon release by glucose.
RESUMO
Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired ß-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic ß-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve ß-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn2+ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn2+ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.
Assuntos
Descoberta de Drogas , Insulina/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Animais , Glicemia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Teste de Tolerância a Glucose , Ensaios de Triagem em Larga Escala , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Ratos , Ratos Zucker , Receptores Acoplados a Proteínas G/genética , Bibliotecas de Moléculas Pequenas , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Antibodies are powerful research tools that can be used in many areas of biology to probe, measure, and perturb various biological structures. Successful drug discovery is dependent on the correct identification of a target implicated in disease, coupled with the successful selection, optimization, and development of a candidate drug. Because of their specific binding characteristics, with regard to specificity, affinity, and avidity, coupled with their amenability to protein engineering, antibodies have become a key tool in drug discovery, enabling the quantification, localization, and modulation of proteins of interest. This review summarizes the application of antibodies and other protein affinity reagents as specific research tools within the drug discovery process.
Assuntos
Anticorpos/química , Descoberta de Drogas/métodos , Proteômica/métodos , Animais , Animais Geneticamente Modificados , Afinidade de Anticorpos , Cristalização , Epitopos/química , Humanos , Imunoglobulina G/química , Chaperonas Moleculares/química , Fenótipo , Engenharia de Proteínas/métodos , RNA/químicaRESUMO
Chronic exposure of skeletal muscle to saturated fatty acids, such as palmitate (C16:0), enhances proinflammatory IKK-NFκB signaling by a mechanism involving the MAP kinase (Raf-MEK-ERK) pathway. Raf activation can be induced by its dissociation from the Raf-kinase inhibitor protein (RKIP) by diacylglycerol (DAG)-sensitive protein kinase C (PKC). However, whether these molecules mediate the proinflammatory action of palmitate, an important precursor for DAG synthesis, is currently unknown. Here, involvement of DAG-sensitive PKCs, RKIP, and the structurally related monounsaturated fatty acid palmitoleate (C16:1) on proinflammatory signaling are investigated. Palmitate, but not palmitoleate, induced phosphorylation/activation of the MEK-ERK-IKK axis and proinflammatory cytokine (IL-6, CINC-1) expression. Palmitate increased intramyocellular DAG and invoked PKC-dependent RKIP(Ser153) phosphorylation, resulting in RKIP-Raf1 dissociation and MEK-ERK signaling. These responses were mimicked by PMA, a DAG mimetic and PKC activator. However, while pharmacological inhibition of PKC suppressed PMA-induced activation of MEK-ERK-IKK signaling, activation by palmitate was upheld, suggesting that DAG-sensitive PKC and RKIP were dispensable for palmitate's proinflammatory action. Strikingly, the proinflammatory effect of palmitate was potently repressed by palmitoleate. This repression was not due to reduced palmitate uptake but linked to increased neutral lipid storage and enhanced cellular oxidative capacity brought about by palmitoleate's ability to restrain palmitate-induced mitochondrial dysfunction.
Assuntos
Diglicerídeos/metabolismo , Ácidos Graxos Monoinsaturados/farmacologia , Mitocôndrias/metabolismo , Palmitatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase I-kappa B/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Oxigênio/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , RatosRESUMO
BACKGROUND: Sustained exposure of pancreatic ß cells to an increase in saturated fatty acids induces pleiotropic effects on ß-cell function, including a reduction in stimulus-induced insulin secretion. The objective of this study was to investigate the effects of chronic over supply of palmitate upon glucose- and amino acid-stimulated insulin secretion (GSIS and AASIS, respectively) and autocrine-dependent insulin signalling with particular focus on the importance of ceramide, ERK and CaMKII signalling. PRINCIPAL FINDINGS: GSIS and AASIS were both stimulated by >7-fold resulting in autocrine-dependent activation of protein kinase B (PKB, also known as Akt). Insulin release was dependent upon nutrient-induced activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) as their pharmacological inhibition suppressed GSIS/AASIS significantly. Chronic (48 h, 0.4 mM) palmitate treatment blunted glucose/AA-induced activation of CaMKII and ERK and caused a concomitant reduction (~75%) in GSIS/AASIS and autocrine-dependent activation of PKB. This inhibition could not be attributed to enhanced mitochondrial fatty acid uptake/oxidation or ceramide synthesis, which were unaffected by palmitate. In contrast, diacylglycerol synthesis was elevated suggesting increased palmitate esterification rather than oxidation may contribute to impaired stimulus-secretion coupling. Consistent with this, 2-bromopalmitate, a non-oxidisable palmitate analogue, inhibited GSIS as effectively as palmitate. CONCLUSIONS: Our results exclude changes in ceramide content or mitochondrial fatty acid handling as factors initiating palmitate-induced defects in insulin release from MIN6 ß cells, but suggest that reduced CaMKII and ERK activation associated with palmitate overload may contribute to impaired stimulus-induced insulin secretion.
Assuntos
Aminoácidos/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Glucose/farmacologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Palmitatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Free fatty acid receptor 2 (Ffar2), also known as GPR43, is activated by short-chain fatty acids (SCFA) and expressed in intestine, adipocytes, and immune cells, suggesting involvement in lipid and immune regulation. In the present study, Ffar2-deficient mice (Ffar2-KO) were given a high-fat diet (HFD) or chow diet and studied with respect to lipid and energy metabolism. On a HFD, Ffar2-KO mice had lower body fat mass and increased lean body mass. The changed body composition was accompanied by improved glucose control and lower HOMA index, indicating improved insulin sensitivity in Ffar2-KO mice. Moreover, the Ffar2-KO mice had higher energy expenditure accompanied by higher core body temperature and increased food intake. The liver weight and content of triglycerides as well as plasma levels of cholesterol were lower in the Ffar2-KO mice fed a HFD. A histological examination unveiled decreased lipid interspersed in brown adipose tissue of the Ffar2-KO mice. Interestingly, no significant differences in white adipose tissue (WAT) cell size were observed, but significantly lower macrophage content was detected in WAT from HFD-fed Ffar2-KO compared with wild-type mice. In conclusion, Ffar2 deficiency protects from HFD-induced obesity and dyslipidemia at least partly via increased energy expenditure.
Assuntos
Gorduras na Dieta/administração & dosagem , Hiperglicemia/prevenção & controle , Hipoglicemia/prevenção & controle , Obesidade/prevenção & controle , Receptores Acoplados a Proteínas G/fisiologia , Tecido Adiposo Marrom/patologia , Tecido Adiposo Branco/patologia , Animais , Composição Corporal , Regulação da Temperatura Corporal , Dieta , Dislipidemias/sangue , Dislipidemias/prevenção & controle , Metabolismo Energético , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Homeostase , Resistência à Insulina , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Obesidade/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of approximately 20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min(-1)) and was decreased during the steady-state responses to insulin (0.18 min(-1)), AICAR (0.16 min(-1)), and A-769662 (0.24 min(-1)). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Animais , Compostos de Bifenilo , Linhagem Celular , Endocitose/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Cinética , Fibras Musculares Esqueléticas/citologia , Transporte Proteico/efeitos dos fármacos , Pironas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tiofenos/farmacologiaRESUMO
GPR40 (G-protein-coupled receptor 40) has been shown to be a physiologically relevant receptor for long-chain fatty acids. It is a family A G-protein-coupled receptor highly expressed in the beta-cell where it increases insulin secretion by signalling via Gq and phospholipase C. Fatty acids are well known to mediate both acute stimulatory effects and chronic detrimental effects on the beta-cell. GPR40-transgenic and GPR40-/- animals have been important tools in studies of the metabolic effects of GPR40. In the present article, we review the literature on transgenic GPR40 models and present some of our own studies on the effects of a high-fat diet on the metabolic phenotype of GPR40-/- mice. GPR40 ligands represent interesting novel therapies for Type 2 diabetes but it is presently unclear whether agonists or antagonists represent the best therapeutic approach.