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1.
Diabetologia ; 57(6): 1182-91, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24663807

RESUMO

AIMS/HYPOTHESIS: The NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/ß-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas. METHODS: A KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of ß-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting ß-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists. RESULTS: ß-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice. CONCLUSIONS/INTERPRETATION: The results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.


Assuntos
Ilhotas Pancreáticas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Secretoras de Somatostatina/metabolismo , Somatostatina/metabolismo , Animais , Camundongos , Camundongos Mutantes , Receptores Acoplados a Proteínas G/genética
2.
Mol Cancer Ther ; 17(11): 2309-2319, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30097489

RESUMO

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kß isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kß inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kß and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kß inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309-19. ©2018 AACR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias/patologia , PTEN Fosfo-Hidrolase/deficiência , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Compostos de Anilina/farmacologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Feminino , Fluordesoxiglucose F18/farmacocinética , Proteína Forkhead Box O3/metabolismo , Glucose/metabolismo , Humanos , Camundongos Nus , Neoplasias/enzimologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
3.
Clin Cancer Res ; 23(24): 7584-7595, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-28972046

RESUMO

Purpose: PTEN-null tumors become dependent on the PI3Kß isoform and can be targeted by molecules such as the selective PI3Kß inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kß are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kß also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kß inhibition in PTEN-null tumors supporting the concept that targeting PI3Kß may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.


Assuntos
Compostos de Anilina/administração & dosagem , Cromonas/administração & dosagem , Classe II de Fosfatidilinositol 3-Quinases/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Compostos de Anilina/efeitos adversos , Animais , Linhagem Celular Tumoral , Cromonas/efeitos adversos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hidroximetilglutaril-CoA Sintase/genética , Redes e Vias Metabólicas/genética , Camundongos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Med Chem ; 60(7): 3187-3197, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28374589

RESUMO

GPR120 agonists have therapeutic potential for the treatment of diabetes, but few selective agonists have been reported. We identified an indazole-6-phenylcyclopropylcarboxylic acid series of GPR120 agonists and conducted SAR studies to optimize GPR120 potency. Furthermore, we identified a (S,S)-cyclopropylcarboxylic acid structural motif which gave selectivity against GPR40. Good oral exposure was obtained with some compounds displaying unexpected high CNS penetration. Increased MDCK efflux was utilized to identify compounds such as 33 with lower CNS penetration, and activity in oral glucose tolerance studies was demonstrated. Differential activity was observed in GPR120 null and wild-type mice indicating that this effect operates through a mechanism involving GPR120 agonism.


Assuntos
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Indazóis/química , Indazóis/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Glicemia/análise , Ácidos Carboxílicos/farmacocinética , Humanos , Indazóis/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares
5.
Peptides ; 77: 16-20, 2016 03.
Artigo em Inglês | MEDLINE | ID: mdl-26144594

RESUMO

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from proglucagon, which is released from intestinal L-cells and increases insulin secretion in a glucose dependent manner. GPR119 is a lipid derivative receptor present in L-cells, believed to play a role in the detection of dietary fat. This study aimed to characterize the responses of primary murine L-cells to GPR119 agonism and assess the importance of GPR119 for the detection of ingested lipid. METHODS: GLP-1 secretion was measured from murine primary cell cultures stimulated with a panel of GPR119 ligands. Plasma GLP-1 levels were measured in mice lacking GPR119 in proglucagon-expressing cells and controls after lipid gavage. Intracellular cAMP responses to GPR119 agonists were measured in single primary L-cells using transgenic mice expressing a cAMP FRET sensor driven by the proglucagon promoter. RESULTS: L-cell specific knockout of GPR119 dramatically decreased plasma GLP-1 levels after a lipid gavage. GPR119 ligands triggered GLP-1 secretion in a GPR119 dependent manner in primary epithelial cultures from the colon, but were less effective in the upper small intestine. GPR119 agonists elevated cAMP in ∼70% of colonic L-cells and 50% of small intestinal L-cells. CONCLUSIONS/INTERPRETATION: GPR119 ligands strongly enhanced GLP-1 release from colonic cultures, reflecting the high proportion of colonic L-cells that exhibited cAMP responses to GPR119 agonists. Less GPR119-dependence could be demonstrated in the upper small intestine. In vivo, GPR119 in L-cells plays a key role in oral lipid-triggered GLP-1 secretion.


Assuntos
Óleo de Milho/farmacologia , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Azeite de Oliva/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Cultivadas , AMP Cíclico/metabolismo , Células Enteroendócrinas/efeitos dos fármacos , Feminino , Masculino , Camundongos Transgênicos , Azeite de Oliva/administração & dosagem , Cultura Primária de Células , Sistemas do Segundo Mensageiro
6.
Mol Cancer Ther ; 14(1): 48-58, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25398829

RESUMO

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kß isoform. Inhibitors of PI3Kß have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kß and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 µmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kß with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs.


Assuntos
Compostos de Anilina/administração & dosagem , Antineoplásicos/administração & dosagem , Cromonas/administração & dosagem , Inibidores de Fosfoinositídeo-3 Quinase , Neoplasias da Próstata/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Taxoides/administração & dosagem , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Docetaxel , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Camundongos , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Br J Pharmacol ; 165(8): 2758-70, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22029844

RESUMO

BACKGROUND AND PURPOSE: ß-cells express a range of fatty acid-responsive G protein-coupled receptors, including GPR119, which regulates insulin secretion and is seen as a potential therapeutic target in type 2 diabetes. The long-chain unsaturated fatty acid derivative oleoylethanolamide (OEA) is an endogenous agonist of GPR119 and, under certain conditions, some long-chain unsaturated fatty acids can promote ß-cell cytoprotection. It is not known, however, if OEA is cytoprotective in ß-cells. The present study has examined this and determined whether GPR119 is involved. METHODS: Clonal rat insulin-secreting cell lines, BRIN-BD11 or INS-1E, were exposed to fatty acids complexed with BSA. cAMP levels, insulin release and cell viability were measured. Protein expression was studied by Western blotting and receptor expression by RT-PCR. KEY RESULTS: GPR119 was expressed in both BRIN-BD11 and INS-1E cells and OEA was cytoprotective in these cells. However, cytoprotection was not reproduced by any of a range of selective, synthetic ligands of GPR119. The cytoprotective response to OEA was lost during exposure to inhibitors of fatty acid amide hydrolase (FAAH) suggesting that OEA per se is not the cytoprotective species but that release of free oleate is required. Similar data were obtained with anandamide, which was cytoprotective only under conditions favouring release of free arachidonate. CONCLUSIONS AND IMPLICATIONS: Activation of GPR119 is not required to mediate the cytoprotective actions of OEA in BRIN-BD11 or INS-1E cells. Rather, OEA is internalised and subjected to hydrolysis by FAAH to release free oleate, which then mediates the cytoprotection.


Assuntos
Citoproteção/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Ácidos Oleicos/farmacologia , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Endocanabinoides , Ácidos Graxos/farmacologia , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Ratos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Soroalbumina Bovina/farmacologia
8.
Diabetes ; 58(11): 2506-15, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19675138

RESUMO

OBJECTIVE: Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity. RESEARCH DESIGN AND METHODS: Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11beta-HSD1 inhibition upon insulin signaling and action. RESULTS: Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer(307) insulin receptor substrate (IRS)-1. 11beta-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11beta-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer(307) IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11beta-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer(307) IRS1 decreased and pThr(308) Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression. CONCLUSIONS: Prereceptor facilitation of glucocorticoid action via 11beta-HSD1 increases pSer(307) IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11beta-HSD1 inhibition decreases pSer(307) IRS1, increases pThr(308) Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , Músculo Esquelético/fisiologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , Animais , Linhagem Celular , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Desoxiglucose/metabolismo , Humanos , Cinética , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Mioblastos/citologia , Mioblastos/enzimologia , Mioblastos/fisiologia , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Biochem J ; 373(Pt 1): 155-65, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12678921

RESUMO

PGC1 alpha is a co-activator involved in adaptive thermogenesis, fatty-acid oxidation and gluconeogenesis. We describe the identification of several isoforms of a new human PGC1 alpha homologue, cloned independently and named PGC1 beta. The human PGC1 beta gene is localized to chromosome 5, has 13 exons and spans more than 78 kb. Two different 5' and 3' ends due to differential splicing were identified by rapid amplification of cDNA ends PCR and screening of human cDNA libraries. We show that PGC1 beta variants in humans, mice and rats are expressed predominantly in heart, brown adipose tissue, brain and skeletal muscle. PGC1 beta expression, unlike PGC1 alpha, is not up-regulated in brown adipose tissue in response to cold or obesity. Fasting experiments showed that PGC1 alpha, but not PGC1 beta, is induced in liver and this suggests that only PGC1 alpha is involved in the hepatic gluconeogenesis. No changes in PGC1 beta gene expression were observed associated with exercise. Human PGC1 beta-1a and -2a isoforms localized to the cell nucleus and, specifically, the isoform PGC1 beta-1a co-activated peroxisome-proliferator-activated receptor-gamma, -alpha and the thyroid hormone receptor beta1. Finally, we show that ectopic expression PGC1 beta leads to increased mitochondrial number and basal oxygen consumption. These results suggest that PGC1 beta may play a role in constitutive adrenergic-independent mitochondrial biogenesis.


Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular , Clonagem Molecular , DNA Complementar , Éxons , Biblioteca Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Oligomicinas/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transfecção
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