RESUMO
Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.
Assuntos
Cromatina , Histona Acetiltransferases , Animais , Antifúngicos , Candida albicans/genética , Candida albicans/metabolismo , Cromatina/genética , Histonas/genética , Homozigoto , Mamíferos , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismoRESUMO
Current therapies for treating heterogeneous cancers such as head and neck squamous cell carcinoma (HNSCC) are non-selective and are administered independent of response biomarkers. Therapy resistance subsequently emerges, resulting in increased cellular proliferation that is associated with loss of differentiation. Whether a cancer cell differentiation potential can dictate therapy responsiveness is still currently unknown. A multi-omic approach integrating whole-genome and whole-transcriptome sequencing with drug sensitivity was employed in a HNSCC mouse model, primary patients' data, and human cell lines to assess the potential of functional differentiation in predicting therapy response. Interestingly, a subset of HNSCC with effective GRHL3-dependent differentiation was the most sensitive to inhibitors of PI3K/mTOR, c-Myc, and STAT3 signaling. Furthermore, we identified the GRHL3-differentiation target gene Filaggrin (FLG) as a response biomarker and more importantly, stratified HNSCC subsets as treatment resistant based on their FLG mutational profile. The loss of FLG in sensitive HNSCC resulted in a dramatic resistance to targeted therapies while the GRHL3-FLG signature predicted a favorable patient prognosis. This study provides evidence for a functional GRHL3-FLG tumor-specific differentiation axis that regulates targeted therapy response in HNSCC and establishes a rationale for clinical investigation of differentiation-paired targeted therapy in heterogeneous cancers.
Assuntos
Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/genética , Proteínas Filagrinas/genética , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Prognóstico , Transdução de Sinais , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
BACKGROUND: DNA methylation is a complex epigenetic marker that can be analyzed using a wide variety of methods. Interpretation and visualization of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, but visualization of massively parallel sequencing results remains a significant challenge. FINDINGS: We created a program called Methpat that facilitates visualization and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 86 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab-delimited text file for each sample that summarizes DNA methylation patterns and their read counts for each amplicon, and a HTML file that summarizes this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing and reduced representation bisulfite sequencing datasets with sufficient read depths. CONCLUSIONS: Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarized and visualized for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and yield further biological insight in existing datasets.
Assuntos
Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodos , Software , Linhagem Celular , Humanos , Neoplasias/genética , Especificidade de ÓrgãosRESUMO
During submaximal exercise fatty acids are a predominant energy source for muscle contractions. An important regulator of fatty acid oxidation is acetyl-CoA carboxylase (ACC), which exists as two isoforms (ACC1 and ACC2) with ACC2 predominating in skeletal muscle. Both ACC isoforms regulate malonyl-CoA production, an allosteric inhibitor of carnitine palmitoyltransferase 1 (CPT-1); the primary enzyme controlling fatty acyl-CoA flux into mitochondria for oxidation. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is activated during exercise or by pharmacological agents such as metformin and AICAR. In resting muscle the activation of AMPK with AICAR leads to increased phosphorylation of ACC (S79 on ACC1 and S221 on ACC2), which reduces ACC activity and malonyl-CoA; effects associated with increased fatty acid oxidation. However, whether this pathway is vital for regulating skeletal muscle fatty acid oxidation during conditions of increased metabolic flux such as exercise/muscle contractions remains unknown. To examine this we characterized mice lacking AMPK phosphorylation sites on ACC2 (S212 in mice/S221 in humans-ACC2-knock-in [ACC2-KI]) or both ACC1 (S79) and ACC2 (S212) (ACC double knock-in [ACCD-KI]) during submaximal treadmill exercise and/or ex vivo muscle contractions. We find that surprisingly, ACC2-KI mice had normal exercise capacity and whole-body fatty acid oxidation during treadmill running despite elevated muscle ACC2 activity and malonyl-CoA. Similar results were observed in ACCD-KI mice. Fatty acid oxidation was also maintained in muscles from ACC2-KI mice contracted ex vivo. These findings indicate that pathways independent of ACC phosphorylation are important for regulating skeletal muscle fatty acid oxidation during exercise/muscle contractions.
RESUMO
AIMS/HYPOTHESIS: Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK-ACC2-malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice. METHODS: Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates. RESULTS: ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance. CONCLUSIONS/INTERPRETATION: These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Resistência à Insulina/fisiologia , Insulina/farmacologia , Músculo Esquelético/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Hipoglicemiantes/farmacologia , Leptina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/fisiologia , Malonil Coenzima A/metabolismo , Camundongos , Músculo Esquelético/efeitos dos fármacos , Obesidade/metabolismo , Oxirredução , Fosforilação/efeitos dos fármacos , Ribonucleotídeos/farmacologiaRESUMO
The obesity epidemic has led to an increased incidence of nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. AMP-activated protein kinase (Ampk) regulates energy homeostasis and is activated by cellular stress, hormones and the widely prescribed type 2 diabetes drug metformin. Ampk phosphorylates mouse acetyl-CoA carboxylase 1 (Acc1; refs. 3,4) at Ser79 and Acc2 at Ser212, inhibiting the conversion of acetyl-CoA to malonyl-CoA. The latter metabolite is a precursor in fatty acid synthesis and an allosteric inhibitor of fatty acid transport into mitochondria for oxidation. To test the physiological impact of these phosphorylation events, we generated mice with alanine knock-in mutations in both Acc1 (at Ser79) and Acc2 (at Ser212) (Acc double knock-in, AccDKI). Compared to wild-type mice, these mice have elevated lipogenesis and lower fatty acid oxidation, which contribute to the progression of insulin resistance, glucose intolerance and NAFLD, but not obesity. Notably, AccDKI mice made obese by high-fat feeding are refractory to the lipid-lowering and insulin-sensitizing effects of metformin. These findings establish that inhibitory phosphorylation of Acc by Ampk is essential for the control of lipid metabolism and, in the setting of obesity, for metformin-induced improvements in insulin action.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Acetiltransferases/metabolismo , Resistência à Insulina , Insulina/farmacologia , Metabolismo dos Lipídeos/fisiologia , Metformina/farmacologia , Animais , Células Cultivadas , Sinergismo Farmacológico , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/fisiologiaRESUMO
INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. METHODS: MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann-Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). RESULTS: Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. CONCLUSIONS: This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal/genética , Proteínas de Homeodomínio/genética , Proteínas Proto-Oncogênicas c-myb/genética , Fatores de Transcrição/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/metabolismo , RNA Interferente Pequeno , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Salt reabsorption is the major energy-requiring process in the kidney, and AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism. Mice with targeted deletion of the ß1-subunit of AMPK (AMPK-ß1(-/-) mice) had significantly increased urinary Na(+) excretion on a normal salt diet. This was associated with reduced expression of the ß-subunit of the epithelial Na(+) channel (ENaC) and increased subapical tubular expression of kidney-specific Na(+)-K(+)-2Cl(-) cotransporter 2 (NKCC2) in the medullary thick ascending limb of Henle. AMPK-ß1(-/-) mice fed a salt-deficient diet were able to conserve Na(+), but renin secretion increased 180% compared with control mice. Cyclooxygenase-2 mRNA also increased in the kidney cortex, indicating greater signaling through the macula densa tubular salt-sensing pathway. To determine whether the increase in renin secretion was due to a change in regulation of fatty acid metabolism by AMPK, mice with a mutation of the inhibitory AMPK phosphosite in acetyl-CoA carboxylase 1 [ACC1-knockin (KI)(S79A) mice] were examined. ACC1-KI(S79A) mice on a normal salt diet had no increase in salt loss or renin secretion, and expression of NKCC2, Na(+)-Cl(-) cotransporter, and ENaC-ß were similar to those in control mice. When mice were placed on a salt-deficient diet, however, renin secretion and cortical expression of cyclooxygenase-2 mRNA increased significantly in ACC1-KI(S79A) mice compared with control mice. In summary, our data suggest that renin synthesis and secretion are regulated by AMPK and coupled to metabolism by phosphorylation of ACC1.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Acetil-CoA Carboxilase/metabolismo , Renina/sangue , Proteínas Quinases Ativadas por AMP/deficiência , Acetil-CoA Carboxilase/genética , Animais , Canais Epiteliais de Sódio/biossíntese , Camundongos , Fosforilação , Renina/biossíntese , Sódio/urina , Cloreto de Sódio na Dieta/administração & dosagemAssuntos
Movimento Celular , Transição Epitelial-Mesenquimal , Metástase Neoplásica/patologia , Animais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Metástase Neoplásica/tratamento farmacológico , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK ß1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK ß1 subunit in mice (referred to herein as ß1(-/-) mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. ß1(-/-) macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK ß1-containing complexes. The effect of AMPK ß1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or ß1(-/-) mice into WT recipients. When challenged with a high-fat diet, mice that received ß1(-/-) bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK ß1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Tecido Adiposo/patologia , Células-Tronco Hematopoéticas/enzimologia , Resistência à Insulina/fisiologia , Macrófagos Peritoneais/enzimologia , Obesidade/enzimologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Animais , Gorduras na Dieta/toxicidade , Diglicerídeos/metabolismo , Ativação Enzimática , Ácidos Graxos/metabolismo , Hepatite/enzimologia , Hepatite/patologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Mitocôndrias/metabolismo , Oxirredução , Fosforilação , Processamento de Proteína Pós-Traducional , Quimera por Radiação , Organismos Livres de Patógenos Específicos , Linfócitos T/patologiaRESUMO
AMP-activated protein kinase (AMPK) ß1 or ß2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, α2ß2γ3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK α2, ß2, and γ3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK ß1 and ß2 isoforms in skeletal muscle (ß1ß2M-KO). ß1ß2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young ß1ß2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle α, ß, or γ subunits.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Condicionamento Físico Animal , Proteínas Quinases Ativadas por AMP/genética , Animais , DNA Mitocondrial/genética , Feminino , Glucose/farmacocinética , Hipoglicemiantes/farmacologia , Immunoblotting , Insulina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Contração Muscular , Músculo Esquelético/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: We assessed changes in intracellular Ca(2+) in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR(TETRA)) and observed significant changes in the potency of ATP (EC(50) 0.175 µM (-EGF) versus 1.731 µM (+EGF), P<0.05), and the nature of the ATP-induced Ca(2+) transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca(2+) signaling, indicating that these alterations are not simply a consequence of changes in global Ca(2+) homeostasis. To determine whether changes in ATP-mediated Ca(2+) signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X(5) ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X(5) leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. CONCLUSIONS: The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores Purinérgicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
AMP-activated protein kinase (AMPK) ß subunits (ß1 and ß2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK ß2 (ß2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that ß2 KO mice are viable and breed normally. ß2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the ß1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in ß2 KO mice. During treadmill running ß2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of ß2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet ß2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK ß2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Deleção de Genes , Camundongos/fisiologia , Músculo Esquelético/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Ácidos Graxos/metabolismo , Feminino , Glucose/metabolismo , Masculino , Camundongos/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/fisiologia , Condicionamento Físico AnimalRESUMO
The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK beta1(-/-) mice on a C57Bl/6 background that are viable, fertile, survived greater than 2 years, and display no visible brain developmental defects. These mice have a 90% reduction in hepatic AMPK activity due to loss of the catalytic alpha subunits, with modest reductions of activity detected in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, beta1(-/-) mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of beta1(-/-) mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of beta1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet.
Assuntos
Apetite , Deleção de Genes , Resistência à Insulina , Fígado/metabolismo , Obesidade/prevenção & controle , Proteínas Quinases/deficiência , Quinases Proteína-Quinases Ativadas por AMP , Animais , Apetite/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Dióxido de Carbono/metabolismo , Escuridão , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacologia , Jejum/sangue , Comportamento Alimentar/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Insulina/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/sangue , Obesidade/fisiopatologia , Especificidade de Órgãos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Oxigênio/metabolismo , Proteínas Quinases/metabolismo , Subunidades Proteicas/metabolismo , Respiração/efeitos dos fármacosRESUMO
Rats selectively bred for high endurance running capacity (HCR) have higher insulin sensitivity and improved metabolic health compared with those bred for low endurance capacity (LCR). We investigated several skeletal muscle characteristics, in vitro and in vivo, that could contribute to the metabolic phenotypes observed in sedentary LCR and HCR rats. After 16 generations of selective breeding, HCR had approximately 400% higher running capacity (P < 0.001), improved insulin sensitivity (P < 0.001), and lower fasting plasma glucose and triglycerides (P < 0.05) compared with LCR. Skeletal muscle ceramide and diacylglycerol content, basal AMP-activated protein kinase (AMPK) activity, and basal lipolysis were similar between LCR and HCR. However, the stimulation of lipolysis in response to 10 mum isoproterenol was 70% higher in HCR (P = 0.004). Impaired isoproterenol sensitivity in LCR was associated with lower basal triacylglycerol lipase activity, Ser660 phosphorylation of HSL, and beta2-adrenergic receptor protein content in skeletal muscle. Expression of the orphan nuclear receptor Nur77, which is induced by beta-adrenergic signaling and is associated with insulin sensitivity, was lower in LCR (P < 0.05). Muscle protein content of Nur77 target genes, including uncoupling protein 3, fatty acid translocase/CD36, and the AMPK gamma3 subunit were also lower in LCR (P < 0.05). Our investigation associates whole-body insulin resistance with impaired beta-adrenergic response and reduced expression of genes that are critical regulators of glucose and lipid metabolism in skeletal muscle. We identify impaired beta-adrenergic signal transduction as a potential mechanism for impaired metabolic health after artificial selection for low intrinsic exercise capacity.
Assuntos
Agonistas Adrenérgicos beta/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Exercício Físico , Resistência à Insulina , Lipólise , Músculo Esquelético/metabolismo , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Modelos Animais de Doenças , Expressão Gênica , Humanos , Hibridização Genética , Isoproterenol , Masculino , Resistência Física , Ratos , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Corrida , Transdução de SinaisRESUMO
The AMP-activated protein kinase (AMPK) is the critical component of a highly conserved signalling pathway found in all eukaryotes that plays a key role in regulating metabolic processes in response to variations in energy supply and demand. AMPK protects cells from stresses that decrease cellular energy charge (i.e increase the AMP:ATP ratio) by initiating a shift in metabolism towards the generation of ATP while simultaneously down regulating pathways that consume ATP. The role of AMPK as an energy sensor extends beyond the cell and it is now apparent that it is a key regulator of whole-body energy homeostasis. These functions have stimulated considerable interest in AMPK as a promising target to treat metabolic disorders such as obesity and Type 2 diabetes. Recently, crystal structures of heterotrimeric core fragments and individual domains of AMPK from mammals, Schizosaccharomyces pombe and Saccharomyces cerevisiae have been solved. Together they provide an impressive insight into the molecular interactions involved in regulating kinase activity, heterotrimeric assembly, glycogen binding, and binding of the regulatory nucleotides AMP and ATP.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP/química , Metabolismo Energético , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas Serina-Treonina Quinases/químicaRESUMO
The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the beta subunit carbohydrate-binding module and residues from the gamma subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the beta1 subunit. Our findings highlight the regulatory role played by the beta subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Pironas/farmacologia , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/química , Monofosfato de Adenosina/metabolismo , Animais , Compostos de Bifenilo , Células COS , Metabolismo dos Carboidratos , Domínio Catalítico , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Glucose/metabolismo , Hepatócitos/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Camundongos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
OBJECTIVE: Insulin resistance associated with obesity and diabetes is ameliorated by specific overexpression of GLUT4 in skeletal muscle. The molecular mechanisms regulating skeletal muscle GLUT4 expression remain to be elucidated. The purpose of this study was to examine these mechanisms. RESEARCH DESIGN AND METHODS AND RESULTS: Here, we report that AMP-activated protein kinase (AMPK) regulates GLUT4 transcription through the histone deacetylase (HDAC)5 transcriptional repressor. Overexpression of HDAC5 represses GLUT4 reporter gene expression, and HDAC inhibition in human primary myotubes increases endogenous GLUT4 gene expression. In vitro kinase assays, site-directed mutagenesis, and site-specific phospho-antibodies establish AMPK as an HDAC5 kinase that targets S259 and S498. Constitutively active but not dominant-negative AMPK and 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR) treatment in human primary myotubes results in HDAC5 phosphorylation at S259 and S498, association with 14-3-3 isoforms, and H3 acetylation. This reduces HDAC5 association with the GLUT4 promoter, as assessed through chromatin immunoprecipitation assays and HDAC5 nuclear export, concomitant with increases in GLUT4 gene expression. Gene reporter assays also confirm that the HDAC5 S259 and S498 sites are required for AICAR induction of GLUT4 transcription. CONCLUSIONS: These data reveal a signal transduction pathway linking cellular energy charge to gene transcription directed at restoring cellular and whole-body energy balance and provide new therapeutic targets for the treatment and management of insulin resistance and type 2 diabetes.
Assuntos
Transportador de Glucose Tipo 4/genética , Histona Desacetilases/metabolismo , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Proteínas Quinases Ativadas por AMP , Adulto , Biópsia por Agulha , Técnicas de Cultura de Células , Humanos , Cinética , Masculino , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Fosforilação , Plasmídeos , Reação em Cadeia da Polimerase , RNA/genética , RNA/isolamento & purificaçãoRESUMO
Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.
Assuntos
Tecido Adiposo/enzimologia , Insulina/farmacologia , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Lipase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Triglicerídeos/metabolismoRESUMO
AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK alpha1beta1gamma1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian alphabetagamma complex shows that beta binds alpha and gamma. Here we show that a beta1(186-270)gamma1 complex can form in the absence of detectable alpha subunit. Moreover, using alanine mutagenesis we show that beta1 Thr-263 and Tyr-267 are required for betagamma association but not alphabeta association.