RESUMO
AMP-activated protein kinase (AMPK) is a heterotrimer of α-catalytic and ß- and γ-regulatory subunits that acts to regulate cellular and whole-body nutrient metabolism. The key role of AMPK in sensing energy status has led to significant interest in AMPK as a therapeutic target for dysfunctional metabolism in type 2 diabetes, insulin resistance and obesity. Despite the actions of AMPK in the liver and skeletal muscle being extensively studied, the role of AMPK in adipose tissue and adipocytes remains less well characterised. Small molecules that selectively influence AMPK heterotrimers containing specific AMPKß subunit isoforms have been developed, including MT47-100, which selectively inhibits complexes containing AMPKß2. AMPKß1 and AMPKß2 are the principal AMPKß subunit isoforms in rodent liver and skeletal muscle, respectively, yet the contribution of specific AMPKß isoforms to adipose tissue function, however, remains largely unknown. This study therefore sought to determine the contribution of AMPKß subunit isoforms to adipocyte biology, focussing on adipogenesis. AMPKß2 was the principal AMPKß isoform in 3T3-L1 adipocytes, isolated rodent adipocytes and human subcutaneous adipose tissue, as assessed by the contribution to total cellular AMPK activity. Down-regulation of AMPKß2 with siRNA inhibited lipid accumulation, cellular adiponectin levels and adiponectin secretion during 3T3-L1 adipogenesis, whereas down-regulation of AMPKß1 had no effect. Incubation of 3T3-L1 cells with MT47-100 selectively inhibited AMPK complexes containing AMPKß2 whilst simultaneously inhibiting cellular lipid accumulation as well as cellular levels and secretion of adiponectin. Taken together, these data indicate that increased expression of AMPKß2 is an important feature of efficient adipogenesis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/enzimologia , Adipogenia , Regulação Enzimológica da Expressão Gênica , Metabolismo dos Lipídeos , Regulação para Cima , Células 3T3-L1 , Animais , Humanos , Isoenzimas/metabolismo , Masculino , Camundongos , Ratos Sprague-DawleyRESUMO
Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.
Assuntos
Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Aorta/enzimologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Pironas/farmacologia , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Aorta/citologia , Compostos de Bifenilo , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Quinase C/metabolismo , Linhagem Celular , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
AMPK (AMP-activated protein kinase) is a key regulator of cellular and whole-body energy balance. AMPK phosphorylates and regulates many proteins concerned with nutrient metabolism, largely acting to suppress anabolic ATP-consuming pathways while stimulating catabolic ATP-generating pathways. This has led to considerable interest in AMPK as a therapeutic target for the metabolic dysfunction observed in obesity and insulin resistance. The role of AMPK in skeletal muscle and the liver has been extensively studied, such that AMPK has been demonstrated to inhibit synthesis of fatty acids, cholesterol and isoprenoids, hepatic gluconeogenesis and translation while increasing fatty acid oxidation, muscle glucose transport, mitochondrial biogenesis and caloric intake. The role of AMPK in the other principal metabolic and insulin-sensitive tissue, adipose, remains poorly characterized in comparison, yet increasing evidence supports an important role for AMPK in adipose tissue function. Obesity is characterized by hypertrophy of adipocytes and the development of a chronic sub-clinical pro-inflammatory environment in adipose tissue, leading to increased infiltration of immune cells. This combination of dysfunctional hypertrophic adipocytes and a pro-inflammatory environment contributes to insulin resistance and the development of Type 2 diabetes. Exciting recent studies indicate that AMPK may not only influence metabolism in adipocytes, but also act to suppress this pro-inflammatory environment, such that targeting AMPK in adipose tissue may be desirable to normalize adipose dysfunction and inflammation. In the present review, we discuss the role of AMPK in adipose tissue, focussing on the regulation of carbohydrate and lipid metabolism, adipogenesis and pro-inflammatory pathways in physiological and pathophysiological conditions.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Tecido Adiposo/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Obesidade/enzimologia , Proteínas Quinases Ativadas por AMP/genética , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Humanos , Inflamação/enzimologia , Inflamação/genética , Obesidade/imunologia , Obesidade/metabolismoRESUMO
GPR84 is a poorly characterized, nominally orphan, proinflammatory G protein-coupled receptor that can be activated by medium chain length fatty acids. It is attracting considerable interest as a potential therapeutic target for antagonist ligands in both inflammatory bowel diseases and idiopathic pulmonary fibrosis. Successful screening of more than 300â¯000 compounds from a small molecule library followed by detailed analysis of some 50 drug-like hits identified 3-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)methyl)-1H-indole as a high affinity and highly selective competitive antagonist of human GPR84. Tritiation of a di-iodinated form of the core structure produced [3H]3-((5,6-diphenyl-1,2,4-triazin-3-yl)methyl)-1H-indole, which allowed effective measurement of receptor levels in both transfected cell lines and lipopolysaccharide-treated THP-1 monocyte/macrophage cells. Although this compound series lacks significant affinity at mouse GPR84, homology modeling and molecular dynamics simulations provided a potential rationale for this difference, and alteration of two residues in mouse GPR84 to the equivalent amino acids in the human orthologue, predicted to open the antagonist binding pocket, validated this model. Sequence alignment of other species orthologues further predicted binding of the compounds as high affinity antagonists at macaque, pig, and dog GPR84 but not at the rat orthologue, and pharmacological experiments confirmed these predictions. These studies provide a new class of GPR84 antagonists that display species selectivity defined via receptor modeling and mutagenesis.
RESUMO
Many members of the G protein-coupled receptor family, including examples with clear therapeutic potential, remain poorly characterised. This often reflects limited availability of suitable tool ligands with which to interrogate receptor function. In the case of GPR84, currently a target for the treatment of idiopathic pulmonary fibrosis, recent times have seen the description of novel orthosteric and allosteric agonists. Using 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) and di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) as exemplars of each class, in cell lines transfected to express either human or mouse GPR84, both ligands acted as effective on-target activators and with high co-operativity in their interactions. This was also the case in lipopolysaccharide-activated model human and mouse immune cell lines. However in mouse bone-marrow-derived neutrophils, where expression of GPR84 is particularly high, the capacity of PSB-16671 but not of 2-HTP to promote G protein activation was predominantly off-target because it was not blocked by an antagonist of GPR84 and was preserved in neutrophils isolated from GPR84 deficient mice. These results illustrate the challenges of attempting to study and define functions of poorly characterised receptors using ligands that have been developed via medicinal chemistry programmes, but where assessed activity has been limited largely to the initially identified target.
Assuntos
Receptores Acoplados a Proteínas G/química , Sítio Alostérico , Animais , Sítios de Ligação , Células da Medula Óssea/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Ligantes , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Células RAW 264.7 , Receptores Acoplados a Proteínas G/agonistas , Células THP-1RESUMO
In addition to the well-characterized role of AMPK in the regulation of nutrient metabolism, it is increasingly clear that AMPK activation has multiple actions on inflammatory signalling. Here we describe methods to identify effects of AMPK activity on pro-inflammatory signalling, specifically (1) the nuclear localization of the key inflammatory mediators nuclear factor-κB (NFκB) and phosphorylated c-Jun N-terminal kinase (JNK), (2) preparation of conditioned medium to analyze the secretion of cytokines/chemokines, and (3) the pro-inflammatory adhesion of leukocytes to cultured cells.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/etiologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Células Cultivadas , Meios de Cultivo Condicionados , Ativadores de Enzimas/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos Mononucleares , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Decompression sickness is a condition that results from an abrupt change from a higher to a lower pressure. It is described most commonly in divers; however, it can occur in aviation incidents, which this case report will discuss.CASE REPORT: Following an acute cabin depressurization incident, 36 patients presented to a small outpatient clinic with multiple symptoms, including fatigue, headache, nausea, vomiting, and dizziness. These patients were evaluated, triaged, and some were able to be successfully treated with supplemental oxygen in clinic. Eight of the patients had symptoms that were either persistent or concerning enough that they were referred to the dive medical clinic, where the dive medical team diagnosed six of the patients with Type II decompression sickness and referred them for hyperbaric oxygen chamber therapy. All patients who received hyperbaric therapy experienced at least some relief of symptoms, with most reporting some residual fatigue after the therapy.DISCUSSION: This case provided both lessons in triage and management of multiple patients in a small outpatient clinic, as well as the challenges in making the diagnosis of decompression sickness.Mancini S, Crotty AM, Cook J. Triage and treatment of mass casualty decompression sickness after depressurization at 6400 m. Aerosp Med Hum Perform. 2018; 89(12):1085-1088.
Assuntos
Aeronaves , Doença da Descompressão/terapia , Oxigenoterapia Hiperbárica , Incidentes com Feridos em Massa , Militares , Triagem , Adolescente , Adulto , Descompressão , Doença da Descompressão/diagnóstico , Humanos , Masculino , Medicina Militar , Adulto JovemRESUMO
Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1ß-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1ß-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1ß-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1ß-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1ß signalling.
Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Anti-Inflamatórios/farmacologia , Canagliflozina/farmacologia , Citocinas/imunologia , Células Endoteliais/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Animais , Células Cultivadas , Quimiocinas/imunologia , Células Endoteliais/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Interleucina-1beta/imunologia , CamundongosRESUMO
Inflammation of adipose tissue in obesity is associated with increased IL-1ß, IL-6 and TNF-α secretion and proposed to contribute to insulin resistance. AMP-activated protein kinase (AMPK) regulates nutrient metabolism and is reported to have anti-inflammatory actions in adipose tissue, yet the mechanisms underlying this remain poorly characterised. The effect of AMPK activation on cytokine-stimulated proinflammatory signalling was therefore assessed in cultured adipocytes. AMPK activation inhibited IL-1ß-stimulated CXCL10 secretion, associated with reduced interleukin-1 receptor associated kinase-4 (IRAK4) phosphorylation and downregulated MKK4/JNK and IKK/IκB/NFκB signalling. AMPK activation inhibited TNF-α-stimulated IKK/IκB/NFκB signalling but had no effect on JNK phosphorylation. The JAK/STAT3 pathway was also suppressed by AMPK after IL-6 stimulation and during adipogenesis. Adipose tissue from AMPKα1-/- mice exhibited increased JNK and STAT3 phosphorylation, supporting suppression of these distinct proinflammatory pathways by AMPK in vivo. The inhibition of multiple pro-inflammatory signalling pathways by AMPK may underlie the reported beneficial effects of AMPK activation in adipose tissue.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/enzimologia , Adipócitos/patologia , Inflamação/enzimologia , Inflamação/patologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Compostos de Bifenilo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Pironas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT3/metabolismo , Tiofenos/farmacologiaRESUMO
Canagliflozin, dapagliflozin, and empagliflozin, all recently approved for treatment of type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose reuptake by sodium/glucose cotransporter (SGLT) 2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMPK, an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin, or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of acetyl-CoA carboxylase (ACC) 1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue, or spleen. Because phosphorylation of ACC by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared with other SGLT2 inhibitors.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canagliflozina/farmacologia , Glucose/metabolismo , Mitocôndrias/metabolismo , Transportador 2 de Glucose-Sódio/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Fosforilação , Transportador 2 de Glucose-Sódio/genética , Inibidores do Transportador 2 de Sódio-GlicoseRESUMO
Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Rα/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.