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1.
Clin Sci (Lond) ; 138(4): 173-187, 2024 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-38315575

RESUMO

Semaglutide is an anti-diabetes and weight loss drug that decreases food intake, slows gastric emptying, and increases insulin secretion. Patients begin treatment with low-dose semaglutide and increase dosage over time as efficacy plateaus. With increasing dosage, there is also greater incidence of gastrointestinal side effects. One reason for the plateau in semaglutide efficacy despite continued low food intake is due to compensatory actions whereby the body becomes more metabolically efficient to defend against further weight loss. Mitochondrial uncoupler drugs decrease metabolic efficiency, therefore we sought to investigate the combination therapy of semaglutide with the mitochondrial uncoupler BAM15 in diet-induced obese mice. Mice were fed high-fat western diet (WD) and stratified into six treatment groups including WD control, BAM15, low-dose semaglutide without or with BAM15, and high-dose semaglutide without or with BAM15. Combining BAM15 with either semaglutide dose decreased body fat and liver triglycerides, which was not achieved by any monotherapy, while high-dose semaglutide with BAM15 had the greatest effect on glucose homeostasis. This study demonstrates a novel approach to improve weight loss without loss of lean mass and improve glucose control by simultaneously targeting energy intake and energy efficiency. Such a combination may decrease the need for semaglutide dose escalation and hence minimize potential gastrointestinal side effects.


Assuntos
Ingestão de Energia , Redução de Peso , Humanos , Animais , Camundongos , Camundongos Obesos , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo
2.
Mol Cell ; 57(3): 537-51, 2015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25658205

RESUMO

Ras is mutated in up to 30% of cancers, including 90% of pancreatic ductal adenocarcinomas, causing it to be constitutively GTP-bound, and leading to activation of downstream effectors that promote a tumorigenic phenotype. As targeting Ras directly is difficult, there is a significant effort to understand the downstream biological processes that underlie its protumorigenic activity. Here, we show that expression of oncogenic Ras or direct activation of the MAPK pathway leads to increased mitochondrial fragmentation and that blocking this phenotype, through knockdown of the mitochondrial fission-mediating GTPase Drp1, inhibits tumor growth. This fission is driven by Erk2-mediated phosphorylation of Drp1 on Serine 616, and both this phosphorylation and mitochondrial fragmentation are increased in human pancreatic cancer. Finally, this phosphorylation is required for Ras-associated mitochondrial fission, and its inhibition is sufficient to block xenograft growth. Collectively, these data suggest mitochondrial fission may be a target for treating MAPK-driven malignancies.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Benzamidas/farmacologia , Linhagem Celular Tumoral , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Dinaminas , GTP Fosfo-Hidrolases/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/genética , Neoplasias Experimentais/metabolismo , Fosforilação , Serina/metabolismo , Proteínas ras/metabolismo
3.
Bioorg Med Chem Lett ; 73: 128912, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35907607

RESUMO

We report new mitochondrial uncouplers derived from the conversion of [1,2,5]oxadiazolo[3,4-b]pyrazines to 1H-imidazo[4,5-b]pyrazines. The in situ Fe-mediated reduction of the oxadiazole fragment followed by cyclization gave access to imidazopyrazines in moderate to good yields. A selection of orthoesters also allowed functionalization on the 2-position of the imidazole ring. This method afforded a variety of imidazopyrazine derivatives with varying substitution on the 2, 5 and 6 positions. Our studies suggest that both a 2-trifluoromethyl group and N-methylation are crucial for mitochondrial uncoupling capacity.


Assuntos
Mitocôndrias , Pirazinas , Ciclização , Mitocôndrias/metabolismo , Oxidiazóis/metabolismo , Pirazinas/metabolismo
4.
Cell Mol Life Sci ; 78(21-22): 7025-7041, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34626204

RESUMO

Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.


Assuntos
Biomarcadores Tumorais/metabolismo , Metabolismo Energético/fisiologia , Glucosilceramidas/metabolismo , Fígado/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Resistência a Múltiplos Medicamentos/fisiologia , Glucosiltransferases/metabolismo , Glicólise/fisiologia , Glicoesfingolipídeos/metabolismo , Neoplasias Hepáticas/metabolismo , Camundongos , Mitocôndrias/metabolismo , Transdução de Sinais/fisiologia
5.
Int J Mol Sci ; 23(9)2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35562868

RESUMO

Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer. Low numbers of HCC patients being suitable for liver resection or transplantation and multidrug resistance development during pharmacotherapy leads to high death rates for HCC patients. Understanding the molecular mechanisms of HCC etiology may contribute to the development of novel therapeutic strategies for prevention and treatment of HCC. UDP-glucose ceramide glycosyltransferase (UGCG), a key enzyme in glycosphingolipid metabolism, generates glucosylceramide (GlcCer), which is the precursor for all glycosphingolipids (GSLs). Since UGCG gene expression is altered in 0.8% of HCC tumors, GSLs may play a role in cellular processes in liver cancer cells. Here, we discuss the current literature about GSLs and their abundance in normal liver cells, Gaucher disease and HCC. Furthermore, we review the involvement of UGCG/GlcCer in multidrug resistance development, globosides as a potential prognostic marker for HCC, gangliosides as a potential liver cancer stem cell marker, and the role of sulfatides in tumor metastasis. Only a limited number of molecular mechanisms executed by GSLs in HCC are known, which we summarize here briefly. Overall, the role GSLs play in HCC progression and their ability to serve as biomarkers or prognostic indicators for HCC, requires further investigation.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/genética , Resistência a Múltiplos Medicamentos , Glucosilceramidas/metabolismo , Glucosiltransferases/metabolismo , Glicoesfingolipídeos/metabolismo , Glicosiltransferases/metabolismo , Humanos , Neoplasias Hepáticas/genética
6.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077188

RESUMO

Obesity-related insulin resistance is a highly prevalent and growing health concern, which places stress on the pancreatic islets of Langerhans by increasing insulin secretion to lower blood glucose levels. The glucose transporters GLUT1 and GLUT3 play a key role in glucose-stimulated insulin secretion in human islets, while GLUT2 is the key isoform in rodent islets. However, it is unclear whether other glucose transporters also contribute to insulin secretion by pancreatic islets. Herein, we show that SLC2A6 (GLUT6) is markedly upregulated in pancreatic islets from genetically obese leptin-mutant (ob/ob) and leptin receptor-mutant (db/db) mice, compared to lean controls. Furthermore, we observe that islet SLC2A6 expression positively correlates with body mass index in human patients with type 2 diabetes. To investigate whether GLUT6 plays a functional role in islets, we crossed GLUT6 knockout mice with C57BL/6 ob/ob mice. Pancreatic islets isolated from ob/ob mice lacking GLUT6 secreted more insulin in response to high-dose glucose, compared to ob/ob mice that were wild type for GLUT6. The loss of GLUT6 in ob/ob mice had no adverse impact on body mass, body composition, or glucose tolerance at a whole-body level. This study demonstrates that GLUT6 plays a role in pancreatic islet insulin secretion in vitro but is not a dominant glucose transporter that alters whole-body metabolic physiology in ob/ob mice.


Assuntos
Diabetes Mellitus Tipo 2 , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Obesidade/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/genética , Humanos , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos
7.
J Immunol ; 202(6): 1826-1832, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30700586

RESUMO

The polarization processes for M1 versus M2 macrophages are quite distinct in the context of changes in cellular metabolism. M1 macrophages are highly glycolytic, whereas M2 macrophages require a more oxidative nutrient metabolism. An important part of M1 polarization involves upregulation of the glucose transporter (GLUT) GLUT1 to facilitate increased glucose uptake and glycolytic metabolism; however, the role of other glucose transporters in this process is largely unknown. In surveying the Functional Annotation of the Mammalian Genome and Gene Expression Omnibus Profiles databases, we discovered that the glucose transporter GLUT6 is highly upregulated in LPS-activated macrophages. In our previous work, we have not detected mouse GLUT6 protein expression in any noncancerous tissue; therefore, in this study, we investigated the expression and significance of GLUT6 in bone marrow-derived macrophages from wild-type and GLUT6 knockout C57BL/6 mice. We show that LPS-induced M1 polarization markedly upregulated GLUT6 protein, whereas naive macrophages and IL-4-induced M2 macrophages do not express GLUT6 protein. However, despite strong upregulation of GLUT6 in M1 macrophages, the absence of GLUT6 did not alter M1 polarization in the context of glucose uptake, glycolytic metabolism, or cytokine production. Collectively, these data show that GLUT6 is dispensable for LPS-induced M1 polarization and function. These findings are important because GLUT6 is an anticancer drug target, and this study suggests that inhibition of GLUT6 may not impart detrimental side effects on macrophage function to interfere with their antitumor properties.


Assuntos
Diferenciação Celular/imunologia , Proteínas Facilitadoras de Transporte de Glucose/imunologia , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Knockout
8.
J Cell Mol Med ; 24(21): 12491-12503, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32954656

RESUMO

Thrombocyte formation from megakaryocyte and their progenitor cells is tightly regulated by thrombopoietin (TPO) and its receptor c-MPL, thereby maintaining physiological functionality and numbers of circulating platelets. In patients, dysfunction of this regulation could cause thrombocytopenia or myeloproliferative syndromes. Since regulation of this pathway is still not completely understood, we investigated the role of the ubiquitin ligase c-Cbl which was previously shown to negatively regulated c-MPL signalling. We developed a new conditional mouse model using c-Cblfl/fl Pf4Cre mice and demonstrated that platelet-specific knockout of c-Cbl led to severe microthrombocytosis and impaired uptake of TPO and c-MPL receptor internalization. Furthermore, we characterized a constitutive STAT5 activation c-Cbl KO platelets. This study identified c-Cbl as a potential player in causing megakaryocytic and thrombocytic disorders.


Assuntos
Endocitose , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Receptores de Trombopoetina/metabolismo , Animais , Integrases/metabolismo , Linfocitose , Megacariócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transporte Proteico , Transdução de Sinais , Trombocitose , Trombopoese , Trombopoetina/metabolismo
9.
J Biol Chem ; 294(45): 16740-16755, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548309

RESUMO

Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. However, the molecular mechanisms governing the biogenesis and growth of LDs remain poorly understood. Phosphatidic acid (PA) is a precursor of phospholipids and triacylglycerols and substrate of CDP-diacylglycerol (CDP-DAG) synthase 1 (CDS1) and CDS2, which catalyze the formation of CDP-DAG. Here, using siRNA-based gene knockdowns and CRISPR/Cas9-mediated gene knockouts, along with immunological, molecular, and fluorescence microscopy approaches, we examined the role of CDS1 and CDS2 in LD biogenesis and growth. Knockdown of either CDS1 or CDS2 expression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (CIDEC), encoding a prominent regulator of LD growth in adipocytes, restored LD size in CDS1- but not in CDS2-deficient cells. On the other hand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol O-acyltransferase 2 (DGAT2) and glycerol-3-phosphate acyltransferase 4 (GPAT4), rescued the LD phenotype in CDS2-deficient, but not CDS1-deficient, cells. Moreover, CDS2 deficiency, but not CDS1 deficiency, promoted the LD association of DGAT2 and GPAT4 and impaired initial LD maturation. Finally, although both CDS1 and CDS2 appeared to regulate PA levels on the LD surface, CDS2 had a stronger effect. We conclude that CDS1 and CDS2 regulate LD dynamics through distinct mechanisms.


Assuntos
Diacilglicerol Colinofosfotransferase/metabolismo , Gotículas Lipídicas/metabolismo , Linhagem Celular , Diacilglicerol Colinofosfotransferase/deficiência , Diacilglicerol Colinofosfotransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Ácidos Fosfatídicos/metabolismo
10.
Bioorg Med Chem Lett ; 30(8): 127057, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113842

RESUMO

Mitochondrial protonophores transport protons through the mitochondrial inner membrane into the matrix to uncouple nutrient oxidation from ATP production thereby decreasing the proton motive force. Mitochondrial uncouplers have beneficial effects of decrease reactive oxygen species generation and have the potential for treating diseases such as obesity, neurodegenerative diseases, non-alcoholic fatty liver disease (NAFLD), diabetes, and many others. In this study, we report the structure-activity relationship profile of the pyrazine scaffold bearing substituted aniline rings. Our work indicates that a trifluoromethyl group is best at the para position while the trifluoromethoxy group is preferred in the meta position of the aniline rings of 2,3-substituted pyrazines. As proton transport and cycling requires the formation of a negative charge that has to traverse the mitochondrial membrane, a stabilizing internal hydrogen bond is a key feature for efficient mitochondrial uncoupling activity.


Assuntos
Compostos de Anilina/farmacologia , Mitocôndrias/efeitos dos fármacos , Pirazinas/farmacologia , Desacopladores/farmacologia , Compostos de Anilina/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Pirazinas/química , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Desacopladores/química
11.
Bioessays ; 40(10): e1800098, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30132936

RESUMO

Despite promising early work into the role of immune cells such as eosinophils in adipose tissue (AT) homeostasis, recent findings revealed that elevating the number of eosinophils in AT alone is insufficient for improving metabolic impairments in obese mice. Eosinophils are primarily recognized for their role in allergic immunity and defence against parasitic worms. They have also been detected in AT and appear to contribute to adipose homeostasis and drive energy expenditure, but the underlying mechanisms remain elusive. It has long been recognized that immune cells such as macrophages respond to external signals to regulate adipose homeostasis and energy balance, however, less is known about the relevance of eosinophil activity in AT. As the authors propose in this review, given recent debate over the relative importance of their tissue-specific abundance, the stage is now set for exploring the functionality and activation states of AT eosinophils.


Assuntos
Tecido Adiposo/citologia , Eosinófilos/fisiologia , Obesidade/metabolismo , Redução de Peso/fisiologia , Tecido Adiposo/fisiologia , Adiposidade/fisiologia , Animais , Homeostase , Humanos , Camundongos Obesos , Obesidade/patologia
12.
Diabetologia ; 62(1): 99-111, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30334081

RESUMO

AIMS/HYPOTHESIS: Pancreatic beta cells secrete insulin to maintain glucose homeostasis, and beta cell failure is a hallmark of type 2 diabetes. Glucose triggers insulin secretion in beta cells via oxidative mitochondrial pathways. However, it also feeds mitochondrial anaplerotic pathways, driving citrate export and cytosolic malonyl-CoA production by the acetyl-CoA carboxylase 1 (ACC1) enzyme. This pathway has been proposed as an alternative glucose-sensing mechanism, supported mainly by in vitro data. Here, we sought to address the role of the beta cell ACC1-coupled pathway in insulin secretion and glucose homeostasis in vivo. METHODS: Acaca, encoding ACC1 (the principal ACC isoform in islets), was deleted in beta cells of mice using the Cre/loxP system. Acaca floxed mice were crossed with Ins2cre mice (ßACC1KO; life-long beta cell gene deletion) or Pdx1creER mice (tmx-ßACC1KO; inducible gene deletion in adult beta cells). Beta cell function was assessed using in vivo metabolic physiology and ex vivo islet experiments. Beta cell mass was analysed using histological techniques. RESULTS: ßACC1KO and tmx-ßACC1KO mice were glucose intolerant and had defective insulin secretion in vivo. Isolated islet studies identified impaired insulin secretion from beta cells, independent of changes in the abundance of neutral lipids previously implicated as amplification signals. Pancreatic morphometry unexpectedly revealed reduced beta cell size in ßACC1KO mice but not in tmx-ßACC1KO mice, with decreased levels of proteins involved in the mechanistic target of rapamycin kinase (mTOR)-dependent protein translation pathway underpinning this effect. CONCLUSIONS/INTERPRETATION: Our study demonstrates that the beta cell ACC1-coupled pathway is critical for insulin secretion in vivo and ex vivo and that it is indispensable for glucose homeostasis. We further reveal a role for ACC1 in controlling beta cell growth prior to adulthood.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Acetil-CoA Carboxilase/genética , Animais , Feminino , Secreção de Insulina/genética , Secreção de Insulina/fisiologia , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismo
13.
J Biol Chem ; 293(15): 5731-5745, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29440390

RESUMO

Obesity is associated with metabolic dysfunction, including insulin resistance and hyperinsulinemia, and with disorders such as cardiovascular disease, osteoporosis, and neurodegeneration. Typically, these pathologies are examined in discrete model systems and with limited temporal resolution, and whether these disorders co-occur is therefore unclear. To address this question, here we examined multiple physiological systems in male C57BL/6J mice following prolonged exposure to a high-fat/high-sucrose diet (HFHSD). HFHSD-fed mice rapidly exhibited metabolic alterations, including obesity, hyperleptinemia, physical inactivity, glucose intolerance, peripheral insulin resistance, fasting hyperglycemia, ectopic lipid deposition, and bone deterioration. Prolonged exposure to HFHSD resulted in morbid obesity, ectopic triglyceride deposition in liver and muscle, extensive bone loss, sarcopenia, hyperinsulinemia, and impaired short-term memory. Although many of these defects are typically associated with aging, HFHSD did not alter telomere length in white blood cells, indicating that this diet did not generally promote all aspects of aging. Strikingly, glucose homeostasis was highly dynamic. Glucose intolerance was evident in HFHSD-fed mice after 1 week and was maintained for 24 weeks. Beyond 24 weeks, however, glucose tolerance improved in HFHSD-fed mice, and by 60 weeks, it was indistinguishable from that of chow-fed mice. This improvement coincided with adaptive ß-cell hyperplasia and hyperinsulinemia, without changes in insulin sensitivity in muscle or adipose tissue. Assessment of insulin secretion in isolated islets revealed that leptin, which inhibited insulin secretion in the chow-fed mice, potentiated glucose-stimulated insulin secretion in the HFHSD-fed mice after 60 weeks. Overall, the excessive calorie intake was accompanied by deteriorating function of numerous physiological systems.


Assuntos
Carboidratos da Dieta/efeitos adversos , Gorduras na Dieta/efeitos adversos , Doenças Metabólicas , Sacarose/efeitos adversos , Homeostase do Telômero/efeitos dos fármacos , Animais , Carboidratos da Dieta/farmacologia , Gorduras na Dieta/farmacologia , Masculino , Doenças Metabólicas/induzido quimicamente , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Camundongos , Sacarose/farmacologia , Fatores de Tempo
14.
J Enzyme Inhib Med Chem ; 34(1): 728-739, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30822267

RESUMO

The most challenging issue facing peptide drug development is producing a molecule with optimal physical properties while maintaining target binding affinity. Masking peptides with protecting groups that can be removed inside the cell, produces a cell-permeable peptide, which theoretically can maintain its biological activity. Described are series of prodrugs masked using: (a) O-alkyl, (b) N-alkyl, and (c) acetyl groups, and their binding affinity for Hsp90. Alkyl moieties increased compound permeability, Papp, from 3.3 to 5.6, however alkyls could not be removed by liver microsomes or in-vivo and their presence decreased target binding affinity (IC50 of ≥10 µM). Thus, unlike small molecules, peptide masking groups cannot be predictably removed; their removal is related to the 3-D conformation. O-acetyl groups were cleaved but are labile, increasing challenges during synthesis. Utilising acetyl groups coupled with mono-methylated amines may decrease the polarity of a peptide, while maintaining binding affinity.


Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Pró-Fármacos/farmacologia , Animais , Relação Dose-Resposta a Droga , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Relação Estrutura-Atividade
15.
Am J Physiol Endocrinol Metab ; 315(2): E286-E293, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29664675

RESUMO

Glucose transporter 6 (GLUT6) is a member of the facilitative glucose transporter family. GLUT6 is upregulated in several cancers but is not widely expressed in normal tissues. Previous studies have shown that GLUT6 knockdown kills endometrial cancer cells that express elevated levels of the protein. However, whether GLUT6 represents a viable anticancer drug target is unclear because the role of GLUT6 in normal metabolic physiology is unknown. Herein we generated GLUT6 knockout mice to determine how loss of GLUT6 affected whole body glucose homeostasis and metabolic physiology. We found that the mouse GLUT6 ( Slc2a6) gene expression pattern was similar to humans with mRNA found primarily in brain and spleen. CRISPR-Cas9-mediated deletion of Slc2a6 did not alter mouse development, growth, or whole body glucose metabolism in male or female mice fed either a chow diet or Western diet. GLUT6 deletion did not impact glucose tolerance or blood glucose and insulin levels in male or female mice fed either diet. However, compared with wild-type littermate controls, GLUT6 null female mice had a relatively minor decrease in fat accumulation when fed Western diet and had a lower respiratory exchange ratio when fed chow diet. Collectively, these data show that GLUT6 is not a major regulator of whole body metabolic physiology; therefore, GLUT6 inhibition may have minimal adverse effects if targeted for cancer therapy.


Assuntos
Proteínas Facilitadoras de Transporte de Glucose/genética , Metabolismo/genética , Metabolismo/fisiologia , Adiposidade/genética , Animais , Glicemia/metabolismo , Peso Corporal/genética , Sistemas CRISPR-Cas , Dieta , Metabolismo Energético/genética , Feminino , Genótipo , Glucose/metabolismo , Teste de Tolerância a Glucose , Homeostase/genética , Insulina/sangue , Masculino , Camundongos , Camundongos Knockout
16.
Biochim Biophys Acta ; 1861(7): 671-9, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27106139

RESUMO

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.


Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células Endoteliais/enzimologia , Junções Comunicantes/química , Miócitos de Músculo Liso/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Transporte Biológico , Bradicinina/farmacologia , Sinalização do Cálcio , Comunicação Celular/efeitos dos fármacos , Técnicas de Cocultura , GMP Cíclico/metabolismo , Diglicerídeos/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Humanos , Inositol 1,4,5-Trifosfato , Compostos Macrocíclicos/farmacologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/classificação , Óxido Nítrico Sintase Tipo III/genética , Oxazóis/farmacologia , Fenilefrina/farmacologia , Fosfatidilserinas/metabolismo , Fosforilação , Cultura Primária de Células , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tapsigargina/farmacologia
17.
Gynecol Oncol ; 147(3): 654-662, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29050779

RESUMO

Endometrial cancer is the most common gynecological malignancy in the developed world. It is the fifth most common cancer and accounts for 4.8% of all cancers in women. Long intergenic non-coding RNAs (lincRNAs), a subclass of long non-coding RNAs, are pervasively transcribed throughout the human genome. OBJECTIVE: LincRNA expression patterns in endometrial cancer compared to normal healthy tissue are poorly characterised. In this study, the lincRNA transcriptome of endometrial cancers and adjacent normal endometrium from the same patients was sequenced and compared with transcriptomes of other gynaecologic malignancies including ovarian and cervical cancers. METHODS: RNA was isolated from malignant and adjacent non-affected endometrial tissue from 6 patients with low grade and stage Type I endometrial cancer. Subsequently, Illumina paired-end RNA sequencing was performed, followed by bioinformatics analysis, to determine differential transcriptome expression patterns. RESULTS: LINC00958 was upregulated in all three cancers, and four lincRNAs including LINC01480, LINC00645, LINC00891 and LINC00702 demonstrated exquisite specificity for malignant endometrium compared to normal endometrium while also distinguishing endometrial cancer from ovarian and cervical cancers. Furthermore, LINC01480 has features required to express a micropeptide. CONCLUSIONS: The lincRNAs, characterised in this study, represent high priority genes to be tested for functional significance in the pathogenesis and/or progression of endometrial cancer. Furthermore, lincRNAs have potential to be released into the bloodstream and therefore the four lincRNAs identified here may represent biomarkers for early detection of endometrial cancer without biopsy.


Assuntos
Neoplasias do Endométrio/genética , RNA Longo não Codificante/genética , Estudos de Casos e Controles , Neoplasias do Endométrio/patologia , Feminino , Humanos , Estadiamento de Neoplasias , Oligopeptídeos/biossíntese , Oligopeptídeos/genética , Especificidade de Órgãos , RNA Neoplásico/genética , Transcriptoma , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
18.
J Pathol ; 239(1): 3-5, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26880235

RESUMO

Fatty liver, also termed hepatic steatosis or fatty liver disease, is a condition characterized by excess fat accumulation in the liver. Common causes of fatty liver include obesity, ageing, medications, genetic disorders, viral hepatitis, excess alcohol or toxins. This diversity in pathogenesis is matched by an equally diverse spectrum of consequences, whereby some individuals remain asymptomatic yet others progress through a series of inflammatory, fibrotic and metabolic disorders that can lead to liver failure, cancer or diabetes. Current treatment approaches for fatty liver do not differ by disease aetiology and primarily involve weight loss strategies or management of co-morbidities. In a recent paper published in this journal, Urasaki et al used capillary isoelectric focusing (cIEF) to create profiles of protein post-translational modifications that distinguish four different models of fatty liver in mice. Importantly, this new cIEF approach has the potential to provide rapid individualized diagnosis of fatty liver pathogenesis that may enable more accurate and personalized treatment strategies. Further testing and optimization of cIEF as a diagnostic screening tool in humans is warranted.


Assuntos
Fígado Gorduroso/metabolismo , Ensaios de Triagem em Larga Escala , Fígado/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteômica/métodos , Animais
19.
Nature ; 477(7363): 220-4, 2011 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-21857682

RESUMO

Rapid and efficient removal of apoptotic cells by phagocytes is important during development, tissue homeostasis and in immune responses. Efficient clearance depends on the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the corpse-derived cellular material. However, the factors that influence continued clearance by phagocytes are not known. Here we show that the mitochondrial membrane potential of the phagocyte critically controls engulfment capacity, with lower potential enhancing engulfment and vice versa. The mitochondrial membrane protein Ucp2, which acts to lower the mitochondrial membrane potential, was upregulated in phagocytes engulfing apoptotic cells. Loss of Ucp2 reduced phagocytic capacity, whereas Ucp2 overexpression enhanced engulfment. Mutational and pharmacological studies indicated a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice were impaired in phagocytosis in vitro, and Ucp2-deficient mice showed profound in vivo defects in clearing dying cells in the thymus and testes. Collectively, these data indicate that mitochondrial membrane potential and Ucp2 are key molecular determinants of apoptotic cell clearance. As Ucp2 is linked to metabolic diseases and atherosclerosis, this newly discovered role for Ucp2 in apoptotic cell clearance has implications for the complex aetiology and pathogenesis of these diseases.


Assuntos
Apoptose , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , Fagócitos/citologia , Fagócitos/metabolismo , Fagocitose/fisiologia , Animais , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Canais Iônicos/deficiência , Canais Iônicos/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Fagócitos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Timo/citologia , Proteína Desacopladora 2
20.
J Immunol ; 192(7): 3190-9, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24567531

RESUMO

Fatty acids (FAs) are essential constituents of cell membranes, signaling molecules, and bioenergetic substrates. Because CD8(+) T cells undergo both functional and metabolic changes during activation and differentiation, dynamic changes in FA metabolism also occur. However, the contributions of de novo lipogenesis to acquisition and maintenance of CD8(+) T cell function are unclear. In this article, we demonstrate the role of FA synthesis in CD8(+) T cell immunity. T cell-specific deletion of acetyl coenzyme A carboxylase 1 (ACC1), an enzyme that catalyzes conversion of acetyl coenzyme A to malonyl coenzyme A, a carbon donor for long-chain FA synthesis, resulted in impaired peripheral persistence and homeostatic proliferation of CD8(+) T cells in naive mice. Loss of ACC1 did not compromise effector CD8(+) T cell differentiation upon listeria infection but did result in a severe defect in Ag-specific CD8(+) T cell accumulation because of increased death of proliferating cells. Furthermore, in vitro mitogenic stimulation demonstrated that defective blasting and survival of ACC1-deficient CD8(+) T cells could be rescued by provision of exogenous FA. These results suggest an essential role for ACC1-mediated de novo lipogenesis as a regulator of CD8(+) T cell expansion, and may provide insights for therapeutic targets for interventions in autoimmune diseases, cancer, and chronic infections.


Assuntos
Acetil-CoA Carboxilase/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Ácidos Graxos/imunologia , Homeostase/imunologia , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Ácidos Graxos/metabolismo , Citometria de Fluxo , Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Antígenos Comuns de Leucócito/imunologia , Antígenos Comuns de Leucócito/metabolismo , Lipogênese/genética , Lipogênese/imunologia , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Listeria monocytogenes/fisiologia , Listeriose/genética , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/genética , Ovalbumina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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