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1.
JCI Insight ; 7(14)2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35866481

RESUMO

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.


Assuntos
Anexina A6 , Cálcio , Animais , Anexina A6/genética , Anexina A6/metabolismo , Anexinas , Cálcio/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo
2.
Sci Transl Med ; 13(610): eabf0376, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34516828

RESUMO

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor ß (TGFß) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFß. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein's hinge region, was linked to increased sequestration of latent TGFß, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-ß, and together, these data underscored the functional role of LTBP4's hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4's hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mdx mice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy.


Assuntos
Distrofias Musculares , Distrofia Muscular de Duchenne , Proteínas de Transporte , Fibrose , Humanos , Proteínas de Ligação a TGF-beta Latente/metabolismo , Músculo Esquelético/metabolismo , Músculos/metabolismo , Distrofias Musculares/patologia , Distrofias Musculares/terapia , Distrofia Muscular de Duchenne/patologia , Fator de Crescimento Transformador beta/metabolismo
3.
JCI Insight ; 6(9)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33755598

RESUMO

BACKGROUNDEstimates of seroprevalence to SARS-CoV-2 vary widely and may influence vaccination response. We ascertained IgG levels across a single US metropolitan site, Chicago, from June 2020 through December 2020.METHODSParticipants (n = 7935) were recruited through electronic advertising and received materials for a self-sampled dried-blood spot assay through the mail or a minimal contact in-person method. IgG against the receptor-binding domain of SARS-CoV-2 was measured using an established highly sensitive and highly specific assay.RESULTSOverall seroprevalence was 17.9%, with no significant difference between method of contact. Only 2.5% of participants reported having had a diagnosis of COVID-19 based on virus detection, consistent with a 7-fold greater exposure to SARS-CoV-2 measured by serology than that detected by viral testing. The range of IgG level observed in seropositive participants from this community survey overlapped with the range of IgG levels associated with COVID-19 cases having a documented positive PCR test. From a subset of those who participated in repeat testing, half of seropositive individuals retained detectable antibodies for 3 to 4 months.CONCLUSIONQuantitative IgG measurements with a highly specific and sensitive assay indicated more widespread exposure to SARS-CoV-2 than observed by viral testing. The range of IgG concentrations produced from these asymptomatic exposures was similar to IgG levels occurring after documented nonhospitalized COVID-19, which were considerably lower than those produced from hospitalized COVID-19 cases. The differing ranges of IgG response, coupled with the rate of decay of antibodies, may influence response to subsequent viral exposure and vaccine.FundingNational Science Foundation grant 2035114, NIH grant 3UL1TR001422-06S4, NIH National Center for Advancing Translational Sciences grants UL1 TR001422 and UL1 TR002389, Dixon Family Foundation, Northwestern University Cancer Center (NIH grant P30 CA060553), and Walder Foundation's Chicago Coronavirus Assessment Network.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/epidemiologia , Pandemias , SARS-CoV-2/imunologia , Adolescente , Adulto , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/virologia , Teste Sorológico para COVID-19/estatística & dados numéricos , Chicago/epidemiologia , Estudos de Coortes , Teste em Amostras de Sangue Seco/métodos , Teste em Amostras de Sangue Seco/estatística & dados numéricos , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Adulto Jovem
4.
medRxiv ; 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33236031

RESUMO

Background: Estimates of seroprevalence to SARS-CoV-2 vary widely and may influence vaccination response. We ascertained IgG levels across a single US metropolitan site, Chicago, from June 2020 through December 2020. Methods: Participants (n=7935) were recruited through electronic advertising and received materials for a self-sampled dried blood spot assay through the mail or a minimal contact in person method. IgG to the receptor binding domain of SARS-CoV-2 was measured using an established highly sensitive and highly specific assay. Results: Overall seroprevalence was 17.9%, with no significant difference between method of contact. Only 2.5% of participants reported having had a diagnosis of COVID-19 based on virus detection, consistent with a 7-fold greater exposure to SARS-CoV-2 measured by serology than detected by viral testing. The range of IgG level observed in seropositive participants from this community survey overlapped with the range of IgG levels associated with COVID-19 cases having a documented positive PCR positive test. From a subset of those who participated in repeat testing, half of seropositive individuals retained detectable antibodies for 3-4 months. Conclusions: Quantitative IgG measurements with a highly specific and sensitive assay indicate more widespread exposure to SARS-CoV-2 than observed by viral testing. The range of IgG concentration produced from these asymptomatic exposures is similar to IgG levels occurring after documented non-hospitalized COVID-19, which is considerably lower than that produced from hospitalized COVID-19 cases. The differing ranges of IgG response, coupled with the rate of decay of antibodies, may influence response to subsequent viral exposure and vaccine.

5.
PLoS One ; 15(8): e0237833, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32797108

RESUMO

OBJECTIVE: Serological testing is needed to investigate the extent of transmission of SARS-CoV-2 from front-line essential workers to their household members. However, the requirement for serum/plasma limits serological testing to clinical settings where it is feasible to collect and process venous blood. To address this problem we developed a serological test for SARS-CoV-2 IgG antibodies that requires only a single drop of finger stick capillary whole blood, collected in the home and dried on filter paper (dried blood spot, DBS). We describe assay performance and demonstrate its utility for remote sampling with results from a community-based study. METHODS: An ELISA to the receptor binding domain of the SARS-CoV-2 spike protein was optimized to quantify IgG antibodies in DBS. Samples were self-collected from a community sample of 232 participants enriched with health care workers, including 30 known COVID-19 cases and their household members. RESULTS: Among 30 individuals sharing a household with a virus-confirmed case of COVID-19, 80% were seropositive. Of 202 community individuals without prior confirmed acute COVID-19 diagnoses, 36% were seropositive. Of documented convalescent COVID-19 cases from the community, 29 of 30 (97%) were seropositive for IgG antibodies to the receptor binding domain. CONCLUSION: DBS ELISA provides a minimally-invasive alternative to venous blood collection. Early analysis suggests a high rate of transmission among household members. High rates of seroconversion were also noted following recovery from infection. Serological testing for SARS-CoV-2 IgG antibodies in DBS samples can facilitate seroprevalence assessment in community settings to address epidemiological questions, monitor duration of antibody responses, and assess if antibodies against the spike protein correlate with protection from reinfection.


Assuntos
Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Teste em Amostras de Sangue Seco , Características da Família , Pessoal de Saúde , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Anticorpos Antivirais/sangue , COVID-19 , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/transmissão , Pneumonia Viral/virologia , SARS-CoV-2 , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto Jovem
6.
IUBMB Life ; 72(4): 601-606, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32027092

RESUMO

The sodium channel NaX (encoded by the SCN7A gene) was originally identified in the heart and skeletal muscle and is structurally similar to the other voltage-gated sodium channels but does not appear to be voltage gated. Although NaX is expressed at high levels in cardiac and skeletal muscle, little information exists on the function of NaX in these tissues. Transcriptional profiling of ion channels in the heart in a subset of patients with Brugada syndrome revealed an inverse relationship between the expression of NaX and NaV 1.5 suggesting that, in cardiac myocytes, the expression of these channels may be linked. We propose that NaX plays a role in excitation-contraction coupling based on our experimental observations. Here we show that in cardiac myocytes, NaX is expressed in a striated pattern on the sarcolemma in regions corresponding to the sarcomeric M-line. Knocking down NaX expression decreased NaV 1.5 mRNA and protein and reduced the inward sodium current (INa+ ) following cell depolarization. When the expression of NaV 1.5 was knocked down, ~85% of the INa+ was reduced consistent with the observations that NaV 1.5 is the main voltage-gated sodium channel in cardiac muscle and that NaX likely does not directly participate in mediating the INa+ following depolarization. Silencing NaV 1.5 expression led to significant upregulation of NaX mRNA. Similar to NaV 1.5, NaX protein levels were rapidly downregulated when the intracellular [Ca2+ ] was increased either by CaCl2 or caffeine. These data suggest that a relationship exists between NaX and NaV 1.5 and that NaX may play a role in excitation-contraction coupling.


Assuntos
Miócitos Cardíacos/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Síndrome de Brugada/genética , Cálcio/metabolismo , Células Cultivadas , Cães , Técnicas de Silenciamento de Genes , Humanos , Contração Miocárdica/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ratos , Sarcômeros/metabolismo , Canais de Sódio Disparados por Voltagem/genética
7.
J Clin Invest ; 129(11): 4657-4670, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545299

RESUMO

Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.


Assuntos
Anexina A6/farmacologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Músculo Esquelético/lesões , Distrofia Muscular Animal/prevenção & controle , Animais , Anexina A6/genética , Membrana Celular/patologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
8.
Endocrinology ; 159(4): 1658-1677, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29370351

RESUMO

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 hours to induce insulin resistance. Insulin-resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in insulin receptor substrate (IRS)-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, Akt Ser473 and Thr308 phosphorylation, accompanied by impaired glucose transporter 4 translocation. In contrast, protein kinase C (PKC)-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell-permeable peptide reversed the signaling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative, kinase-inactive PKC-ζ blocked insulin resistance, whereas constitutively active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1 Ser318 and Akt Thr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1 S318A and Akt T34A each partially corrected insulin signaling, whereas combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats for 48 hours similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and Akt Thr34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.


Assuntos
Adipócitos/metabolismo , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Resistência à Insulina/fisiologia , Proteína Quinase C/metabolismo , Transdução de Sinais/fisiologia , Adipócitos/efeitos dos fármacos , Animais , Glucose/farmacologia , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
9.
Sci Rep ; 6: 36694, 2016 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-27841282

RESUMO

Phase III clinical trials evaluating bevacizumab (an antibody to the angiogenic ligand, VEGF-A) in breast cancer have found improved responses in the presurgical neoadjuvant setting but no benefits in the postsurgical adjuvant setting. The objective of this study was to evaluate alternative antiangiogenic therapies, which target multiple VEGF family members or differentially modulate the Angiopoietin/Tie2 pathway, in a mouse model of resectable triple-negative breast cancer (TNBC). Neoadjuvant therapy experiments involved treating established orthotopic xenografts of an aggressive metastatic variant of the MDA-MB-231 human TNBC cell line, LM2-4. Adjuvant therapies were given after primary tumor resections to treat postsurgical regrowths and distant metastases. Aflibercept ('VEGF Trap', which neutralizes VEGF-A, VEGF-B and PlGF) showed greater efficacy than nesvacumab (an anti-Ang2 antibody) as an add-on to neoadjuvant/adjuvant chemotherapy. Concurrent inhibition of Ang1 and Ang2 signaling (through an antagonistic anti-Tie2 antibody) was not more efficacious than selective Ang2 inhibition. In contrast, short-term perioperative BowAng1 (a recombinant Ang1 variant) improved the efficacy of adjuvant chemotherapy. In conclusion, concurrent VEGF pathway inhibition is more likely than Ang/Tie2 pathway inhibition (e.g., anti-Ang2, anti-Ang2/Ang1, anti-Tie2) to improve neoadjuvant/adjuvant chemotherapies for TNBC. Short-term perioperative Ang1 supplementation may also have therapeutic potential in conjunction with adjuvant chemotherapy for TNBC.


Assuntos
Angiopoietina-1/farmacologia , Terapia Neoadjuvante , Neoplasias Experimentais/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Feminino , Humanos , Camundongos , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Biochim Biophys Acta ; 1850(9): 1781-5, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25960387

RESUMO

BACKGROUND: Since the initial discovery of mutations in the isocitrate dehydrogenase 1 (IDH1) gene in a large subset of human low-grade gliomas and acute myelogenous leukemia (AML), much interest focused on the function of IDH1 and on the relationship between mutations in IDH1 and tumor progression. To date, mutations in the IDH1 gene have been found in numerous cancers with the highest frequencies occurring in gliomas, chondrosarcomas/enchondromas and cholangiocarcinomas. SCOPE OF REVIEW: IDH1 was first described in the scientific literature as early as 1950. Early researchers proposed that the enzyme likely functions in cellular lipid metabolism based on the observation that the enzymatic reaction produces NADPH and partially localizes to peroxisomes. This article highlights the studies implicating IDH1 in cytoplasmic and peroxisomal lipid metabolism from the early researchers to the recent studies examining mutant IDH1(R132), the most common IDH1 mutation found in cancer. MAJOR CONCLUSIONS: While a role for IDH1 in lipid biosynthesis in the liver and adipose tissue is now established, a role in lipid metabolism in the brain and tumors is beginning to be examined. The recent discoveries that IDH1(R132H) interferes with the metabolism of phospholipids in gliomas and that IDH1 activity could participate in the synthesis of acetyl-CoA from glutamine in hypoxic tumors highlight roles for IDH1 in lipid metabolism in a broad spectrum of tissues. GENERAL SIGNIFICANCE: Interferences in cytoplasmic and peroxisomal lipid metabolism by IDH1(R132) may contribute to the more favorable clinical outcome in patients whose tumors express mutations in the IDH1 gene.


Assuntos
Isocitrato Desidrogenase/fisiologia , Metabolismo dos Lipídeos , Neoplasias/metabolismo , Animais , Desenvolvimento Ósseo , Humanos , Peroxissomos/metabolismo
11.
EMBO Mol Med ; 7(6): 770-87, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25851538

RESUMO

Angiopoietin-1 (Ang1) activation of Tie2 receptors on endothelial cells (ECs) reduces adhesion by tumor cells (TCs) and limits junctional permeability to TC diapedesis. We hypothesized that systemic therapy with Vasculotide (VT)-a purported Ang1 mimetic, Tie2 agonist-can reduce the extravasation of potentially metastatic circulating TCs by similarly stabilizing the host vasculature. In vitro, VT and Ang1 treatments impeded endothelial hypermeability and the transendothelial migration of MDA-MB-231∙LM2-4 (breast), HT29 (colon), or SN12 (renal) cancer cells to varying degrees. In mice, VT treatment inhibited the transit of TCs through the pulmonary endothelium, but not the hepatic or lymphatic endothelium. In the in vivo LM2-4 model, VT monotherapy had no effect on primary tumors, but significantly delayed distant metastatic dissemination to the lungs. In the post-surgical adjuvant treatment setting, VT therapeutically complemented sunitinib therapy, an anti-angiogenic tyrosine kinase inhibitor which limited the local growth of residual disease. Unexpectedly, detailed investigations into the putative mechanism of action of VT revealed no evidence of Tie2 agonism or Tie2 binding; alternative mechanisms have yet to be determined.


Assuntos
Angiopoietina-1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/secundário , Células Endoteliais/efeitos dos fármacos , Metástase Neoplásica/prevenção & controle , Receptor TIE-2/agonistas , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Camundongos , Permeabilidade/efeitos dos fármacos
12.
J Cell Mol Med ; 19(1): 82-91, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25381905

RESUMO

The endoplasmic reticulum (ER) is an organelle important for protein synthesis and folding, lipid synthesis and Ca(2+) homoeostasis. Consequently, ER stress or dysfunction affects numerous cellular processes and has been implicated as a contributing factor in several pathophysiological conditions. Tunicamycin induces ER stress in various cell types in vitro as well as in vivo. In mice, a hallmark of tunicamycin administration is the development of fatty livers within 24-48 hrs accompanied by hepatic ER stress. We hypothesized that tunicamycin would induce ER stress in adipose tissue that would lead to increased lipolysis and subsequently to fatty infiltration of the liver and hepatomegaly. Our results show that intraperitoneal administration of tunicamycin rapidly induced an ER stress response in adipose tissue that correlated with increased circulating free fatty acids (FFAs) and glycerol along with decreased adipose tissue mass and lipid droplet size. Furthermore, we found that in addition to fatty infiltration of the liver as well as hepatomegaly, lipid accumulation was also present in the heart, skeletal muscle and kidney. To corroborate our findings to a clinical setting, we examined adipose tissue from burned patients where increases in lipolysis and the development of fatty livers have been well documented. We found that burned patients displayed significant ER stress within adipose tissue and that ER stress augments lipolysis in cultured human adipocytes. Our results indicate a possible role for ER stress induced lipolysis in adipose tissue as an underlying mechanism contributing to increases in circulating FFAs and fatty infiltration into other organs.


Assuntos
Tecido Adiposo/patologia , Estresse do Retículo Endoplasmático , Lipólise , Tecido Adiposo/efeitos dos fármacos , Animais , Queimaduras/patologia , Queimaduras/cirurgia , Separação Celular , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ácidos Graxos/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Humanos , Lipólise/efeitos dos fármacos , Masculino , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/efeitos dos fármacos , Tunicamicina/farmacologia
13.
Neurosci Lett ; 582: 87-92, 2014 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-25218477

RESUMO

Isocitrate dehydrogenase 1 (IDH1) is an evolutionarily conserved enzyme that catalyzes the interconversion of isocitrate to α-ketoglutarate with the concomitant reduction of NADP(+) to NADPH. IDH1 has previously been shown to participate in lipid biosynthesis in various tissues such as the liver and adipose tissue. We examined the potential role of IDH1 in phospholipid metabolism in the brain. Here we show that IDH1 is highly expressed in the brain and astrocytes during embryonic development and the postnatal period and subsequently declines in adulthood. Silencing of IDH1 expression using siRNA in astrocytes isolated from E18.5 mouse cortices led to increased incorporation of [(3)H]-palmitate into the phosphatidylcholines (PCs) and decreased the incorporation of [(3)H]-palmitate into sphingomyelin and the phosphatidylethanolamines (PEs). In pulse-chase experiments, knock-down of IDH1 expression impaired the turnover of PCs and decreased the synthesis of PEs. The decrease in [(3)H]-palmitate incorporation into PEs when IDH1 was knocked-down in astrocytes was not due to impairments within the CDP-ethanolamine pathway or in the rate of decarboxylation of phosphatidylserine (PS). In conclusion, our results reveal a role for IDH1 in the synthesis/turnover of phospholipids in developing astrocytes and highlight the lipid alterations resulting from the loss of wild-type IDH1 activity.


Assuntos
Astrócitos/metabolismo , Encéfalo/citologia , Isocitrato Desidrogenase/metabolismo , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Técnicas de Silenciamento de Genes , Isocitrato Desidrogenase/genética , Camundongos Endogâmicos BALB C , Especificidade de Órgãos
14.
Shock ; 41(2): 138-44, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24434416

RESUMO

Extensively burned patients often suffer from sepsis (especially caused by Pseudomonas aeruginosa), which may prolong metabolic derangement, contribute to multiple organ failure, and increase mortality. The molecular and cellular mechanisms of such infection-related metabolic derangement and organ dysfunction are unclear. We have previously shown that severely burned patients have significant and persisting hepatic endoplasmic reticulum (ER) stress. We hypothesized that ER stress and the unfolded protein response correlate with NOD-like receptor, pyrin domain containing 3 (NLRP3) inflammasome activation in burn. These may trigger profound metabolic changes in the liver, which form the pathological basis of liver damage and liver dysfunction after burn injury. A two-hit rat model was established by a 60% total body surface area scald burn and intraperitoneal injection of P. aeruginosa-derived lipopolysaccharide (LPS) 3 days after burn. One day later, animals were killed, and liver tissue samples were collected for gene expression and protein analysis of NLRP3 inflammasome activation, ER stress, and glucose and lipid metabolism. Liver damage was assessed by plasma markers (alanine aminotransferase and aspartate aminotransferase) and liver immunohistochemical analysis. Our results showed that burn injury and LPS injection induced inflammasome activation in liver and augmented hepatic ER stress and liver damage. Although there was an increased metabolic demand after burn, hepatic NLRP3 inflammasome activation corresponded to inhibition of PGC-1α (peroxisome proliferator-activated receptor γ-coactivator 1α) and its upstream regulators protein kinase A catalyst unit, AMP-activated protein kinase α, and sirtuin-1 may provide a mechanism for the enhanced metabolic derangement after major burn injury plus sepsis. In conclusion, burn + LPS augments inflammasome activation and ER stress in liver, which in turn contribute to postburn metabolic derangement.


Assuntos
Queimaduras/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Inflamassomos/fisiologia , Lipopolissacarídeos/toxicidade , Hepatopatias/fisiopatologia , Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas de Transporte , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Pseudomonas aeruginosa/química , Ratos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo
15.
Crit Care ; 16(3): 125, 2012 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-22587777

RESUMO

Critical illness, trauma and burns are associated with profound metabolic abnormalities, of which protein catabolism, hyperglycemia and insulin resistance are hallmarks of these conditions. Increased protein breakdown and loss results in muscle wasting, weakness and diminished functioning. Interestingly, hyperglycemia and insulin resistance augment catabolic responses. Insulin, which is routinely administered to critically ill patients to prevent excessive hyperglycemia, also stimulates protein synthesis and prevents whole-body protein loss. The present commentary highlights the results of a recent study published in Critical Care and discusses whether moderate insulin therapy is equally as beneficial as conventional insulin therapy in preventing protein catabolism and loss.


Assuntos
Glicemia/metabolismo , Estado Terminal , Insulina/uso terapêutico , Nitrogênio/metabolismo , Feminino , Humanos , Masculino
16.
Mol Cancer Res ; 10(3): 428-40, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22232519

RESUMO

Aberrations in epidermal growth factor receptor (EGFR/ErbB1) are the most common oncogenic alterations in glioblastoma multiforme (GBM), the most common primary brain tumor. Interactions between wild-type (wt) and mutant EGFRs and their subsequent activation are of biologic and potential therapeutic importance in GBMs. We recently showed that in situ proximity ligation assay (PLA) allows for quantitative evaluation of EGFR dimerization and activation in intact cells. Using this in situ platform, we show the aberrant homo-/heterodimeric properties of EGFRvIII and EGFRc958 mutants, the two most common EGFR mutants in GBMs. In addition, dimer phosphoactivation status could be detected by PLA with superior signal-noise ratio (>17-fold) and sensitivity (>16-fold) than immunofluorescence-based phospho-EGFR measurements. Dimer activation analysis indicated quantitative activation differences of mutant dimers. These aberrant features were not overexpression dependent but appeared independent of cellular expression levels, suggesting inherent properties of the mutant receptors. Moreover, we observed in situ detection of EGFRwt-EGFRvIII heterodimerization in GBM specimens, supporting our cell line observations. Notably, currently used anti-EGFR therapeutics, such as cetuximab, matuzumab, and panitumumab, could effectively block EGFRwt dimerization and activation but did not equally impair EGFRvIII homodimers, EGFRwt-EGFRvIII, or EGFRvIII-EGFRc958 heterodimers. EGFRvIII appears to have intrinsic phosphoactivation independent of dimerization as matuzumab blockade of homodimerization had no effect on receptor phosphorylation levels. These data suggest differences in the dimerization-blocking efficacy of EGFR monoclonal antibodies as mutant EGFR dimer configurations prevalent in GBMs can evade blockade by anti-EGFR treatments. Further studies are warranted to evaluate whether this evasion contributes to poor therapeutic response or resistance.


Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Glioblastoma/enzimologia , Terapia de Alvo Molecular , Proteínas Mutantes/metabolismo , Multimerização Proteica , Animais , Anticorpos Antineoplásicos/imunologia , Bioensaio , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática , Epitopos/imunologia , Receptores ErbB/imunologia , Humanos , Immunoblotting , Fosforilação , Razão Sinal-Ruído
17.
Histochem Cell Biol ; 132(2): 225-37, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19424712

RESUMO

The tyrosine kinase receptor Tie2 is expressed on endothelial cells, and together with its ligand angiopoietin-1 (Ang1), is important for angiogenesis and vascular stability. Upon activation by Ang1, Tie2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold electron microscopy, we show that on the surface of endothelial cells, Tie2 is arranged in variably sized clusters containing dimers and higher order oligomers. Clusters of Tie2 were expressed on the apical and basolateral plasma membranes, and on the tips of microvilli. Upon activation by Ang1, Tie2 co-localized with the clathrin heavy chain at the apical and basolateral plasma membranes and within endothelial cells indicating that Tie2 internalizes through clathrin-coated pits. Inhibiting cellular endocytosis by depleting cellular potassium or by acidifying the cytosol blocked the internalization of Tie2 in response to Ang1. Our results suggest that one pathway mediating the internalization of Tie2 in response to Ang1 is through clathrin-coated pits.


Assuntos
Angiopoietina-1/metabolismo , Invaginações Revestidas da Membrana Celular/enzimologia , Endotélio Vascular/enzimologia , Receptor TIE-2/metabolismo , Angiopoietina-1/agonistas , Células Cultivadas , Invaginações Revestidas da Membrana Celular/ultraestrutura , Endotélio Vascular/ultraestrutura , Humanos
18.
Cancer Res ; 68(2): 521-9, 2008 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-18199548

RESUMO

Vascular endothelial growth factor (VEGF) is a potent proangiogenic protein that activates VEGF receptor (VEGFR) tyrosine kinases expressed by vascular endothelial cells. We previously showed that one of these receptors, VEGFR-2, has a truncated soluble form (sVEGFR-2) that can be detected in mouse and human plasma. Because activation of VEGFR-2 plays an important role in tumor angiogenesis, clinical interest in monitoring plasma sVEGFR-2 levels in cancer patients has focused on its potential exploitation as a surrogate biomarker for disease progression as well as assessing efficacy/activity of antiangiogenic drugs, particularly those that target VEGF or VEGFR-2. However, no preclinical studies have been done to study sVEGFR-2 during tumor growth or the mechanisms involved in its modulation. Using spontaneously growing tumors and both localized and metastatic human tumor xenografts, we evaluated the relationship between sVEGFR-2 and tumor burden as well as underlying factors governing protein level modulation in vivo. Our results show an inverse relationship between the levels of sVEGFR-2 and tumor size. Furthermore, using various methods of VEGF overexpression in vivo, including cell transfection and adenoviral delivery, we found plasma sVEGFR-2 decreases to be mediated largely by tumor-derived VEGF. Finally, in vitro studies indicate VEGF-mediated sVEGFR-2 modulation is the result of ligand-induced down-regulation of the VEGFR-2 from the cell surface. Taken together, these findings may be pertinent to further clinical exploitation of plasma sVEGFR-2 levels as a surrogate biomarker of VEGF-dependent tumor growth as well as an activity indicator of antiangiogenic drugs that target the VEGFR system.


Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/sangue , Proliferação de Células , Neoplasias Mamárias Experimentais/patologia , Neoplasias da Próstata/patologia , Carga Tumoral , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangue , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoviridae , Animais , Feminino , Células HT29 , Humanos , Masculino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Neoplasias , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Solubilidade , Transdução Genética , Transplante Heterólogo , Carga Tumoral/genética , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
J Cell Sci ; 119(Pt 17): 3551-60, 2006 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16895971

RESUMO

The receptor tyrosine kinase Tie2 is highly expressed in endothelial cells and is crucial for angiogenesis and vascular maintenance. The ligands for Tie2 are the angiopoietins, of which angiopoietin-1 and angiopoietin-2 have been the most studied. Angiopoietin-1 has been characterized as the primary activating ligand for Tie2 whereas the role of angiopoietin-2 remains controversial; activating Tie2 in some studies and inhibiting Tie2 in others. Our studies were aimed at understanding the regulation of Tie2 in endothelial cells by angiopoietin-1 and angiopoietin-2 and revealed that both ligands activated Tie2 in a concentration-dependent manner. Angiopoietin-2 was considerably weaker at activating Tie2 compared with angiopoietin-1 suggesting that angiopoietin-2 may be a partial agonist. Activation of Tie2 by these ligands resulted in differential turnover of the receptor where binding of angiopoietin-1, and to a lesser extent angiopoietin-2, induced rapid internalization and degradation of Tie2. Furthermore, our binding studies demonstrate that both ligands are differentially released from the endothelial cell surface after receptor activation and accumulate in the surrounding medium. Altogether, these data begin our understanding of the regulation of Tie2 and the activity of the angiopoietins after engaging the endothelial cell surface.


Assuntos
Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Endocitose/fisiologia , Células Endoteliais/fisiologia , Receptor TIE-2/metabolismo , Angiopoietina-1/genética , Angiopoietina-2/genética , Animais , Células Cultivadas , Células Endoteliais/citologia , Ativação Enzimática , Humanos , Ligantes , Ensaio Radioligante , Receptor TIE-2/genética
20.
Am J Physiol Endocrinol Metab ; 285(4): E744-53, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12799318

RESUMO

Exposure to high concentrations of glucose and insulin results in insulin resistance of metabolic target tissues, a characteristic feature of type 2 diabetes. High glucose has also been associated with oxidative stress, and increased levels of reactive oxygen species have been proposed to cause insulin resistance. To determine whether oxidative stress contributes to insulin resistance induced by hyperglycemia in vivo, nondiabetic rats were infused with glucose for 6 h to maintain a circulating glucose concentration of 15 mM with and without coinfusion of the antioxidant N-acetylcysteine (NAC), followed by a 2-h hyperinsulinemic-euglycemic clamp. High glucose (HG) induced a significant decrease in insulin-stimulated glucose uptake [tracer-determined disappearance rate (Rd), control 41.2 +/- 1.7 vs. HG 32.4 +/- 1.9 mg. kg-1. min-1, P < 0.05], which was prevented by NAC (HG + NAC 45.9 +/- 3.5 mg. kg-1. min-1). Similar results were obtained with the antioxidant taurine. Neither NAC nor taurine alone altered Rd. HG caused a significant (5-fold) increase in soleus muscle protein carbonyl content, a marker of oxidative stress that was blocked by NAC, as well as elevated levels of malondialdehyde and 4-hydroxynonenal, markers of lipid peroxidation, which were reduced by taurine. In contrast to findings after long-term hyperglycemia, there was no membrane translocation of novel isoforms of protein kinase C in skeletal muscle after 6 h. These data support the concept that oxidative stress contributes to the pathogenesis of hyperglycemia-induced insulin resistance.


Assuntos
Acetilcisteína/farmacologia , Glucose/administração & dosagem , Resistência à Insulina/fisiologia , Insulina/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , Taurina/farmacologia , Animais , Glicemia/análise , Glicemia/metabolismo , Insulina/sangue , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
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