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1.
Diabetes ; 70(9): 2014-2025, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34233931

RESUMO

Persons living with HIV (PLWH) manifest chronic disorders of brown and white adipose tissues that lead to diabetes and metabolic syndrome. The mechanisms that link viral factors to defective adipose tissue function and abnormal energy balance in PLWH remain incompletely understood. Here, we explored how the HIV accessory protein viral protein R (Vpr) contributes to adaptive thermogenesis in two mouse models and human adipose tissues. Uncoupling protein 1 (UCP1) gene expression was strongly increased in subcutaneous white adipose tissue (WAT) biopsy specimens from PLWH and in subcutaneous WAT of the Vpr mice, with nearly equivalent mRNA copy number. Histology and functional studies confirmed beige transformation in subcutaneous but not visceral WAT in the Vpr mice. Measurements of energy balance indicated Vpr mice displayed metabolic inflexibility and could not shift efficiently from carbohydrate to fat metabolism during day-night cycles. Furthermore, Vpr mice showed a marked inability to defend body temperature when exposed to 4°C. Importantly, Vpr couples higher tissue catecholamine levels with UCP1 expression independent of ß-adrenergic receptors. Our data reveal surprising deficits of adaptive thermogenesis that drive metabolic inefficiency in HIV-1 Vpr mouse models, providing an expanded role for viral factors in the pathogenesis of metabolic disorders in PLWH.


Assuntos
Tecido Adiposo Branco/metabolismo , Obesidade/metabolismo , Termogênese/fisiologia , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Tecido Adiposo Marrom/metabolismo , Adulto , Temperatura Corporal/fisiologia , Metabolismo Energético/fisiologia , Feminino , Humanos , Pessoa de Meia-Idade , Proteína Desacopladora 1/metabolismo
2.
J Tissue Eng Regen Med ; 14(2): 306-318, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31821703

RESUMO

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, ß-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, ß2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/ß, PCG 1α/ß, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Receptores Adrenérgicos beta/metabolismo , Adipogenia , Adrenérgicos , Reatores Biológicos , Diferenciação Celular/fisiologia , Proliferação de Células , Condutividade Elétrica , Epinefrina/farmacologia , Humanos , Hipóxia , Cinética , Miócitos Cardíacos/citologia , Transdução de Sinais , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Alicerces Teciduais , Fatores de Transcrição/metabolismo
3.
AIDS ; 29(6): 667-74, 2015 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-25849830

RESUMO

OBJECTIVE: The objective of this study is to determine whether adipose tissue functions as a reservoir for HIV-1. DESIGN: We examined memory CD4(+) T cells and HIV DNA in adipose tissue-stromal vascular fraction (AT-SVF) of five patients [four antiretroviral therapy (ART)-treated and one untreated]. To determine whether adipocytes stimulate CD4(+) T cells and regulate HIV production, primary human adipose cells were cocultured with HIV-infected CD4(+) T cells. METHODS: AT-SVF T cells were studied by flow cytometry, and AT-SVF HIV DNA (Gag and Env) was examined by nested PCR and sequence analyses. CD4(+) T-cell activation and HIV production were measured by flow cytometry and ELISA. RESULTS: AT-SVF CD3(+) T cells were activated (>60% CD69(+)) memory CD4(+) and CD8(+) T cells in uninfected and HIV-infected persons, but the AT-SVF CD4(+)/CD8(+) ratio was lower in HIV patients. HIV DNA (Gag and Env) was detected in AT-SVF of all five patients examined by nested PCR, comparably to other tissues [peripheral blood mononuclear cell (PBMC), lymph node or thymus]. In coculture experiments, adipocytes increased CD4(+) T-cell activation and HIV production approximately two to three-fold in synergy with gamma-chain cytokines interleukin (IL)-2, IL7 or IL15. These effects were mitigated by neutralizing antibodies against IL6 and integrin-α1ß1. Adipocytes also enhanced T-cell viability. CONCLUSION: Adipose tissues of ART-treated patients harbour activated memory CD4(+) T cells and HIV DNA. Adipocytes promote CD4(+) T-cell activation and HIV production in concert with intrinsic adipose factors. Adipose tissue may be an important reservoir for HIV.


Assuntos
Adipócitos/fisiologia , Tecido Adiposo/imunologia , Tecido Adiposo/virologia , Linfócitos T CD4-Positivos/virologia , HIV/crescimento & desenvolvimento , Subpopulações de Linfócitos T/virologia , Linfócitos T CD4-Positivos/química , Células Cultivadas , Técnicas de Cocultura , DNA Viral/análise , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , HIV/isolamento & purificação , Humanos , Ativação Linfocitária , Subpopulações de Linfócitos T/química
4.
Sci Transl Med ; 5(213): 213ra164, 2013 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-24285483

RESUMO

Viral infections, such as HIV, have been linked to obesity, but mechanistic evidence that they cause adipose dysfunction in vivo is lacking. We investigated a pathogenic role for the HIV-1 accessory protein viral protein R (Vpr), which can coactivate the glucocorticoid receptor (GR) and co-repress peroxisome proliferator-activated receptor γ (PPARγ) in vitro, in HIV-associated adipose dysfunction. Vpr circulated in the blood of most HIV-infected patients tested, including those on antiretroviral therapy (ART) with undetectable viral load. Vpr-mediated mechanisms were dissected in vivo using mouse models expressing the Vpr transgene in adipose tissues and liver (Vpr-Tg) or infused with synthetic Vpr. Both models demonstrated accelerated whole-body lipolysis, hyperglycemia and hypertriglyceridemia, and tissue-specific findings. Fat depots in these mice had diminished mass, macrophage infiltration, and blunted PPARγ target gene expression but increased GR target gene expression. In liver, we observed blunted PPARα target gene expression, steatosis with decreased adenosine monophosphate-activated protein kinase activity, and insulin resistance. Similar to human HIV-infected patients, Vpr circulated in the serum of Vpr-Tg mice. Vpr blocked differentiation in preadipocytes through cell cycle arrest, whereas in mature adipocytes, it increased lipolysis with reciprocally altered association of PPARγ and GR with their target promoters. These results delineate a distinct pathogenic sequence: Vpr, released from HIV-1 in tissue reservoirs after ART, can disrupt PPAR/GR co-regulation and cell cycle control to produce adipose dysfunction and hepatosteatosis. Confirmation of these mechanisms in HIV patients could lead to targeted treatment of the metabolic complications with Vpr inhibitors, GR antagonists, or PPARγ/PPARα agonists.


Assuntos
Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Receptores de Glucocorticoides/metabolismo , Células 3T3-L1 , Animais , Cromatografia em Camada Fina , Ensaio de Imunoadsorção Enzimática , Produtos do Gene vpr/genética , HIV-1/genética , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Transgênicos , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/metabolismo , Receptores de Glucocorticoides/agonistas
5.
Arterioscler Thromb Vasc Biol ; 33(7): 1714-21, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23640486

RESUMO

OBJECTIVE: HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non-HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. APPROACH AND RESULTS: Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[(3)H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). CONCLUSIONS: Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.


Assuntos
Antirretrovirais/efeitos adversos , Infecções por HIV/tratamento farmacológico , Hiperlipidemias/induzido quimicamente , Lipoproteínas HDL/sangue , Biomarcadores/sangue , Linhagem Celular Tumoral , Ésteres do Colesterol/metabolismo , Terapia Combinada , Dieta , Exercício Físico , Ácidos Fíbricos/uso terapêutico , Infecções por HIV/sangue , Infecções por HIV/diagnóstico , Hepatócitos/metabolismo , Humanos , Hiperlipidemias/sangue , Hiperlipidemias/terapia , Hipolipemiantes/uso terapêutico , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Niacina/uso terapêutico , Estabilidade Proteica , Receptores de Lipoproteínas/metabolismo , Fatores de Tempo , Resultado do Tratamento , Triglicerídeos/sangue
6.
Proc Natl Acad Sci U S A ; 109(32): 13016-21, 2012 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-22826236

RESUMO

Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.


Assuntos
Transdiferenciação Celular/fisiologia , Fibroblastos/citologia , Mioblastos Cardíacos/citologia , Proteína Proto-Oncogênica c-ets-2/metabolismo , Pele/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Western Blotting , Transdiferenciação Celular/genética , Citometria de Fluxo , Imunofluorescência , Técnicas de Inativação de Genes , Humanos , Camundongos , Mioblastos Cardíacos/fisiologia , Reação em Cadeia da Polimerase , Proteína Proto-Oncogênica c-ets-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Obesity (Silver Spring) ; 20(2): 253-62, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21869759

RESUMO

Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation.


Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Síndrome de Lipodistrofia Associada ao HIV/metabolismo , Monócitos/metabolismo , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Adipócitos , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Western Blotting , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular , Proliferação de Células , Quimiocina CXCL1/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/genética , Síndrome de Lipodistrofia Associada ao HIV/genética , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Obesidade/genética , Reação em Cadeia da Polimerase em Tempo Real
8.
Obesity (Silver Spring) ; 14(10): 1716-23, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17062800

RESUMO

OBJECTIVE: Ghrelin is postulated to be an orexigenic signal that promotes weight regain after weight loss (WL). However, it is not known whether this putative effect of ghrelin is sustained after weight stabilization. The objective of this study was to investigate the relationship of plasma ghrelin concentrations to active WL and weight maintenance in obese subjects. RESEARCH METHODS AND PROCEDURES: This study was a randomized clinical trial, with a 12-month follow-up period. Obese Mexican-American women matched for age and BMI were randomized to a 12-month WL program (n = 25) or no intervention (controls, n = 23). Interventions included diet, exercise, and orlistat. Body weight and fasting ghrelin, leptin, insulin, and glucose concentrations were measured at baseline and 6 and 12 months. RESULTS: The WL group lost 8.5% of body weight after 6 months and maintained the new weight for the next 6 months. Ghrelin concentrations increased significantly at 6 months but returned to baseline at 12 months. Baseline ghrelin concentrations were directly related to the degree of WL achieved after 12 months. Controls experienced no change in BMI or ghrelin levels. There were no associations between plasma ghrelin and leptin or insulin concentrations. DISCUSSION: Consistent with previous results, ghrelin rises in response to WL, perhaps as a counterregulatory mechanism. However, the present results indicate that ghrelin concentrations return to baseline with sustained weight maintenance, suggesting that its effects are unlikely to regulate long-term energy balance. Baseline ghrelin concentrations are related to the degree of WL that can be achieved by active weight reduction.


Assuntos
Peso Corporal/fisiologia , Americanos Mexicanos , Obesidade/terapia , Hormônios Peptídicos/sangue , Redução de Peso/fisiologia , Adulto , Análise de Variância , Índice de Massa Corporal , Feminino , Seguimentos , Grelina , Promoção da Saúde/métodos , Humanos , Insulina/sangue , Resistência à Insulina/fisiologia , Leptina/sangue , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/fisiopatologia , Radioimunoensaio/métodos , Fatores de Tempo , Relação Cintura-Quadril
9.
Proc Natl Acad Sci U S A ; 103(12): 4516-21, 2006 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-16537394

RESUMO

Phosphorylation of a cluster of amino acids in the serum response factor (SRF) "MADS box" alphaI coil DNA binding domain regulated the transcription of genes associated with proliferation or terminal muscle differentiation. Mimicking phosphorylation of serine-162, a target of protein kinase C-alpha, with an aspartic acid substitution (SRF-S162D) completely inhibited SRF-DNA binding and blocked alpha-actin gene transcription even in the presence of potent myogenic cofactors, while preserving c-fos promoter activity because of stabilization of the ternary complex via Elk-1. Introduction of SRF-S162D into SRF null ES cells permitted transcription of the c-fos gene but was unable to rescue expression of myogenic contractile genes. Transition of proliferating C2C12 myoblasts to postfusion myocytes after serum withdrawal was associated with a progressive decline in SRF-S162 phosphorylation and an increase in alpha-actin gene expression. Hence, the phosphorylation status of serine-162 in the alphaI coil may constitute a novel switch that directs target gene expression into proliferation or differentiation programs.


Assuntos
Actinas/genética , Genes fos/genética , Desenvolvimento Muscular/genética , Mioblastos Esqueléticos/citologia , Fator de Resposta Sérica/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Ácido Aspártico/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Camundongos , Dados de Sequência Molecular , Contração Muscular/genética , Músculo Liso/metabolismo , Mioblastos Esqueléticos/metabolismo , Regiões Promotoras Genéticas/genética , Conformação Proteica , Proteína Quinase C-alfa/metabolismo , Serina/genética , Serina/metabolismo , Fator de Resposta Sérica/química , Fator de Resposta Sérica/genética , Proteínas Elk-1 do Domínio ets/metabolismo
10.
Dev Cell ; 4(1): 107-18, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12530967

RESUMO

Cysteine-rich LIM-only proteins, CRP1 and CRP2, expressed during cardiovascular development act as bridging molecules that associate with serum response factor and GATA proteins. SRF-CRP-GATA complexes strongly activated smooth muscle gene targets. CRP2 was found in the nucleus during early stages of coronary smooth muscle differentiation from proepicardial cells. A dominant-negative CRP2 mutant blocked proepicardial cells from differentiating into smooth muscle cells. Together with SRF and GATA proteins, CRP1 and CRP2 converted pluripotent 10T1/2 fibroblasts into smooth muscle cells, while muscle LIM protein CRP3 inhibited the conversion. Thus, LIM-only proteins of the CRP family play important roles in organizing multiprotein complexes, both in the cytoplasm, where they participate in cytoskeletal remodeling, and in the nucleus, where they strongly facilitate smooth muscle differentiation.


Assuntos
Proteínas Aviárias , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Sistema Cardiovascular/citologia , Sistema Cardiovascular/embriologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Embrião de Galinha , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Fatores de Ligação de DNA Eritroide Específicos , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Proteínas com Domínio LIM , Substâncias Macromoleculares , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional
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