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1.
Circ Res ; 135(2): 335-349, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38828596

RESUMO

BACKGROUND: Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. METHODS: We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. RESULTS: Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. CONCLUSIONS: Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.


Assuntos
Apolipoproteína A-I , Aterosclerose , Diabetes Mellitus Tipo 1 , Receptores de LDL , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Apolipoproteína A-I/sangue , Apolipoproteína A-I/metabolismo , Apolipoproteína B-100/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/sangue , Aterosclerose/metabolismo , Aterosclerose/genética , Aterosclerose/sangue , Aterosclerose/patologia , Proteínas de Transferência de Ésteres de Colesterol/genética , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/sangue , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/sangue , Modelos Animais de Doenças , Lipoproteínas HDL/sangue , Lipoproteínas HDL/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de LDL/genética , Receptores de LDL/deficiência , Receptores de LDL/metabolismo
2.
J Lipid Res ; 65(4): 100531, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38490635

RESUMO

Altered apolipoprotein kinetics play a critical role in promoting dyslipidemia and atherogenesis. Human apolipoprotein kinetics have been extensively evaluated, but similar studies in mice are hampered by the lack of robust methods suitable for the small amounts of blood that can be collected at sequential time points from individual mice. We describe a targeted liquid chromatography tandem mass spectrometry method for simultaneously quantifying the stable isotope enrichment of several apolipoproteins represented by multiple peptides in serial blood samples (15 µl each) obtained after retro-orbital injection of 13C6,15N2-lysine (Lys8) in mice. We determined apolipoprotein fractional clearance rates (FCRs) and production rates (PRs) in WT mice and in two genetic models widely used for atherosclerosis research, LDL receptor-deficient (Ldlr-/-) and apolipoprotein E-deficient (Apoe-/-) mice. Injection of Lys8 produced a unique and readily detectable mass shift of labeled compared with unlabeled peptides with sensitivity allowing robust kinetics analyses. Ldlr-/- mice showed slower FCRs of APOA1, APOA4, total APOB, APOB100, APOCs, APOE and APOM, while FCRs of APOA1, APOB100, APOC2, APOC3, and APOM were not lower in Apoe-/- mice versus WT mice. APOE PR was increased in Ldlr-/- mice, and APOB100 and APOA4 PRs were reduced in Apoe-/- mice. Thus, our method reproducibly quantifies plasma apolipoprotein kinetics in different mouse models. The method can easily be expanded to include a wide range of proteins in the same biospecimen and should be useful for determining the kinetics of apolipoproteins in animal models of human disease.


Assuntos
Apolipoproteínas , Marcação por Isótopo , Proteômica , Animais , Camundongos , Proteômica/métodos , Apolipoproteínas/sangue , Cinética , Receptores de LDL/genética , Receptores de LDL/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/sangue , Cromatografia Líquida/métodos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Masculino
3.
Mol Pharmacol ; 99(5): 342-357, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33574048

RESUMO

In recent years, highly sensitive mass spectrometry-based phosphoproteomic analysis is beginning to be applied to identification of protein kinase substrates altered downstream of increased cAMP. Such studies identify a very large number of phosphorylation sites regulated in response to increased cAMP. Therefore, we now are tasked with the challenge of determining how many of these altered phosphorylation sites are relevant to regulation of function in the cell. This minireview describes the use of phosphoproteomic analysis to monitor the effects of cyclic nucleotide phosphodiesterase (PDE) inhibitors on cAMP-dependent phosphorylation events. More specifically, it describes two examples of this approach carried out in the authors' laboratories using the selective PDE inhibitor approach. After a short discussion of several likely conclusions suggested by these analyses of cAMP function in steroid hormone-producing cells and also in T-cells, it expands into a discussion about some newer and more speculative interpretations of the data. These include the idea that multiple phosphorylation sites and not a single rate-limiting step likely regulate these and, by analogy, many other cAMP-dependent pathways. In addition, the idea that meaningful regulation requires a high stoichiometry of phosphorylation to be important is discussed and suggested to be untrue in many instances. These new interpretations have important implications for drug design, especially for targeting pathway agonists. SIGNIFICANCE STATEMENT: Phosphoproteomic analyses identify thousands of altered phosphorylation sites upon drug treatment, providing many possible regulatory targets but also highlighting questions about which phosphosites are functionally important. These data imply that multistep processes are regulated by phosphorylation at not one but rather many sites. Most previous studies assumed a single step or very few rate-limiting steps were changed by phosphorylation. This concept should be changed. Previous interpretations also assumed substoichiometric phosphorylation was not of regulatory importance. This assumption also should be changed.


Assuntos
AMP Cíclico/metabolismo , Fosforilação/fisiologia , Proteoma/metabolismo , Animais , Humanos , Proteômica/métodos , Transdução de Sinais/fisiologia
4.
J Lipid Res ; 61(1): 33-44, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31722970

RESUMO

Chronic inflammation contributes to cardiovascular disease. Increased levels of the inflammatory cytokine, TNF-α, are often present in conditions associated with cardiovascular disease risk, and TNF-α induces a number of pro-atherogenic effects in macrovascular endothelial cells, including expression of adhesion molecules and chemokines, and lipoprotein uptake and transcytosis to the subendothelial tissue. However, little is known about the roles of acyl-CoA synthetases (ACSLs), enzymes that esterify free fatty acids into their acyl-CoA derivatives, or about the effects of TNF-α on ACSLs in endothelial cells. Therefore, we investigated the effects of TNF-α on ACSLs and downstream lipids in cultured human coronary artery endothelial cells and human umbilical vein endothelial cells. We demonstrated that TNF-α induces ACSL1, ACSL3, and ACSL5, but not ACSL4, in both cell types. TNF-α also increased oleoyl-CoA levels, consistent with the increased ACSL3 expression. RNA-sequencing demonstrated that knockdown of ACSL3 had no marked effects on the TNF-α transcriptome. Instead, ACSL3 was required for TNF-α-induced lipid droplet formation in cells exposed to oleic acid. These results demonstrate that increased acyl-CoA synthesis as a result of ACSL3 induction is part of the TNF-α response in human macrovascular endothelial cells.


Assuntos
Coenzima A Ligases/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Células Cultivadas , Coenzima A Ligases/genética , Células Endoteliais/enzimologia , Feminino , Humanos , Gotículas Lipídicas/metabolismo , Masculino
5.
J Proteome Res ; 19(3): 1235-1247, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32037842

RESUMO

Kinase-catalyzed protein phosphorylation is fundamental to eukaryotic signal transduction, regulating most cellular processes. Kinases are frequently dysregulated in cancer, inflammation, and degenerative diseases, and because they can be inhibited with small molecules, they became important drug targets. Accordingly, analytical approaches that determine kinase activation states are critically important to understand kinase-dependent signal transduction and to identify novel drug targets and predictive biomarkers. Multiplexed inhibitor beads (MIBs or kinobeads) efficiently enrich kinases from cell lysates for liquid chromatography-mass spectrometry (LC-MS) analysis. When combined with phosphopeptide enrichment, kinobead/LC-MS can also quantify the phosphorylation state of kinases, which determines their activation state. However, an efficient kinobead/LC-MS kinase phospho-profiling protocol that allows routine analyses of cell lines and tissues has not yet been developed. Here, we present a facile workflow that quantifies the global phosphorylation state of kinases with unprecedented sensitivity. We also found that our kinobead/LC-MS protocol can measure changes in kinase complex composition and show how these changes can indicate kinase activity. We demonstrate the utility of our approach in specifying kinase signaling pathways that control the acute steroidogenic response in Leydig cells; this analysis establishes the first comprehensive framework for the post-translational control of steroid biosynthesis.


Assuntos
Transdução de Sinais , Espectrometria de Massas em Tandem , Cromatografia Líquida , Humanos , Masculino , Fosforilação , Proteínas Quinases/metabolismo
7.
Proc Natl Acad Sci U S A ; 113(38): E5685-93, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27601673

RESUMO

Luteinizing hormone (LH) stimulates steroidogenesis largely through a surge in cyclic AMP (cAMP). Steroidogenic rates are also critically dependent on the availability of cholesterol at mitochondrial sites of synthesis. This cholesterol is provided by cellular uptake of lipoproteins, mobilization of intracellular lipid, and de novo synthesis. Whether and how these pathways are coordinated by cAMP are poorly understood. Recent phosphoproteomic analyses of cAMP-dependent phosphorylation sites in MA10 Leydig cells suggested that cAMP regulates multiple steps in these processes, including activation of the SCAP/SREBP pathway. SCAP [sterol-regulatory element-binding protein (SREBP) cleavage-activating protein] acts as a cholesterol sensor responsible for regulating intracellular cholesterol balance. Its role in cAMP-mediated control of steroidogenesis has not been explored. We used two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associated protein 9) knockout approaches to test the role of SCAP in steroidogenesis. Our results demonstrate that SCAP is required for progesterone production induced by concurrent inhibition of the cAMP phosphodiesterases PDE4 and PDE8. These inhibitors increased SCAP phosphorylation, SREBP2 activation, and subsequent expression of cholesterol biosynthetic genes, whereas SCAP deficiency largely prevented these effects. Reexpression of SCAP in SCAP-deficient cells restored SREBP2 protein expression and partially restored steroidogenic responses, confirming the requirement of SCAP-SREBP2 in steroidogenesis. Inhibitors of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase and isoprenylation attenuated, whereas exogenously provided cholesterol augmented, PDE inhibitor-induced steroidogenesis, suggesting that the cholesterol substrate needed for steroidogenesis is provided by both de novo synthesis and isoprenylation-dependent mechanisms. Overall, these results demonstrate a novel role for LH/cAMP in SCAP/SREBP activation and subsequent regulation of steroidogenesis.


Assuntos
AMP Cíclico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Esteroides/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Proteínas de Transporte , Colesterol/metabolismo , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Intersticiais do Testículo/metabolismo , Lipoproteínas/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Proteínas de Membrana/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fosforilação , Esteroides/química , Proteína de Ligação a Elemento Regulador de Esterol 2/genética
8.
Proc Natl Acad Sci U S A ; 110(16): E1533-42, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23509299

RESUMO

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Western Blotting , Primers do DNA/genética , Drosophila melanogaster , Deleção de Genes , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Sistema de Sinalização das MAP Quinases/genética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mutagênese Sítio-Dirigida , Fosforilação , Ressonância de Plasmônio de Superfície
9.
Int J Cancer ; 134(4): 778-88, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23929039

RESUMO

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet-derived growth factor C (PDGF-C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF-CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast-like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell-derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte-derived PDGF-C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF-CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF-C transgenic mice provide a unique model for the in vivo study of tumor-stromal interactions in the liver.


Assuntos
Carcinoma Hepatocelular/patologia , Fígado Gorduroso/patologia , Células Estreladas do Fígado/patologia , Neoplasias Hepáticas/patologia , Linfocinas/metabolismo , Comunicação Parácrina , Fator de Crescimento Derivado de Plaquetas/metabolismo , Células Estromais/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Estudos de Coortes , Citocinas/genética , Citocinas/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Linfocinas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais/metabolismo
10.
Mol Pharmacol ; 81(4): 556-66, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22232524

RESUMO

Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(-/-), PDE8B(-/-) and PDE8A(-/-)/B(-/-) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (-/-)/B(-/-) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (-/-)/B(-/-) cells had higher testosterone production than cells from either PDE8A(-/-) or PDE8B(-/-) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(-/-)/B(-/-) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Isoenzimas/metabolismo , Células Intersticiais do Testículo/metabolismo , Esteroides/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Imunoprecipitação , Isoenzimas/genética , Células Intersticiais do Testículo/enzimologia , Masculino , Camundongos , Camundongos Knockout
11.
Mol Pharmacol ; 79(4): 639-48, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21187369

RESUMO

The functions of the phosphodiesterase 8B (PDE8) family of phosphodiesterases have been largely unexplored because of the unavailability of selective pharmacological inhibitors. Here, we report a novel function of PDE8B as a major regulator of adrenal steroidogenesis using a genetically ablated PDE8B mouse model as well as cell lines treated with either a new PDE8-selective inhibitor or a short hairpin RNA (shRNA) construct against PDE8B. We demonstrate that PDE8B is highly enriched in mouse adrenal fasciculata cells, and show that PDE8B knockout mice have elevated urinary corticosterone as a result of adrenal hypersensitivity toward adrenocorticotropin. Likewise, ablation of PDE8B mRNA transcripts by an shRNA construct potentiates steroidogenesis in the commonly used Y-1 adrenal cell line. We also observed that the PDE8-selective inhibitor (PF-04957325) potentiates adrenocorticotropin stimulation of steroidogenesis by increasing cAMP-dependent protein kinase activity in both primary isolated adrenocortical cells and Y-1 cells. It is noteworthy that PDE8s have their greatest control under low adrenocorticotropin-stimulated conditions, whereas other higher K(m) PDE(s) modulate steroidogenesis more effectively when cells are fully stimulated. Finally, both genetic ablation of PDE8B and long-term pharmacological inhibition of PDE8s cause increased expression of steroidogenic enzymes. We conclude that PDE8B is a major regulator of one or more pools of cAMP that promote steroidogenesis via both short- and long-term mechanisms. These findings further suggest PDE8B as a potential therapeutic target for the treatment of several different adrenal diseases.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Córtex Suprarrenal/enzimologia , Esteroides/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/deficiência , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Córtex Suprarrenal/metabolismo , Animais , Células Cultivadas , AMP Cíclico/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/genética
12.
J Clin Invest ; 131(22)2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34491909

RESUMO

Loss-of-function mutations in the transcription factor CREB3L3 (CREBH) associate with severe hypertriglyceridemia in humans. CREBH is believed to lower plasma triglycerides by augmenting the activity of lipoprotein lipase (LPL). However, by using a mouse model of type 1 diabetes mellitus (T1DM), we found that greater liver expression of active CREBH normalized both elevated plasma triglycerides and cholesterol. Residual triglyceride-rich lipoprotein (TRL) remnants were enriched in apolipoprotein E (APOE) and impoverished in APOC3, an apolipoprotein composition indicative of increased hepatic clearance. The underlying mechanism was independent of LPL, as CREBH reduced both triglycerides and cholesterol in LPL-deficient mice. Instead, APOE was critical for CREBH's ability to lower circulating remnant lipoproteins because it failed to reduce TRL cholesterol in Apoe-/- mice. Importantly, individuals with CREB3L3 loss-of-function mutations exhibited increased levels of remnant lipoproteins that were deprived of APOE. Recent evidence suggests that impaired clearance of TRL remnants promotes cardiovascular disease in patients with T1DM. Consistently, we found that hepatic expression of CREBH prevented the progression of diabetes-accelerated atherosclerosis. Our results support the proposal that CREBH acts through an APOE-dependent pathway to increase hepatic clearance of remnant lipoproteins. They also implicate elevated levels of remnants in the pathogenesis of atherosclerosis in T1DM.


Assuntos
Aterosclerose/prevenção & controle , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Diabetes Mellitus Tipo 1/complicações , Dislipidemias/prevenção & controle , Lipoproteínas/sangue , Triglicerídeos/sangue , Animais , Apolipoproteína C-III/sangue , Apolipoproteínas E/sangue , Aterosclerose/etiologia , Remanescentes de Quilomícrons/sangue , Dislipidemias/etiologia , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
13.
Lab Invest ; 90(12): 1704-17, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20548286

RESUMO

In nonalcoholic fatty liver disease (NAFLD), depletion of hepatic antioxidants may contribute to the progression of steatosis to nonalcoholic steatohepatitis (NASH) by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. We investigated whether depletion of glutathione (GSH) increases NASH-associated hepatic pathology in mice fed a diet deficient in methionine and choline (MCD diet). Wild-type (wt) mice and genetically GSH-deficient mice lacking the modifier subunit of glutamate cysteine ligase (Gclm null mice), the rate-limiting enzyme for de novo synthesis of GSH, were fed the MCD diet, a methionine/choline-sufficient diet, or standard chow for 21 days. We assessed NASH-associated hepatic pathology, including steatosis, fibrosis, inflammation, and hepatocyte ballooning, and used the NAFLD Scoring System to evaluate the extent of changes. We measured triglyceride levels, determined the level of lipid peroxidation products, and measured by qPCR the expression of mRNAs for several proteins associated with lipid metabolism, oxidative stress, and fibrosis. MCD-fed GSH-deficient Gclm null mice were to a large extent protected from MCD diet-induced excessive fat accumulation, hepatocyte injury, inflammation, and fibrosis. Compared with wt animals, MCD-fed Gclm null mice had much lower levels of F2-isoprostanes, lower expression of acyl-CoA oxidase, carnitine palmitoyltransferase 1a, uncoupling protein-2, stearoyl-coenzyme A desaturase-1, transforming growth factor-ß, and plasminogen activator inhibitor-1 mRNAs, and higher activity of catalase, indicative of low oxidative stress, inhibition of triglyceride synthesis, and lower expression of profibrotic proteins. Global gene analysis of hepatic RNA showed that compared with wt mice, the livers of Gclm null mice have a high capacity to metabolize endogenous and exogenous compounds, have lower levels of lipogenic proteins, and increased antioxidant activity. Thus, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis.


Assuntos
Dieta/efeitos adversos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Glutationa/metabolismo , Acil Coenzima A/metabolismo , Acil-CoA Oxidase/metabolismo , Animais , Antioxidantes/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Colina/metabolismo , Progressão da Doença , Fígado Gorduroso/complicações , Fígado Gorduroso/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Inflamação/complicações , Inflamação/metabolismo , Inflamação/patologia , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Peroxidação de Lipídeos/fisiologia , Fígado/metabolismo , Fígado/patologia , Masculino , Metionina/deficiência , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/fisiologia , Proteína Desacopladora 2
14.
J Clin Invest ; 129(10): 4165-4179, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31295146

RESUMO

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.


Assuntos
Aterosclerose/metabolismo , Doença da Artéria Coronariana/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Células Espumosas/metabolismo , Calcificação Vascular/metabolismo , Adulto , Animais , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Doença da Artéria Coronariana/tratamento farmacológico , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patologia , Feminino , Células Espumosas/patologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos Antissenso/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/genética , Calcificação Vascular/patologia
15.
JCI Insight ; 3(11)2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29875324

RESUMO

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor-deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.


Assuntos
Aterosclerose/imunologia , Transportador de Glucose Tipo 1/metabolismo , Glicólise/imunologia , Síndrome Metabólica/complicações , Monócitos/imunologia , Animais , Artérias/citologia , Artérias/imunologia , Artérias/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Dicarbetoxi-Di-Hidrocolidina/administração & dosagem , Modelos Animais de Doenças , Progressão da Doença , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/imunologia , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Receptores de LDL/genética
16.
Diabetes ; 67(5): 946-959, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29483182

RESUMO

Cardiovascular disease caused by atherosclerosis is the leading cause of mortality associated with type 2 diabetes and metabolic syndrome. Insulin therapy is often needed to improve glycemic control, but it does not clearly prevent atherosclerosis. Upon binding to the insulin receptor (IR), insulin activates distinct arms of downstream signaling. The IR-Akt arm is associated with blood glucose lowering and beneficial effects, whereas the IR-Erk arm might exert less desirable effects. We investigated whether selective activation of the IR-Akt arm, leaving the IR-Erk arm largely inactive, would result in protection from atherosclerosis in a mouse model of metabolic syndrome. The insulin mimetic peptide S597 lowered blood glucose and activated Akt in insulin target tissues, mimicking insulin's effects, but only weakly activated Erk and even prevented insulin-induced Erk activation. Strikingly, S597 retarded atherosclerotic lesion progression through a process associated with protection from leukocytosis, thereby reducing lesional accumulation of inflammatory Ly6Chi monocytes. S597-mediated protection from leukocytosis was accompanied by reduced numbers of the earliest bone marrow hematopoietic stem cells and reduced IR-Erk activity in hematopoietic stem cells. This study provides a conceptually novel treatment strategy for advanced atherosclerosis associated with metabolic syndrome and type 2 diabetes.


Assuntos
Aterosclerose/prevenção & controle , Glicemia/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Receptor de Insulina/efeitos dos fármacos , Animais , Aterosclerose/etiologia , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Angiopatias Diabéticas/etiologia , Angiopatias Diabéticas/prevenção & controle , Modelos Animais de Doenças , Masculino , Síndrome Metabólica/complicações , Camundongos , Camundongos Knockout , Monócitos , Placa Aterosclerótica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/agonistas , Receptor de Insulina/metabolismo , Receptores de LDL/genética , Transdução de Sinais
17.
PLoS One ; 11(3): e0151063, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27002851

RESUMO

BACKGROUND & AIMS: The gut microbiota significantly influences hepatic immunity. Little is known on the precise mechanism by which liver cells mediate recognition of gut microbes at steady state. Here we tested the hypothesis that a specific liver cell population was the sensor and we aimed at deciphering the mechanism by which the activation of TLR4 pathway would mediate liver response to gut microbiota. METHODS: Using microarrays, we compared total liver gene expression in WT versus TLR4 deficient mice. We performed in situ localization of the major candidate protein, CXCL1. With an innovative technique based on cell sorting, we harvested enriched fractions of KCs, LSECs and HSCs from the same liver. The cytokine secretion profile was quantified in response to low levels of LPS (1ng/mL). Chemotactic activity of stellate cell-derived CXCL1 was assayed in vitro on neutrophils upon TLR4 activation. RESULTS: TLR4 deficient liver had reduced levels of one unique chemokine, CXCL1 and subsequent decreased of neutrophil counts. Depletion of gut microbiota mimicked TLR4 deficient phenotype, i.e., decreased neutrophils counts in the liver. All liver cells were responsive to low levels of LPS, but hepatic stellate cells were the major source of chemotactic levels of CXCL1. Neutrophil migration towards secretory hepatic stellate cells required the TLR4 dependent secretion of CXCL1. CONCLUSIONS: Showing the specific activation of TLR4 and the secretion of one major functional chemokine-CXCL1, the homolog of human IL-8-, we elucidate a new mechanism in which Hepatic Stellate Cells play a central role in the recognition of gut microbes by the liver at steady state.


Assuntos
Quimiocina CXCL1/imunologia , Microbioma Gastrointestinal/imunologia , Células Estreladas do Fígado/imunologia , Fígado/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Interleucina-8/imunologia , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Transdução de Sinais/imunologia
18.
Cell Signal ; 28(7): 764-78, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26643407

RESUMO

Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increases cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B and PDE4C as well as PDE8A and PDE8B are expressed in rodent Leydig cells and that combined inhibition of PDE4 and PDE8 leads to dramatically increased steroid biosynthesis. Here we investigated the effect of PDE4 and PDE8 inhibition on the molecular mechanisms of cAMP actions in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE4 family specific inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180min. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests that cAMP, PDE4 and PDE8 coordinate steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that may be regulated to assure timely and sufficient testosterone secretion in response to LH.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Células Intersticiais do Testículo/enzimologia , Fosfoproteínas/metabolismo , Proteômica/métodos , Transdução de Sinais , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Análise por Conglomerados , Bases de Dados como Assunto , Endocitose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ontologia Genética , Insulina/metabolismo , Marcação por Isótopo , Células Intersticiais do Testículo/efeitos dos fármacos , Masculino , Camundongos , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Proteoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo
19.
J Neurosci ; 23(16): 6452-9, 2003 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-12878685

RESUMO

The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed. Although expression of guanylate cyclase and/or cGMP-dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cells in vivo has been demonstrated. To determine if changes in PKG activation and PDE5 regulation occur in vivo we have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylation in vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as an in vivo indicator for PKG activation.


Assuntos
3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Cerebelo/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Células de Purkinje/metabolismo , 3',5'-GMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/química , Animais , Especificidade de Anticorpos , Cerebelo/química , Cerebelo/citologia , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5 , Citosol/química , Citosol/enzimologia , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos , Imuno-Histoquímica , Injeções Intraventriculares , Depressão Sináptica de Longo Prazo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitroprussiato/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , Células de Purkinje/enzimologia
20.
PLoS One ; 9(3): e92925, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24667490

RESUMO

Chronic liver injury leads to fibrosis, cirrhosis, and loss of liver function. Liver cirrhosis is the 12th leading cause of death in the United States, and it is the primary risk factor for developing liver cancer. Fibrosis and cirrhosis result from activation of hepatic stellate cells (HSCs), which are the primary collagen producing cell type in the liver. Here, we show that platelet-derived growth factor receptor α (PDGFRα) is expressed by human HSCs, and PDGFRα expression is elevated in human liver disease. Using a green fluorescent protein (GFP) reporter mouse strain, we evaluated the role of PDGFRα in liver disease in mice and found that mouse HSCs express PDGFRα and expression is upregulated during carbon tetrachloride (CCl4) induced liver injury and fibrosis injection. This fibrotic response is reduced in Pdgfrα heterozygous mice, consistent with the hypothesis that liver fibrosis requires upregulation and activation of PDGFRα. These results indicate that Pdgfrα expression is important in the fibrotic response to liver injury in humans and mice, and suggest that blocking PDGFRα-specific signaling pathways in HSCs may provide therapeutic benefit for patients with chronic liver disease.


Assuntos
Cirrose Hepática/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Alelos , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética
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