RESUMO
Eif2ak4, a susceptibility gene for type 2 diabetes, encodes GCN2, a molecule activated by amino acid deficiency. Mutations or deletions in GCN2 in pancreatic ß-cells increase mTORC1 activity by decreasing Sestrin2 expression in a TSC2-independent manner. In this study, we searched for molecules downstream of GCN2 that suppress mTORC1 activity in a TSC2-dependent manner. To do so, we used a pull-down assay to identify molecules that competitively inhibit the binding of the T1462 phosphorylation site of TSC2 to 14-3-3. l-asparaginase was identified. Although l-asparaginase is frequently used as an anticancer drug for acute lymphoblastic leukemia, little is known about endogenous l-asparaginase. l-Asparaginase, which is expressed downstream of GCN2, was found to bind 14-3-3 and thereby to inhibit its binding to the T1462 phosphorylation site of TSC2 and contribute to TSC2 activation and mTORC1 inactivation upon TSC2 dephosphorylation. Further investigation of the regulation of mTORC1 activity in pancreatic ß-cells by l-asparaginase should help to elucidate the mechanism of diabetes and insulin secretion failure during anticancer drug use.
Assuntos
Antineoplásicos , Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Asparaginase , Células Secretoras de Insulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Overexpression of sterol regulatory element-binding protein-1c (SREBP-1c) in ß cells causes impaired insulin secretion and ß cell dysfunction associated with diminished pancreatic duodenal homeodomain transcription factor-1 (PDX-1) expression in vitro and in vivo. To identify the molecular mechanism responsible for this effect, the mouse Pdx-1 gene promoter (2.7 kb) was analyzed in ß cell and non-ß cell lines. Despite no apparent sterol regulatory element-binding protein-binding sites, the Pdx-1 promoter was suppressed by SREBP-1c in ß cells in a dose-dependent manner. PDX-1 activated its own promoter. The E-box (-104/-99 bp) in the proximal region, occupied by ubiquitously expressed upstream stimulatory factors (USFs), was crucial for the PDX-1-positive autoregulatory loop through direct PDX-1·USF binding. This positive feedback activation was a prerequisite for SREBP-1c suppression of the promoter in non-ß cells. SREBP-1c and PDX-1 directly interact through basic helix-loop-helix and homeobox domains, respectively. This robust SREBP-1c·PDX-1 complex interferes with PDX-1·USF formation and inhibits the recruitment of PDX-1 coactivators. SREBP-1c also inhibits PDX-1 binding to the previously described PDX-1-binding site (-2721/-2646 bp) in the distal enhancer region of the Pdx-1 promoter. Endogenous up-regulation of SREBP-1c in INS-1 cells through the activation of liver X receptor and retinoid X receptor by 9-cis-retinoic acid and 22-hydroxycholesterol inhibited PDX-1 mRNA and protein expression. Conversely, SREBP-1c RNAi restored Pdx-1 mRNA and protein levels. Through these multiple mechanisms, SREBP-1c, when induced in a lipotoxic state, repressed PDX-1 expression contributing to the inhibition of insulin expression and ß cell dysfunction.
Assuntos
Proteínas de Homeodomínio/biossíntese , Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Elementos de Resposta/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transativadores/biossíntese , Regulação para Cima/fisiologia , Alitretinoína , Animais , Antineoplásicos/farmacologia , Células Hep G2 , Proteínas de Homeodomínio/genética , Humanos , Insulina/genética , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Transativadores/genética , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacos , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismoRESUMO
During musculoskeletal development, Sry-type HMG box 9 (Sox9) has a crucial role in mesenchymal condensation and chondrogenesis. On the other hand, a tissue-specific basic helix-loop-helix (bHLH) transcription factor Scleraxis (Scx) regulates the differentiation of tendon and ligament progenitors. Whereas these two transcription factors cooperatively participate in the determination of cellular lineages, the precise interaction between Sox9 and Scx remains unclear. We have previously demonstrated that the Sox9-dependent transcription is synergistically activated by several Sox9-associating molecules, such as p300 and Smad3, on chromatin. In this study, we investigated the function of Scx in the Sox9-dependent transcription. The expression of alpha1(II) collagen (Col2a1) gene was stimulated by an appropriate transduction of Sox9 and Scx. Scx and its partner E47, which dimerizes with other bHLH proteins, cooperatively enhanced the Sox9-dependent transcription in luciferase reporter assays. Coactivator p300 synergistically increased the activity of Sox9-regulated reporter gene, which contains promoter and enhancer regions of Col2a1, in the presence of Scx and E47. Immunoprecipitation analyses revealed that Scx and E47 formed a transcriptional complex with Sox9 and p300. Scx/E47 heterodimer also associated with a conserved E-box sequence (CAGGTG) in the Col2a1 promoter on chromatin. These findings suggest that Scx and E47 might modulate the primary chondrogenesis by associating with the Sox9-related transcriptional complex, and by binding to the conserved E-box on Col2a1 promoter.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição TCF/metabolismo , Transcrição Gênica , Animais , Linhagem Celular Tumoral , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Sequência Conservada , Elementos E-Box/genética , Proteína p300 Associada a E1A/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Camundongos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Ratos , Proteína 1 Semelhante ao Fator 7 de Transcrição , Ativação Transcricional/genéticaRESUMO
Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.
Assuntos
Aterosclerose/enzimologia , Ésteres do Colesterol/metabolismo , Células Espumosas/enzimologia , Esterol Esterase/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/prevenção & controle , Linhagem Celular , Ésteres do Colesterol/genética , Inativação Gênica , Humanos , Hidrólise , Camundongos , Camundongos Knockout , Esterol Esterase/genéticaRESUMO
Type 2 diabetes mellitus is associated with a marked increase of coronary heart disease (CHD). We aimed to assess the impact of elevated serum lipoprotein (a) (Lp[a]) concentrations on the risk of CHD in patients with type 2 diabetes mellitus. A consecutive series of 352 outpatients was investigated. We determined the serum lipid profile and checked the patients for a history of CHD and of its traditional risk factors. Furthermore, the patients were divided into 3 groups according to the degree of elevation of the serum Lp(a) concentration: serum Lp(a) concentrations greater than 50 mg/dL, between 30 and 50 mg/dL, and less than 30 mg/dL, a presumed high normal value; and the prevalence of CHD was compared among the 3 groups. The serum Lp(a) concentrations in the subjects varied widely from 0.4 to 163.6 mg/dL. Patients with CHD had significantly higher serum Lp(a) concentrations than those without CHD (P = .0045). Logistic regression analysis to identify factors associated with the presence of CHD revealed that elevated serum Lp(a) is a significant risk factor (P = .0246). The prevalence of CHD increased with increasing serum Lp(a) concentrations (P = .048). Patients with serum Lp(a) concentrations greater than 50 mg/dL had a significantly higher prevalence of CHD than those with serum Lp(a) concentrations less than 30 mg/dL: the odds ratio of an elevated serum Lp(a) concentration was 3.346 (P = .039). In conclusion, elevated serum Lp(a) is a significant risk factor; and the risk of CHD appears to increase with increasing serum Lp(a) concentrations. Serum Lp(a) concentration of 50 mg/dL might represent a threshold level in relation to the risk of CHD in patients with type 2 diabetes mellitus.
Assuntos
Doença das Coronárias/etiologia , Diabetes Mellitus Tipo 2/complicações , Lipoproteína(a)/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , HDL-Colesterol/sangue , Doença das Coronárias/sangue , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
The serum lipoprotein(a) [Lp(a)] concentration is under genetic control, and most humans have values lower than 30 mg/dL. Subjects with markedly elevated serum Lp(a) concentrations, that is, > or =100 mg/dL, are rarely encountered, and these subjects have not yet been fully characterized from the clinical point of view. In the present investigation, we studied a total of 223 subjects, comprising 123 males and 100 females, with serum Lp(a) values of more than 100 mg/dL. Many of these subjects had a variety of underlying diseases, including metabolic disorders, renal diseases, and hypertension. We focused our attention on the patients with metabolic disorders, namely, familial hypercholesterolemia (FH), primary non-FH hypercholesterolemia (HC), and type 2 diabetes mellitus (DM), and conducted a comparative study of the patients of these 3 disease groups with the corresponding disease controls with serum Lp(a) levels of less than 30 mg/dL, a presumed high normal value. The frequency of markedly elevated serum Lp(a) levels in the general population has not been reported previously. We determined the frequencies in a consecutive series of patients at our Diabetes and Lipid Outpatient Clinic; the results revealed that the frequencies were 6.4% (8/125), 2.6% (6/232), and 0.9% (3/352) in patients with FH, HC, and type 2 DM, respectively. In an attempt to further demonstrate the impact of markedly elevated serum Lp(a) concentrations on the risk of coronary heart disease (CHD), we compared the prevalence of CHD among the study subjects with that among the corresponding disease controls. The results revealed a significantly higher CHD prevalence in the study subjects of all the 3 groups as compared with that in the corresponding disease controls: the odds ratios of a markedly elevated serum Lp(a) level were 5.429 (95% confidence interval [CI], 1.353-21.782), 8.243 (95% CI, 2.793-24.327), and 5.981 (95% CI, 2.530-14.139) for FH, HC, and type 2 DM, respectively. In the present study, we examined some characteristics of this rare population of subjects with markedly elevated serum Lp(a) levels and demonstrated a very high prevalence of CHD among these patients with FH, HC, and type 2 DM, strongly suggesting the significance of Lp(a) as a risk factor for CHD.
Assuntos
Doença das Coronárias/sangue , Doença das Coronárias/etiologia , Lipoproteína(a)/sangue , Idoso , Doença das Coronárias/epidemiologia , Diabetes Mellitus Tipo 2/sangue , Feminino , Humanos , Hipercolesterolemia/sangue , Hiperlipoproteinemia Tipo II/sangue , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de RiscoRESUMO
Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with alpha/beta hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.
Assuntos
Adipócitos/enzimologia , Carboxilesterase/metabolismo , Triglicerídeos/metabolismo , Células 3T3 , Adipócitos/citologia , Tecido Adiposo/enzimologia , Sequência de Aminoácidos , Animais , Diferenciação Celular , Sequência Conservada , DNA Complementar , Dipeptídeos/química , Humanos , Hidrólise , Lipólise , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Insulin gene expression is regulated by pancreatic beta cell-specific factors, PDX-1 and BETA2/E47. Here we have demonstrated that the insulin promoter is a novel target for SREBPs established as lipid-synthetic transcription factors. Promoter analyses of rat insulin I gene in non-beta cells revealed that nuclear SREBP-1c activates the insulin promoter through three novel SREBP-binding sites (SREs), two of which overlap with E-boxes, binding sites for BETA2/E47. SREBP-1c activation of the insulin promoter was markedly enhanced by co-expression of BETA2/E47. This synergistic activation by SREBP-1c/BETA2/E47 was not mediated through SREs but through the E-boxes on which BETA2/E47 physically interacts with SREBP-1c, suggesting a novel function of SREBP as a co-activator. These two cis-DNA regions, E1 and E2, with an appropriate distance separating them, were mandatory for the synergism, which implicates formation of SREBP-1c.BETA2.E47 complex in a DNA looping structure for efficient recruitment of CREB-binding protein/p300. However, in the presence of PDX1, the synergistic action of SREBP-1c with BETA2/E47 was canceled. SREBP-1c-mediated activation of the insulin promoter and expression became overt in beta cell lines and isolated islets when endogenous PDX-1 expression was low. This cryptic SREBP-1c action might play a compensatory role in insulin expression in diabetes with beta cell lipotoxicity.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica , Insulina/genética , Regiões Promotoras Genéticas , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologia , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Fatores de Transcrição TCF/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Western Blotting , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , DNA/química , Relação Dose-Resposta a Droga , Genes Reporter , Glutationa Transferase/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutagênese Sítio-Dirigida , Pâncreas/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição TCF/metabolismo , Transativadores/metabolismo , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
Sterol regulatory element-binding proteins (SREBPs) are transcription factors that are predominately involved in the regulation of lipogenic and cholesterogenic enzyme gene expression. To identify unknown proteins that interact with SREBP, we screened nuclear extract proteins with 35S-labeled SREBP-1 bait in Far Western blotting analysis. Using this approach, high mobility group protein-B1 (HMGB1), a chromosomal protein, was identified as a novel SREBP interacting protein. In vitro glutathione S-transferase pull-down and in vivo coimmunoprecipitation studies confirmed an interaction between HMGB1 and both SREBP-1 and -2. The protein-protein interaction was mediated through the helix-loop-helix domain of SREBP-1, residues 309-344, and the A box of HMGB1. Furthermore, an electrophoretic mobility shift assay demonstrated that HMGB1 enhances SREBPs binding to their cognate DNA sequences. Moreover, luciferase reporter analyses, including RNA interference technique showed that HMGB1 potentiates the transcriptional activities of SREBP in cultured cells. These findings raise the intriguing possibility that HMGB1 is potentially involved in the regulation of lipogenic and cholesterogenic gene transcription.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteína HMGB1/química , Proteína HMGB1/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Glutationa Transferase/metabolismo , Humanos , Imunoprecipitação , Fígado/metabolismo , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Oligonucleotídeos/química , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Proteínas Recombinantes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , TransfecçãoRESUMO
Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Experimental/genética , Fígado/metabolismo , Animais , Colesterol/metabolismo , Jejum , Ácidos Graxos não Esterificados/sangue , Regulação da Expressão Gênica/genética , Glucoquinase/genética , Glibureto/farmacologia , Cinética , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Fatores de Transcrição/genética , Triglicerídeos/metabolismoRESUMO
Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are members of nuclear receptors that form obligate heterodimers with retinoid X receptors (RXRs). These nuclear receptors play crucial roles in the regulation of fatty acid metabolism: LXRs activate expression of sterol regulatory element-binding protein 1c (SREBP-1c), a dominant lipogenic gene regulator, whereas PPARalpha promotes fatty acid beta-oxidation genes. In the current study, effects of PPARs on the LXR-SREBP-1c pathway were investigated. Luciferase assays in human embryonic kidney 293 cells showed that overexpression of PPARalpha and gamma dose-dependently inhibited SREBP-1c promoter activity induced by LXR. Deletion and mutation studies demonstrated that the two LXR response elements (LXREs) in the SREBP-1c promoter region are responsible for this inhibitory effect of PPARs. Gel shift assays indicated that PPARs reduce binding of LXR/RXR to LXRE. PPARalpha-selective agonist enhanced these inhibitory effects. Supplementation with RXR attenuated these inhibitions by PPARs in luciferase and gel shift assays, implicating receptor interaction among LXR, PPAR, and RXR as a plausible mechanism. Competition of PPARalpha ligand with LXR ligand was observed in LXR/RXR binding to LXRE in gel shift assay, in LXR/RXR formation in nuclear extracts by coimmunoprecipitation, and in gene expression of SREBP-1c by Northern blot analysis of rat primary hepatocytes and mouse liver RNA. These data suggest that PPARalpha activation can suppress LXR-SREBP-1c pathway through reduction of LXR/RXR formation, proposing a novel transcription factor cross-talk between LXR and PPARalpha in hepatic lipid homeostasis.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ácidos Graxos/metabolismo , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocarbonetos Fluorados , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenômenos Fisiológicos da Nutrição , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/metabolismo , Elementos de Resposta/genética , Receptores X de Retinoides , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1 , Sulfonamidas , Fatores de Transcrição/agonistas , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Fatty acid metabolism is transcriptionally regulated by two reciprocal systems: peroxisome proliferator-activated receptor (PPAR) alpha controls fatty acid degradation, whereas sterol regulatory element-binding protein-1c activated by liver X receptor (LXR) regulates fatty acid synthesis. To explore potential interactions between LXR and PPAR, the effect of LXR activation on PPARalpha signaling was investigated. In luciferase reporter gene assays, overexpression of LXRalpha or beta suppressed PPARalpha-induced peroxisome proliferator response element-luciferase activity in a dose-dependent manner. LXR agonists, T0901317 and 22(R)-hydroxycholesterol, dose dependently enhanced the suppressive effects of LXRs. Gel shift assays demonstrated that LXR reduced binding of PPARalpha/retinoid X receptor (RXR) alpha to peroxisome proliferator response element. Addition of increasing amounts of RXRalpha restored these inhibitory effects in both luciferase and gel shift assays, suggesting the presence of RXRalpha competition. In vitro protein binding assays demonstrated that activation of LXR by an LXR agonist promoted formation of LXR/RXRalpha and, more importantly, LXR/PPARalpha heterodimers, leading to a reduction of PPARalpha/RXRalpha formation. Supportively, in vivo administration of the LXR ligand to mice and rat primary hepatocytes substantially decreased hepatic mRNA levels of PPARalpha-targeted genes in both basal and PPARalpha agonist-induced conditions. The amount of nuclear PPARalpha/RXR heterodimers in the mouse livers was induced by treatment with PPARalpha ligand, and was suppressed by superimposed LXR ligand. Taken together with data from the accompanying paper (Yoshikawa, T., T. Ide, H. Shimano, N. Yahagi, M. Amemiya-Kudo, T. Matsuzaka, S. Yatoh, T. Kitamine, H. Okazaki, Y. Tamura, M. Sekiya, A. Takahashi, A. H. Hasty, R. Sato, H. Sone, J. Osuga, S. Ishibashi, and N. Yamada, Endocrinology 144:1240-1254) describing PPARalpha suppression of the LXR-sterol regulatory element-binding protein-1c pathway, we propose the presence of an intricate network of nutritional transcription factors with mutual interactions, resulting in efficient reciprocal regulation of lipid degradation and lipogenesis.
Assuntos
Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Anticolesterolemiantes/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/efeitos dos fármacos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocarbonetos Fluorados , Hidroxicolesteróis/farmacologia , Fígado/efeitos dos fármacos , Fígado/fisiologia , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenômenos Fisiológicos da Nutrição , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1 , Sulfonamidas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
Recent studies on the in vivo roles of the sterol regulatory element binding protein (SREBP) family indicate that SREBP-2 is more specific to cholesterogenic gene expression whereas SREBP-1 targets lipogenic genes. To define the molecular mechanism involved in this differential regulation, luciferase-reporter gene assays were performed in HepG2 cells to compare the transactivities of nuclear SREBP-1a, -1c, and -2 on a battery of SREBP-target promoters containing sterol regulatory element (SRE), SRE-like, or E-box sequences. The results show first that cholesterogenic genes containing classic SREs in their promoters are strongly and efficiently activated by both SREBP-1a and SREBP-2, but not by SREBP-1c. Second, an E-box containing reporter gene is much less efficiently activated by SREBP-1a and -1c, and SREBP-2 was inactive in spite of its ability to bind to the E-box. Third, promoters of lipogenic enzymes containing variations of SRE (SRE-like sequences) are strongly activated by SREBP-1a, and only modestly and equally by both SREBP-1c and -2. Finally, substitution of the unique tyrosine residue within the basic helix-loop-helix (bHLH) portion of nuclear SREBPs with arginine, the conserved residue found in all other bHLH proteins, abolishes the transactivity of all SREBPs for SRE, and conversely results in markedly increased activity of SREBP-1 but not activity of SREBP-2 for E-boxes. These data demonstrate the different specificity and affinity of nuclear SREBP-1 and -2 for different target DNAs, explaining a part of the mechanism behind the differential in vivo regulation of cholesterogenic and lipogenic enzymes by SREBP-1 and -2, respectively.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Colesterol/genética , Proteínas de Ligação a DNA/fisiologia , Lipídeos/biossíntese , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , ATP Citrato (pro-S)-Liase/genética , Animais , Sequência de Bases , Primers do DNA , Elementos Facilitadores Genéticos , Ácido Graxo Sintases/genética , Glucoquinase/genética , Glucosefosfato Desidrogenase/genética , Humanos , Malato Desidrogenase/genética , Camundongos , Camundongos Transgênicos , Piruvato Quinase/genética , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Células Tumorais CultivadasRESUMO
The mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified. In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs. Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue. This refeeding response is significantly reduced in SREBP-1 deficient mice. Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice. Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression. These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase.
Assuntos
Acil Coenzima A/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fígado/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar , Ácidos Graxos Insaturados/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Ligantes , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fenômenos Fisiológicos da Nutrição , Alinhamento de Sequência , Proteína de Ligação a Elemento Regulador de Esterol 1 , Proteína de Ligação a Elemento Regulador de Esterol 2 , Especificidade por Substrato , Distribuição TecidualRESUMO
Obesity is a common nutritional problem often associated with diabetes, insulin resistance, and fatty liver (excess fat deposition in liver). Leptin-deficient Lep(ob)/Lep(ob) mice develop obesity and those obesity-related syndromes. Increased lipogenesis in both liver and adipose tissue of these mice has been suggested. We have previously shown that the transcription factor sterol regulatory element-binding protein-1 (SREBP-1) plays a crucial role in the regulation of lipogenesis in vivo. To explore the possible involvement of SREBP-1 in the pathogenesis of obesity and its related syndromes, we generated mice deficient in both leptin and SREBP-1. In doubly mutant Lep(ob/ob) x Srebp-1(-/-) mice, fatty livers were markedly attenuated, but obesity and insulin resistance remained persistent. The mRNA levels of lipogenic enzymes such as fatty acid synthase were proportional to triglyceride accumulation in liver. In contrast, the mRNA abundance of SREBP-1 and lipogenic enzymes in the adipose tissue of Lep(ob)/Lep(ob) mice was profoundly decreased despite sustained fat, which could explain why the SREBP-1 disruption had little effect on obesity. In conclusion, SREBP-1 regulation of lipogenesis is highly involved in the development of fatty livers but does not seem to be a determinant of obesity in Lep(ob)/Lep(ob) mice.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fígado Gorduroso/terapia , Resistência à Insulina/genética , Fígado/metabolismo , Obesidade/genética , Fatores de Transcrição , Tecido Adiposo/metabolismo , Animais , Northern Blotting , Cruzamentos Genéticos , Genótipo , Leptina/biossíntese , Metabolismo dos Lipídeos , Lipase Lipoproteica/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Triglicerídeos/metabolismoRESUMO
In the process of seeking sterol regulatory element-binding protein 1a (SREBP-1a) target genes, we identified and cloned a cDNA clone encoding mouse Delta(5)-desaturase (D5D). The hepatic expression of D5D as well as Delta(6)-desaturase (D6D) was highly activated in transgenic mice overexpressing nuclear SREBP-1a, -1c, and -2. Disruption of the SREBP-1 gene significantly reduced the expression of both desaturases in the livers of SREBP-1-deficient mice refed after fasting. The hepatic expression of both desaturases was downregulated by dietary PUFA, which were reported to suppress SREBP-1c gene expression. Sustained expression of hepatic nuclear SREBP-1c protein in the transgenic mice abolished the PUFA suppression of both desaturases. Although these data suggested that SREBP-1c regulates D5D and D6D expression, there was no difference in either the D5D or D6D mRNA level between fasted and refed normal mouse livers, indicating a mechanism for fasting induction of both desaturases. Administration of fibrate, a pharmacological ligand for peroxisome proliferator activating receptor alpha (PPARalpha), caused a significant increase in expression of both desaturases. The data suggested that D5D and D6D expression is dually regulated by SREBP-1c and PPARalpha, two reciprocal transcription factors for fatty acid metabolism, and could be involved in lipogenic gene regulation by producing PUFA.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/fisiologia , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Insaturados/farmacologia , Regulação Enzimológica da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Clonagem Molecular , Proteínas de Ligação a DNA/deficiência , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Fenofibrato/farmacologia , Regulação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipolipemiantes/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1 , Distribuição TecidualRESUMO
DNA microarray analysis on upregulated genes in the livers from transgenic mice overexpressing nuclear sterol regulatory element-binding protein (SREBP)-1a, identified an expressed sequence tag (EST) encoding a part of murine cytosolic acetyl-coenzyme A synthetase (ACAS). Northern blot analysis of the livers from transgenic mice demonstrated that this gene was highly induced by SREBP-1a, SREBP-1c, and SREBP-2. DNA sequencing of the 5' flanking region of the murine ACAS gene identified a sterol regulatory element with an adjacent Sp1 site. This region was shown to be responsible for SREBP binding and activation of the ACAS gene by gel shift and luciferase reporter gene assays. Hepatic and adipose tissue ACAS mRNA levels in normal mice were suppressed at fasting and markedly induced by refeeding, and this dietary regulation was nearly abolished in SREBP-1 knockout mice, suggesting that the nutritional regulation of the ACAS gene is controlled by SREBP-1. The ACAS gene was downregulated in streptozotocin-induced diabetic mice and was restored after insulin replacement, suggesting that diabetic status and insulin also regulate this gene. When acetate was administered, hepatic ACAS mRNA was negatively regulated. These data on dietary regulation and SREBP-1 control of ACAS gene expression demonstrate that ACAS is a novel hepatic lipogenic enzyme, providing further evidence that SREBP-1 and insulin control the supply of acetyl-CoA directly from cellular acetate for lipogenesis. However, its high conservation among different species and the wide range of its tissue distribution suggest that this enzyme might also play an important role in basic cellular energy metabolism.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Proteínas de Ligação a DNA/fisiologia , Metabolismo Energético/fisiologia , Lipídeos/biossíntese , Fatores de Transcrição , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Sequência de Aminoácidos/genética , Ração Animal , Animais , Sequência de Bases/genética , Clonagem Molecular , DNA/genética , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/metabolismo , Jejum/fisiologia , Expressão Gênica , Insulina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteína de Ligação a Elemento Regulador de Esterol 1RESUMO
Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) suppress sterol regulatory element-binding protein 1c (SREBP-1c) expression and, thus, lipogenesis. In the current study, the molecular mechanism for this suppressive effect was investigated with luciferase reporter gene assays using the SREBP-1c promoter in HEK293 cells. Consistent with previous data, the addition of PUFAs to the medium in the assays robustly inhibited the SREBP-1c promoter activity. Deletion and mutation of the two liver X receptor (LXR)-responsive elements (LXREs) in the SREBP-1c promoter region eliminated this suppressive effect, indicating that both LXREs are important PUFA-suppressive elements. The luciferase activities of both SREBP-1c promoter and LXRE enhancer constructs induced by co-expression of LXRalpha or -beta were strongly suppressed by the addition of various PUFAs (arachidonic acid > eicosapentaenoic acid > docosahexaenoic acid > linoleic acid), whereas saturated or mono-unsaturated fatty acids had minimal effects. Gel shift mobility and ligand binding domain activation assays demonstrated that PUFA suppression of SREBP-1c expression is mediated through its competition with LXR ligand in the activation of the ligand binding domain of LXR, thereby inhibiting binding of LXR/retinoid X receptor heterodimer to the LXREs in the SREBP-1c promoter. These data suggest that PUFAs could be deeply involved in nutritional regulation of cellular fatty acid levels by inhibiting an LXR-SREBP-1c system crucial for lipogenesis.