RESUMEN

Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.

Asunto(s)

Dipiridamol/farmacología , Retículo Endoplásmico/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipogénesis/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Células CHO , Línea Celular , Cricetulus , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética

RESUMEN

Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2 dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid-dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP, and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice, both systems of elevated SREBP-1c-driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition of SREBP processing. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions.

Asunto(s)

Glucocorticoides/farmacología , N-Metiltransferasa de Histona-Lisina/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Animales , Cromatina/metabolismo , Dexametasona/farmacología , Elementos de Facilitación Genéticos/genética , Conducta Alimentaria/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Sitios Genéticos , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Lisina/metabolismo , Masculino , Metilación/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Obesos , Regiones Promotoras Genéticas , Unión Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

Circadian rhythms are intimately linked to cellular metabolism. Specifically, the NAD(+)-dependent deacetylase SIRT1, the founding member of the sirtuin family, contributes to clock function. Whereas SIRT1 exhibits diversity in deacetylation targets and subcellular localization, SIRT6 is the only constitutively chromatin-associated sirtuin and is prominently present at transcriptionally active genomic loci. Comparison of the hepatic circadian transcriptomes reveals that SIRT6 and SIRT1 separately control transcriptional specificity and therefore define distinctly partitioned classes of circadian genes. SIRT6 interacts with CLOCK:BMAL1 and, differently from SIRT1, governs their chromatin recruitment to circadian gene promoters. Moreover, SIRT6 controls circadian chromatin recruitment of SREBP-1, resulting in the cyclic regulation of genes implicated in fatty acid and cholesterol metabolism. This mechanism parallels a phenotypic disruption in fatty acid metabolism in SIRT6 null mice as revealed by circadian metabolome analyses. Thus, genomic partitioning by two independent sirtuins contributes to differential control of circadian metabolism.

Asunto(s)

Hígado/metabolismo , Sirtuinas/metabolismo , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/metabolismo , Cromatina , Ritmo Circadiano , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Sirtuina 1/genética , Sirtuina 1/metabolismo , Sirtuinas/genética , Transcripción Genética

RESUMEN

Sterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA "operon" analogous to the classic model for genetic control in bacterial regulatory systems.

Asunto(s)

Genes Reguladores/genética , Homeostasis/genética , Metabolismo de los Lípidos/genética , MicroARNs/genética , Operón/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genética , Animales , Células Cultivadas , Proteínas F-Box/genética , Proteínas F-Box/fisiología , Proteína 7 que Contiene Repeticiones F-Box-WD , Genes Reguladores/fisiología , Homeostasis/fisiología , Metabolismo de los Lípidos/fisiología , Hígado/citología , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , MicroARNs/fisiología , Modelos Animales , Operón/fisiología , Proteína 2 de Unión a Elementos Reguladores de Esteroles/fisiología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/fisiología

RESUMEN

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

Asunto(s)

Acrosoma/metabolismo , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Omega-3/metabolismo , Espermatogénesis , Acrosina/metabolismo , Acrosoma/ultraestructura , Animales , Animales no Consanguíneos , Citoplasma/metabolismo , Citoplasma/ultraestructura , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Ácidos Docosahexaenoicos/deficiencia , Ácidos Docosahexaenoicos/metabolismo , Ácidos Docosahexaenoicos/uso terapéutico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Ácido Graso Desaturasas/genética , Ácidos Grasos Omega-3/uso terapéutico , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Masculino , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , Espermátides/metabolismo , Espermátides/ultraestructura , Sintaxina 1/metabolismo

RESUMEN

OBJECTIVE: To analyze the possible role of arachidonic acid (AA) in macrophage cholesterol biosynthesis and in PON2 expression. METHODS AND RESULTS: We used peritoneal macrophages (MPM) from the 6-DS KO mice that were fed a diet without or with AA. Macrophage cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels were substantially increased, by 98% and 67%, respectively, in MPM from 6-DS KO vs. control (C57BL/6) mice. Furthermore, in the 6-DS KO vs. control mice MPM PON2 expression (mRNA and lactonase activity) was substantially decreased. In line with the above results, AA supplementation to 6-DS KO mice significantly decreased MPM cholesterol biosynthesis rate and HMGCoA-reductase mRNA levels, by 45% and by 4-fold respectively, and increased MPM PON2 lactonase activity and PON2 mRNA, by 119% and 2.3-fold, respectively. Similarly, incubation of control mice MPM or J774A.1 with AA, significantly and dose-dependently decreased cellular cholesterol biosynthesis rate, and increased PON2 expression. These effects were specific for AA since incubation of the cells with docosahexaenoic acid (DHA, another product of 6-DS) had no significant effects on cholesterol biosynthesis rate, and on PON2 activity. CONCLUSIONS: AA decreased macrophage cholesterol biosynthesis rate, and increased PON2 expression. These effects could protect the cells from cholesterol accumulation and oxidation, and from foam cell formation, the hallmark of early atherogenesis.

Asunto(s)

Ácido Araquidónico/metabolismo , Arildialquilfosfatasa/metabolismo , Colesterol/metabolismo , Linoleoil-CoA Desaturasa/genética , Macrófagos Peritoneales/citología , Macrófagos/metabolismo , Animales , Aterosclerosis , Colesterol/química , Ácidos Docosahexaenoicos/metabolismo , Citometría de Flujo/métodos , Células Espumosas/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos

RESUMEN

Delta-6 desaturase-null mice ((-/-)) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The (-/-) males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type ((+/+)) and (-/-) males at weaning until 16 weeks of age (n = 3-5). A breeding success rate of DHA-supplemented (-/-) was comparable to (+/+). DHA-fed (-/-) showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed (-/-), but DPAn6 remained depleted. In AA-fed (-/-), AA was restored at the (+/+) level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of (+/+) and DHA-fed (-/-), whereas it diminished in (-/-) and AA-fed (-/-), suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA.

Asunto(s)

Suplementos Dietéticos , Ácidos Docosahexaenoicos/farmacología , Fertilidad/efectos de los fármacos , Linoleoil-CoA Desaturasa/deficiencia , Linoleoil-CoA Desaturasa/genética , Espermatogénesis/efectos de los fármacos , Animales , Ácido Araquidónico/administración & dosificación , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Colestadienos/metabolismo , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/metabolismo , Grasas de la Dieta/farmacología , Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Docosahexaenoicos/metabolismo , Femenino , Flagelos/efectos de los fármacos , Flagelos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Recuento de Espermatozoides , Cabeza del Espermatozoide/efectos de los fármacos , Cabeza del Espermatozoide/metabolismo , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/metabolismo

RESUMEN

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (-/-), which enabled us to study HUFA deficiency without depleting their precursors. In -/-, no in vivo AA synthesis was detected after administration of [U-(13)C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the -/- developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The -/- also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male -/- lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in -/- declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in -/-. DPAn-6 was absent in -/- brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (-/-) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.

Asunto(s)

Intestinos/patología , Linoleoil-CoA Desaturasa/deficiencia , Linoleoil-CoA Desaturasa/genética , Reproducción/genética , Úlcera Cutánea/genética , Úlcera/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dermatitis/genética , Suplementos Dietéticos , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Femenino , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Hepatomegalia/genética , Infertilidad Masculina/genética , Linoleoil-CoA Desaturasa/metabolismo , Masculino , Ratones , Especificidad de Órganos , Fenotipo , Úlcera Cutánea/etiología , Úlcera Cutánea/metabolismo , Úlcera Cutánea/patología , Esplenomegalia/genética , Úlcera/etiología , Úlcera/metabolismo , Úlcera/patología