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The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway.
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The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) is recognized as the primary target of the fibrate class of hypolipidemic drugs and mediates lipid lowering in part by activating a transcriptional cascade that induces genes involved in the catabolism of lipids. We report here the characterization of three novel PPARalpha agonists with therapeutic potential for treating dyslipidemia. These structurally related compounds display potent and selective binding to human PPARalpha and support robust recruitment of coactivator peptides in vitro. These compounds markedly potentiate chimeric transcription systems in cell-based assays and strikingly lower serum triglycerides in vivo. The transcription networks induced by these selective PPARalpha agonists were assessed by transcriptional profiling of mouse liver after short- and long-term treatment. The induction of several known PPARalpha target genes involved with fatty acid metabolism were observed, reflecting the expected pharmacology associated with PPARalpha activation. We also noted the down-regulation of a number of genes related to immune cell function, the acute phase response, and glucose metabolism, suggesting that these compounds may have anti-inflammatory action in the mammalian liver. Whereas these compounds are efficacious in acute preclinical models, extended safety studies and further clinical testing will be required before the full therapeutic promise of a selective PPARalpha agonist is realized.
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Metabolismo de los Lípidos/fisiología , PPAR alfa/agonistas , Piperidinas/farmacología , Animales , Perfilación de la Expresión Génica , Humanos , Hipolipemiantes/farmacología , Metabolismo de los Lípidos/genética , Hígado , Ratones , Ratones Transgénicos , PPAR alfa/genética , PPAR alfa/metabolismo , Piperidinas/uso terapéuticoRESUMEN
Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 microM PF-429242 (Bioorg Med Chem Lett 17:4411-4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC(50) of 0.5 microM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome.
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Regulación Enzimológica de la Expresión Génica/fisiología , Lipogénesis/fisiología , Proproteína Convertasas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Animales , Células CHO , Línea Celular , Células Cultivadas , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Lipogénesis/efectos de los fármacos , Masculino , Ratones , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Inhibidores de Proteasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/biosíntesisRESUMEN
The discovery and efficacy of a series of potent aminopyrrolidineamide-based inhibitors of sterol regulatory element binding protein site-1 protease is described.
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Proproteína Convertasas/antagonistas & inhibidores , Pirroles/farmacología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Técnicas Químicas Combinatorias , Regulación de la Expresión Génica , Humanos , Hipolipemiantes/química , Hipolipemiantes/farmacología , Concentración 50 Inhibidora , Masculino , Ratones , Estructura Molecular , Pirroles/química , Serina Endopeptidasas , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.
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Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Transactivadores/biosíntesis , Triglicéridos/sangre , Adenoviridae/genética , Adenoviridae/metabolismo , Alimentación Animal , Animales , Cartilla de ADN/química , Ratones , Ratones Obesos , PPAR gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación , Factores de Transcripción , Triglicéridos/metabolismoRESUMEN
Stearoyl-CoA desaturases (SCDs) catalyze the biosynthesis of monounsaturated fatty acids from saturated fatty acids. Four scd genes have been identified in mice and three in human (including one pseudogene). Among the four mouse SCD isoforms, SCD1 is predominantly expressed in liver and adipose tissue. Mice null for the scd1 gene have reduced adiposity, increased energy expenditure and altered lipid profiles. To further evaluate the specific role of hepatic SCD1 and the potential to achieve similar desirable phenotypic changes in adult obese mice, adenovirus-mediated short hairpin interfering RNA (shRNA) was used to acutely knock down hepatic scd1 expression in ob/ob mice. Robust reductions in hepatic SCD1 mRNA and SCD1 enzymatic activity were achieved, sustained up to 2 weeks. Reduced hepatic content of neutral lipids and robust lowering of lipid desaturation indexes, but increased content of liver phosphotidylcholine were observed with SCD1 knockdown. Increased total plasma cholesterol levels were also observed. No significant changes in body weight were observed. Expression levels of several lipogenic and lipid oxidation genes were not significantly altered by short term SCD1 reduction, but UCP2 expression was increased. Our results demonstrate that significant changes to both hepatic and systemic lipid profiles can be achieved through specific knockdown of liver-expressed SCD1 in the ob/ob mouse model. However, hepatic SCD1 knockdown does not result in significant changes in body weight in the short term.
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Ácidos Grasos/química , Lípidos/química , Hígado/enzimología , Obesidad/enzimología , Interferencia de ARN , Estearoil-CoA Desaturasa/metabolismo , Animales , Ratones , Obesidad/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glycerol-3-phosphate acyltransferase (GPAT) controls the first step of triglyceride (TAG) synthesis. Three distinct GPAT activities have been identified, two localized in mitochondria and one in microsomes. Mitochondrial GPAT1 (mtGPAT1) is abundantly expressed in the liver and constitutes approximately 50% of total GPAT activities in this organ. Hepatic mtGPAT1 activity is elevated in obese rodents. Mice deficient in mtGPAT1 have an improved lipid profile. To investigate if beneficial effects can result from reduced hepatic expression of mtGPAT1 in adult obese mice, adenoviral vector-based short hairpin RNA interference (shRNA) technology was used to knockdown mtGPAT1 expression in livers of ob/ob mice. Reduced expression of mtGPAT1 mRNA in liver of ob/ob mice resulted in dramatic and dose dependent reduction in mtGPAT1 activity. Reduced hepatic TAG, diacylglycerol, and free fatty acid, as well as reduced plasma cholesterol and glucose, were also observed. Fatty acid composition analysis revealed decrease of C16:0 in major lipid species. Our results demonstrate that acute reduction of mtGPAT1 in liver of ob/ob mice reduces TAG synthesis, which points to a role for mtGPAT1 in the correction of obesity and related disorders.
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Glicerol-3-Fosfato O-Aciltransferasa/fisiología , Hígado/metabolismo , Adenoviridae/metabolismo , Animales , Secuencia de Bases , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Obesos , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Obesidad/genética , Triglicéridos/metabolismoRESUMEN
RNA interference (RNAi) is an exciting new tool to effect acute in vivo knockdown of genes for pharmacological target validation. Testing the application of this technology to metabolic disease targets, three RNAi delivery methods were compared in two frequently utilized preclinical models of obesity and diabetes, the diet-induced obese (DIO) and B6.V-Lep
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Signaling through the protein phosphatase calcineurin may play a critical role in cardiac hypertrophy. The gene for Down Syndrome Critical Region-1 (DSCR1) encodes a protein that is an endogenous calcineurin inhibitor. This study was designed to test the hypothesis that DSCR1 is directly induced by biomechanical stimuli. Neonatal rat cardiac myocytes were exposed to biaxial cyclic mechanical strain; mechanical strain upregulated DSCR1 mRNA expression in a time- and amplitude-dependent manner (3.4 +/- 0.2-fold at 8% strain for 6 h, n = 11, P < 0.01), and this induction was angiotensin II and endothelin I independent. Biomechanical induction of DSCR1 mRNA was partially blocked by calcineurin inhibition with cyclosporine A (30 +/- 5%, n = 3, P < 0.01). DSCR1 promoter-reporter experiments showed that mechanical strain induced DSCR1 promoter activity by 2.3-fold and that this induction was completely inhibited by cyclosporin A. Furthermore, DSCR1 gene expression was increased in the left ventricles of mice with pressure-overload hypertrophy induced by transverse aortic banding. These data demonstrate that biomechanical strain directly induces gene expression for the calcineurin inhibitor DSCR1 in cardiac myocytes, indicating that mechanically induced DSCR1 may regulate the hypertrophic response to mechanical overload.