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The amygdala, a critical brain region responsible for emotional behavior, is crucially involved in the regulation of the effects of stress on emotional behavior. In the mammalian forebrain, gastrin-releasing peptide (GRP), a 27-amino-acid mammalian neuropeptide, which is a homolog of the 14-amino-acid amidated amphibian peptide bombesin, is highly expressed in the amygdala. The levels of GRP are markedly increased in the amygdala after acute stress; therefore, it is known as a stress-activated modulator. To determine the role of GRP in emotional behavior under stress, we conducted some behavioral and biochemical experiments with GRP-knockout (KO) mice. GRP-KO mice exhibited a longer freezing response than wild-type (WT) littermates in both contextual and auditory fear (also known as threat) conditioning tests only when they were subjected to acute restraint stress 20 min before the conditioning. To identify the critical neural circuits associated with the regulation of emotional memory by GRP, we conducted Arc/Arg3.1-reporter mapping in the amygdala with an Arc-Venus reporter transgenic mouse line. In the amygdalostriatal transition area (AST) and the lateral side of the basal nuclei, fear conditioning after restraint stress increased neuronal activity significantly in WT mice, and GRP KO was found to negate this potentiation only in the AST. These results indicate that the GRP-activated neurons in the AST are likely to suppress excessive fear expression through the regulation of downstream circuits related to fear learning following acute stress.
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Bombesina , Miedo , Amígdala del Cerebelo/metabolismo , Animales , Bombesina/metabolismo , Bombesina/farmacología , Condicionamiento Clásico/fisiología , Miedo/fisiología , Péptido Liberador de Gastrina/metabolismo , Péptido Liberador de Gastrina/farmacología , Mamíferos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Metformin plus a dipeptidyl peptidase-4 inhibitor (DPP-4i) is the most common therapy for Japanese patients with type 2 diabetes. This 24-week, multicentre, open-label, parallel-group trial randomized patients on dual therapy to add-on tofogliflozin (20 mg/day, n = 33) or glimepiride (0.5 mg/day, n = 31). The primary outcome was change in body fat percentage. The secondary outcomes included changes in HbA1c, fat mass, fat-free mass, liver function variables and uric acid. Tofogliflozin and glimepiride reduced HbA1c to a similar extent. Body fat percentage did not change from baseline in either group. Fat mass was reduced by tofogliflozin but was increased by glimepiride (by -2.0 ± 1.7 kg and +1.6 ± 1.6 kg, P = .002). Fat-free mass was also reduced by tofogliflozin and increased by glimepiride (by -1.3 ± 1.3 kg and +0.9 ± 2.0 kg, P < .001). Alanine aminotransferase and uric acid levels were reduced by tofogliflozin (P = .006 and P < .001, respectively). These data provide novel information useful for selecting the third oral agent for patients whose diabetes is inadequately controlled with metformin plus DPP-4i dual therapy.
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Diabetes Mellitus Tipo 2 , Inhibidores de la Dipeptidil-Peptidasa IV , Metformina , Administración Oral , Compuestos de Bencidrilo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/uso terapéutico , Quimioterapia Combinada , Glucósidos , Hemoglobina Glucada/análisis , Humanos , Hipoglucemiantes/uso terapéutico , Japón/epidemiología , Metformina/uso terapéutico , Compuestos de Sulfonilurea/uso terapéutico , Resultado del TratamientoRESUMEN
Pemafibrate is the first clinically-available selective peroxisome proliferator-activated receptor α modulator (SPPARMα) that has been shown to effectively improve hypertriglyceridemia and low high-density lipoprotein cholesterol (HDL-C) levels. Global gene expression analysis reveals that the activation of PPARα by pemafibrate induces fatty acid (FA) uptake, binding, and mitochondrial or peroxisomal oxidation as well as ketogenesis in mouse liver. Pemafibrate most profoundly induces HMGCS2 and PDK4, which regulate the rate-limiting step of ketogenesis and glucose oxidation, respectively, compared to other fatty acid metabolic genes in human hepatocytes. This suggests that PPARα plays a crucial role in nutrient flux in the human liver. Additionally, pemafibrate induces clinically favorable genes, such as ABCA1, FGF21, and VLDLR. Furthermore, pemafibrate shows anti-inflammatory effects in vascular endothelial cells. Pemafibrate is predicted to exhibit beneficial effects in patients with atherogenic dyslipidemia and diabetic microvascular complications.
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Benzoxazoles/farmacología , Butiratos/farmacología , PPAR alfa/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Modelos Moleculares , Oxidación-Reducción/efectos de los fármacos , PPAR alfa/agonistas , PPAR alfa/químicaRESUMEN
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase involved in many cellular functions including cell growth and migration. EGFR may be activated by EGF family ligands such as EGF and epiregulin (EREG). EREG is overexpressed in human colon and breast cancers, implying that EREG plays roles in tumorigenesis. Although EGF family members share a receptor, it is not well known whether their signaling pathways differ. In order to investigate EREG signaling, we established the anti-EREG antibody that inhibits EGFR downstream signaling stimulated by EREG but not by EGF. While the anti-EREG antibody has little effect on cell growth, it inhibits cell adhesion of EREG-expressing autocrine cancer cell lines. Our results suggest that anti-EREG antibodies represent valuable tools for elucidating EREG-specific signaling pathways, and may serve as therapeutic candidates for the treatment of cancers.
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Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Epirregulina/inmunología , Receptores ErbB/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence.
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Proteína Morfogenética Ósea 2/genética , Senescencia Celular/genética , Epigénesis Genética , Proteína Smad1/metabolismo , Actinina/metabolismo , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Metilación de ADN , Proteína Potenciadora del Homólogo Zeste 2 , Fibroblastos/citología , Regulación de la Expresión Génica , Genes ras/genética , Genoma , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Complejo Represivo Polycomb 2 , Transducción de Señal , Proteína smad6/genética , Proteína smad6/metabolismoRESUMEN
AIMS/INTRODUCTION: We aimed to study the relationships among the copper (Cu)/zinc (Zn) ratio, inflammatory biomarkers, and the prevalence of diabetic kidney disease (DKD) in patients with type 2 diabetes. MATERIALS AND METHODS: A cross-sectional study was performed on 651 patients with type 2 diabetes. DKD was defined as a urinary albumin-to-creatinine ratio of ≥30 mg/g creatinine and/or an estimated glomerular filtration rate using cystatin C of < 60 mL/min/1.73 m2 . Areas under the curves (AUCs), cutoff values, and thresholds for detecting DKD were determined for the Cu/Zn ratio, soluble tumor necrosis factor-α receptor 1 (sTNFαR1), and high-sensitivity C-reactive protein (hsCRP). Patients were categorized by each cutoff value of sTNFαR1 and the Cu/Zn ratio. Odds ratios (ORs) and biological interactions for the prevalence of DKD were determined. RESULTS: DKD was identified in 220 patients. AUC/optimal cutoff values were 0.777/1300 pg/mL for sTNFαR1, 0.603/1.1648 for the Cu/Zn ratio, and 0.582/305 ng/mL for hsCRP. The ORs for DKD were higher, but not significantly, in the sTNFαR1 < 1300 and Cu/Zn ≥ 1.1648 group, significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P < 0.0001), and further synergistically elevated in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group (P < 0.0001) compared with the sTNFαR1 < 1300 and Cu/Zn < 1.1648 group after multivariable adjustment. Levels of sTNFαR1 were significantly higher in the sTNFαR1 ≥ 1300 and Cu/Zn ≥ 1.1648 group than in the sTNFαR1 ≥ 1300 and Cu/Zn < 1.1648 group (P = 0.0006). CONCLUSIONS: Under an inflammatory initiation signal of elevated serum sTNFαR1 levels, an increase in the Cu/Zn ratio may further exacerbate inflammation and is synergistically associated with a high prevalence of DKD in patients with type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Nefropatías Diabéticas , Cobre , Estudios Transversales , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Nefropatías Diabéticas/complicaciones , Nefropatías Diabéticas/epidemiología , Humanos , ZincRESUMEN
Ballooning degeneration of hepatocytes is a major distinguishing histological feature of non-alcoholic steatosis (NASH) progression that can lead to cirrhosis and hepatocellular carcinoma (HCC). In this study, we evaluated the effect of the selective PPARα modulator (SPPARMα) pemafibrate (Pema) and sodium-glucose cotransporter 2 (SGLT2) inhibitor tofogliflozin (Tofo) combination treatment on pathological progression in the liver of a mouse model of NASH (STAM) at two time points (onset of NASH progression and HCC survival). At both time points, the Pema and Tofo combination treatment significantly alleviated hyperglycemia and hypertriglyceridemia. The combination treatment significantly reduced ballooning degeneration of hepatocytes. RNA-seq analysis suggested that Pema and Tofo combination treatment resulted in an increase in glyceroneogenesis, triglyceride (TG) uptake, lipolysis and liberated fatty acids re-esterification into TG, lipid droplet (LD) formation, and Cidea/Cidec ratio along with an increased number and reduced size and area of LDs. In addition, combination treatment reduced expression levels of endoplasmic reticulum stress-related genes (Ire1a, Grp78, Xbp1, and Phlda3). Pema and Tofo treatment significantly improved survival rates and reduced the number of tumors in the liver compared to the NASH control group. These results suggest that SPPARMα and SGLT2 inhibitor combination therapy has therapeutic potential to prevent NASH-HCC progression.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Animales , Compuestos de Bencidrilo/farmacología , Benzoxazoles/farmacología , Butiratos/farmacología , Carcinoma Hepatocelular/prevención & control , Glucósidos/farmacología , Neoplasias Hepáticas/prevención & control , Ratones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , PPAR alfa/antagonistas & inhibidores , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , TriglicéridosRESUMEN
Non-alcoholic steatohepatitis (NASH) is characterized by macrovesicular steatosis with ballooning degeneration of hepatocytes, diffused lobular inflammation, and fibrosis. PPAR ligands are promising therapeutic agents in NASH; accordingly, we evaluated the effects of the first clinically available selective PPARα modulator, pemafibrate. We found that pemafibrate improves F4/80-positive macrophage accumulation, ballooning degeneration of hepatocytes, and the non-alcoholic fatty liver disease (NAFLD) activity score without affecting triglyceride (TG) accumulation in the liver of a mouse model of NASH (STAM). A global gene expression analysis indicated that pemafibrate enhances TG hydrolysis and fatty acid ß-oxidation as well as re-esterification from dihydroxyacetone 3-phosphate and monoacylglycerol to TG. These changes are accompanied by the induction of genes involved in lipolysis and lipid droplet formation, along with an increased number and reduced size of lipid droplets in pemafibrate-treated livers. Pemafibrate reduced the expression of the cell adhesion molecule Vcam-1, myeloid cell markers, and inflammation- and fibrosis-related genes in STAM mice. Furthermore, pemafibrate significantly reduced VCAM-1 expression induced by high glucose in cultured human umbilical vein endothelial cells. These results suggest that pemafibrate prevents NASH development by reducing myeloid cell recruitment via interactions with liver sinusoidal endothelial cells, without altering hepatic TG accumulation.
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Benzoxazoles/farmacología , Butiratos/farmacología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , PPAR alfa/genética , Molécula 1 de Adhesión Celular Vascular/genética , Animales , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Oxidación-Reducción , Triglicéridos/metabolismoRESUMEN
Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation, is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression.
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Regulación hacia Abajo/genética , Etanolaminas/metabolismo , Glutamina/metabolismo , ARN Nucleotidiltransferasas/genética , Inanición/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Células HeLa , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-ets/genética , Transcripción Genética/genéticaRESUMEN
A 76 year-old man had had hypertension, diabetes mellitus and hyperlipidemia since 1985, and bruit in his left neck since 1993. He had abrupt decrease in left visual acuity on November 24, 2005, and visited an ophthalmologist. On November 28, his corrected visual acuity was 1.0 in the right and 0.1 in the left. The examination of optic fundi showed ear-side edema of the left optic disk. Fluorescence examination of the left optic fundus showed delay in early filling and later hyperfluorescence. Goldman visual field examination showed horizontal lower semiblindness. Since he did not complain of eye pain, his blood examination showed no reaction of inflammation, and he had hypertension, diabetes mellitus and hyperlipidemia, anterior ischemic optic neuropathy was diagnosed. The treatment with aspirin, alprostadil and prednisolone transiently improved the optic fundi and visual acuity, but his left visual acuity returned to 0.1. Carotid ultrasonography showed 95 percent stenosis in the left internal carotid artery. As there is no established treatment for ischemic optic neuropathy, the management of risk factors is most important.
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Estenosis Carotídea/complicaciones , Complicaciones de la Diabetes , Hiperlipidemias/complicaciones , Hipertensión/complicaciones , Neuropatía Óptica Isquémica/etiología , Anciano , Humanos , MasculinoRESUMEN
BACKGROUND: The global pandemic of type 2 diabetes mellitus (T2DM) is an enormous clinical and socioeconomic burden. Biguanides and DPP-4 inhibitors (DPP-4i) are the most commonly used therapies in Japanese T2DM patients. When glycemic control is not adequate despite combination of these drugs, there is no consensus on the next step drug. Systematic reviews and meta-analyses of previous trials have indicated that glycemic control with triple combination therapies yields similar results. Thus, beneficial effects on cardiovascular risk factors may be important. The present study was designed to evaluate body fat percentage and several insulin resistance parameters after addition of tofogliflozin or glimepiride to the regimens of patients being treated with metformin and a DPP-4 inhibitor but failing to attain adequate blood glucose control. METHODS: Sodium glucose cotransporter-2 inhibitor, tofogliflozin versus glimepiride, comparative trial in patients with type 2 diabetes on body composition is an ongoing, multicenter, prospective, randomized, open-label, parallel-group trial. T2DM patients treated with metformin/DPP-4 inhibitor dual therapy have been recruited and randomly assigned to 20 mg/day tofogliflozin (n = 32) or 0.5 mg/day glimepiride (n = 32) groups, with either of these drugs being added to pre-existing regimens for 24 weeks. PLANNED OUTCOMES: The primary endpoint is the change in body fat percentage from baseline to 24 weeks. The secondary outcomes are changes in body composition other than fat percentage, body weight, parameters related to glycemic control and ß-cell function, parameters related to lipids and arteriosclerosis, parameters related to liver function, parameters related to diabetic nephropathy, and uric acid levels. Safety parameters will also be analyzed. This is the first trial comparing the effects and safety of adding an SGLT2i and a sulfonylurea as the third-line oral agent to metformin/DPP-4i dual therapy. The results will provide valuable information for choosing third-line oral agents. TRIAL REGISTRATION: UMIN000026161. FUNDING: Kowa Co. Ltd. and Kowa Pharmaceutical Co. Ltd., Tokyo, Japan.
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The non-POU domain-containing octamer binding protein p54nrb/NONO is a multifunctional nuclear protein involved in RNA splicing, processing, and transcriptional regulation of nuclear hormone receptors. Through chromosome copy number analysis via whole-exome sequencing, we revealed amplification of the chromosome Xq11.22-q21.33 locus containing the androgen receptor (AR) and NONO genes in androgen-independent, castration-resistant prostate cancer (CRPC)-like LNCaP-SF cells. Moreover, NONO was frequently amplified and overexpressed in patients with CRPC. RNA sequencing data revealed that a truncated ephrin type-A receptor 6 (EPHA6) splice variant (EPHA6-001) was overexpressed in LNCaP-SF cells, and knockdown of NONO or EPHA6-001 prevented EPHA6-001 expression and reduced proliferation and invasion by LNCaP-SF cells grown under androgen deprivation conditions. Growth inhibition and differential splicing of EPHA6 mRNA by p54nrb/NONO were confirmed in gene silencing experiments in 22Rv1 PCa cells. Importantly, NONO knockdown in LNCaP-SF cells led to reduced tumor growth in castrated mice. These findings indicate that p54nrb/NONO is amplified and overexpressed in CRPC cells and clinical samples, and facilitates CRPC growth by mediating aberrant EPHA6 splicing. We therefore propose that p54nrb/NONO constitutes a novel and attractive therapeutic target for CRPC.
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To elucidate the role of AMPK in hepatic glucose metabolism, dominant negative (DN), constitutively active (CA) forms of the AMPKalpha1 subunit and control vector LacZ were overexpressed by means of adenovirus-mediated gene transfer. Five days after virus injection, hepatic AMPK activity was five-fold higher in CA mice than in DN mice. DN mice were apparently glucose intolerant with a higher fasting plasma glucose level (DN 82.3+/-0.7mg/dl, CA 42.5+/-4.8mg/dl and LacZ 54.3+/-2.4mg/dl). PEPCK, a gluconeogenic key enzyme, mRNA was increased 131.54% and 48.92% in DN mice compared to that of CA and LacZ, respectively. Thus, hepatic AMPK activation plays a role in the suppression of gluconeogenesis and this might be the cause of decreased fasting plasma glucose level in CA mice. We also investigated the effects of dexamethasone on hepatic AMPK expression and activity in rat liver, mice liver, as well as primary cultured hepatocytes. Subcutaneously injecting mice with dexamethasone (1mg/day) for 5 days significantly upregulated hepatic AMPKalpha1 and alpha2 expressions. Similarly, the treatment of primary cultured rat hepatocytes with dexamethasone (1microM) increased expression of the AMPKalpha1 subunit, AICAR-induced AMPK phosphorylation and kinase activity. Although increased AMPK expression cannot be attributed to dexamethasone-induced glucose intolerance, taken together our results raise the possibility that AMPK control liver glucose output and its expression in liver might be modulated by various hormones and growth factors.
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Gluconeogénesis , Glucosa/metabolismo , Hígado/enzimología , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Adenoviridae , Animales , Glucemia/análisis , Dexametasona/farmacología , Activación Enzimática , Vectores Genéticos , Glucocorticoides/farmacología , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Intolerancia a la Glucosa/inducido químicamente , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/genética , Proteínas Serina-Treonina Quinasas/genética , Ratas , Ratas Sprague-DawleyRESUMEN
AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts (MEFs) by infecting retrovirus to express oncogenic Raf (RafV600E). RNA was collected from RafV600E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV600E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by shRNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss. CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.
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Resistin is a hormone secreted by adipocytes that acts on skeletal muscle myocytes, hepatocytes, and adipocytes themselves, reducing their sensitivity to insulin. In the present study, we investigated how the expression of resistin is affected by glucose and by mediators known to affect insulin sensitivity, including insulin, dexamethasone, tumor necrosis factor-alpha (TNF-alpha), epinephrine, and somatropin. We found that resistin expression in 3T3-L1 adipocytes was significantly upregulated by high glucose concentrations and was suppressed by insulin. Dexamethasone increased expression of both resistin mRNA and protein 2.5- to 3.5-fold in 3T3-L1 adipocytes and by approximately 70% in white adipose tissue from mice. In contrast, treatment with troglitazone, a thiazolidinedione antihyperglycemic agent, or TNF-alpha suppressed resistin expression by approximately 80%. Epinephrine and somatropin were both moderately inhibitory, reducing expression of both the transcript and the protein by 30-50% in 3T3-L1 adipocytes. Taken together, these data make it clear that resistin expression is regulated by a variety of hormones and that cytokines are related to glucose metabolism. Furthermore, they suggest that these factors affect insulin sensitivity and fat tissue mass in part by altering the expression and eventual secretion of resistin from adipose cells.
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Adipocitos/metabolismo , Glucosa/farmacología , Hormonas Ectópicas/genética , Insulina/farmacología , Péptidos y Proteínas de Señalización Intercelular , Proteínas , Tiazolidinedionas , Células 3T3 , Tejido Adiposo/metabolismo , Animales , Cromanos/farmacología , Dexametasona/farmacología , Epidídimo , Epinefrina/farmacología , Expresión Génica , Glucocorticoides/farmacología , Hormona de Crecimiento Humana/farmacología , Hipoglucemiantes/farmacología , Masculino , Ratones , Factor de Crecimiento Nervioso , ARN Mensajero/análisis , Resistina , Tiazoles/farmacología , Troglitazona , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Akt is critical in insulin-induced metabolism of glucose and lipids. To investigate functions induced by hepatic Akt activation, a constitutively active Akt, NH(2)-terminally myristoylation signal-attached Akt (myr-Akt), was overexpressed in the liver by injecting its adenovirus into mice. Hepatic myr-Akt overexpression resulted in a markedly hypoglycemic, hypoinsulinemic, and hypertriglyceridemic phenotype with fatty liver and hepatomegaly. To elucidate the sterol regulatory element binding protein (SREBP)-1c contribution to these phenotypic features, myr-Akt adenovirus was injected into SREBP-1 knockout mice. myr-Akt overexpression induced hypoglycemia and hepatomegaly with triglyceride accumulation in SREBP-1 knockout mice to a degree similar to that in normal mice, whereas myr-Akt-induced hypertriglyceridemia in knockout mice was milder than that in normal mice. The myr-Akt-induced changes in glucokinase, phosphofructokinase, glucose-6-phosphatase, and PEPCK expressions were not affected by knocking out SREBP-1, whereas stearoyl-CoA desaturase 1 induction was completely inhibited in knockout mice. Constitutively active SREBP-1-overexpressing mice had fatty livers without hepatomegaly, hypoglycemia, or hypertriglyceridemia. Hepatic acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and glucose-6-phosphate dehydrogenase expressions were significantly increased by overexpressing SREBP-1, whereas glucokinase, phospho-fructokinase, glucose-6-phosphatase, and PEPCK expressions were not or only slightly affected. Thus, SREBP-1 is not absolutely necessary for the hepatic Akt-mediated hypoglycemic effect. In contrast, myr-Akt-induced hypertriglyceridemia and hepatic triglyceride accumulation are mediated by both Akt-induced SREBP-1 expression and a mechanism involving fatty acid synthesis independent of SREBP-1.
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Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas de Unión al ADN/metabolismo , Hepatomegalia/etiología , Hipertrigliceridemia/etiología , Hipoglucemia/etiología , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción , Animales , Enzimas/genética , Hígado Graso/etiología , Gluconeogénesis , Glucólisis , Humanos , Lípidos/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fenotipo , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de EsterolesRESUMEN
TNFalpha, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest.
Asunto(s)
Adipocitos/enzimología , Insulina/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células 3T3 , Animales , Western Blotting , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina , Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular , MAP Quinasa Quinasa 1 , MAP Quinasa Quinasa 6 , MAP Quinasa Quinasa 7 , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Transducción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
AIM: Selective PPARα modulators (SPPARMα) are under development for use as next-generation lipid lowering drugs. In the current study, to predict the pharmacological and toxicological effects of a novel SPPARMα K-877, comprehensive transcriptome analyses of K-877-treated primary human hepatocytes and mouse liver tissue were carried out. METHODS: Total RNA was extracted from the K-877 treated primary human hepatocytes and mouse liver and adopted to the transcriptome analysis. Using a cluster analysis, commonly and species specifically regulated genes were identified. Also, the profile of genes regulated by K-877 and fenofibrate were compared to examine the influence of different SPPARMα on the liver gene expression. RESULTS: Consequently, a cell-based transactivation assay showed that K-877 activates PPARα with much greater potency and selectivity than fenofibric acid, the active metabolite of clinically used fenofibrate. K-877 upregulates the expression of several fatty acid ß-oxidative genes in human hepatocytes and the mouse liver. Almost all genes up- or downregulated by K-877 treatment in the mouse liver were also regulated by fenofibrate treatment. In contrast, the K-877-regulated genes in the mouse liver were not affected by K-877 treatment in the Ppara-null mouse liver. Depending on the species, the peroxisomal biogenesis-related gene expression was robustly induced in the K-877-treated mouse liver, but not human hepatocytes, thus suggesting that the clinical dose of K-877 may not induce peroxisome proliferation or liver toxicity in humans. Notably, K-877 significantly induces the expression of clinically beneficial target genes (VLDLR, FGF21, ABCA1, MBL2, ENPEP) in human hepatocytes. CONCLUSION: These results indicate that changes in the gene expression induced by K-877 treatment are mainly mediated through PPARα activation. K-877 regulates the hepatic gene expression as a SPPARMα and thus may improve dyslipidemia as well as metabolic disorders, such as metabolic syndrome and type 2 diabetes, without untoward side effects.
Asunto(s)
Benzoxazoles/farmacología , Butiratos/farmacología , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Análisis por Conglomerados , Fenofibrato/análogos & derivados , Fenofibrato/farmacología , Perfilación de la Expresión Génica , Humanos , Hipolipemiantes/farmacología , Hígado/patología , Ratones , Técnicas de Cultivo de TejidosRESUMEN
Glucose transporters, or membrane proteins, which incorporate glucose into the cell, can be divided into two groups: the facilitative type glucose transporter (GLUT), and the sodium/glucose cotransporter (SGLT). Among the GLUT family isoforms, GLUT4 is particularly important for maintaining glucose metabolism homeostasis since it is involved in insulin or exercise-induced glucose transport into muscle and adipose tissues via movement from intracellular sites to the plasma membrane in response to stimulation. Thus, agents which induce GLUT4 translocation or improve insulin sensitivity, involved in this insulin-induced step, hold the promise of being potent anti-diabetic drugs. On the other hand, SGLT is expressed specifically in the intestines and kidneys. Oral administration of a SGLT inhibitor, T-1095, lowers the blood glucose concentration via excretion of glucose in the urine, due to suppression of renal SGLT function. In addition to this direct blood glucose lowering effect, T-1095 has been shown to restore impaired insulin secretion from pancreatic beta-cells, as well as to improve insulin resistance in muscle and liver. Thus, this SGLT inhibitor is regarded as a novel and promising agent for the treatment of diabetes mellitus.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Glucosa/metabolismo , Hipoglucemiantes/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Sodio/metabolismo , Animales , Humanos , Hipoglucemiantes/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Proteínas de Transporte de Monosacáridos/antagonistas & inhibidores , Transportador 1 de Sodio-GlucosaRESUMEN
Disruption of histamine H2 receptor and gastrin receptor had different effects growth of gastric mucosa: hypertrophy and atrophy, respectively. To clarify the roles of gastrin and histamine H2 receptors in gastric mucosa, mice deficient in both (double-null mice) were generated and analyzed. Double-null mice exhibited atrophy of gastric mucosae, marked hypergastrinemia and higher gastric pH than gastrin receptor-null mice, which were unresponsive even to carbachol. Comparison of gastric mucosae from 10-week-old wild-type, histamine H2 receptor-null, gastrin receptor-null and double-null mice revealed unique roles of these receptors in gastric mucosal homeostasis. While small parietal cells and increases in the number and mucin contents of mucous neck cells were secondary to impaired acid production, the histamine H2 receptor was responsible for chief cell maturation in terms of pepsinogen expression and type III mucin. In double-null and gastrin receptor-null mice, despite gastric mucosal atrophy, surface mucous cells were significantly increased, in contrast to gastrin-null mice. Thus, it is conceivable that gastrin-gene product(s) other than gastrin-17, in the stimulated state, may exert proliferative actions on surface mucous cells independently of the histamine H2 receptor. These findings provide evidence that different G-protein coupled-receptors affect differentiation into different cell lineages derived from common stem cells in gastric mucosa.