RESUMEN
The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).
Asunto(s)
Estrés del Retículo Endoplásmico , Intestino Delgado/metabolismo , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Proteínas de la Membrana/genética , Animales , Secuencia de Bases , Células CACO-2 , Enterocitos/metabolismo , Hígado Graso/genética , Femenino , Hepatocitos/metabolismo , Homeostasis , Humanos , Intestino Delgado/ultraestructura , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Periodo Posprandial , Triglicéridos/biosíntesis , Triglicéridos/sangre , Tunicamicina , Pez CebraRESUMEN
BACKGROUND: Apelin is a peptide ligand of the G-protein-coupled receptor APJ and exhibits anti-diabetes and anti-heart failure activities. However, short serum half-life of the apelin peptide limits its potential clinical applications. This study aimed to develop a long-acting apelin analog. METHODS: To extend apelin's in vivo half-life, we made a recombinant protein by fusing the IgG Fc fragment to apelin-13 (Fc-apelin-13), conducted pharmacokinetics studies in mice, and determined in vitro biological activities in suppressing cyclic adenosine monophosphate and activating extracellular signal-regulated kinase signalling by reporter assays. We investigated the effects of Fc-apelin-13 on food intake, body weight, fasting blood glucose and insulin levels, glucose tolerance test, hepatic steatosis, and cardiac function and fibrosis by subcutaneous administration of Fc-apelin-13 in diet-induced obese mice for 4 weeks. RESULTS: The estimated half-life of Fc-apelin-13 in blood was approximately 33 hours. Reporter assays showed that Fc-apelin-13 was active in suppressing cyclic adenosine monophosphate response element and activating serum response element activities. Four weeks of Fc-apelin-13 treatment in obese mice did not affect food intake and body weight, but resulted in a significant improvement of glucose tolerance, and a decrease in hepatic steatosis and fibrosis, as well as in serum alanine transaminase levels. Moreover, cardiac stroke volume and output were increased and cardiac fibrosis was decreased in the treated mice. CONCLUSIONS: Fc-apelin-13 fusion protein has an extended in vivo half-life and exerts multiple benefits on obese mice with respect to the improvement of glucose disposal, amelioration of liver steatosis and heart fibrosis, and increase of cardiac output. Hence, Fc-apelin-13 is potentially a therapeutic for obesity-associated disease conditions.
Asunto(s)
Dieta/efectos adversos , Hígado Graso/tratamiento farmacológico , Insuficiencia Cardíaca/tratamiento farmacológico , Fragmentos Fc de Inmunoglobulinas/química , Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Obesidad/complicaciones , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Hígado Graso/etiología , Hígado Graso/patología , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Obesidad/etiologíaRESUMEN
Lipoprotein (a) [Lp(a)] is an independent risk factor for atherosclerosis-related events that is under strong genetic control (heritability = 0.68-0.98). However, causal mutations and functional validation of biological pathways modulating Lp(a) metabolism are lacking. We performed a genome-wide association scan to identify genetic variants associated with Lp(a)-cholesterol levels in the Old Order Amish. We confirmed a previously known locus on chromosome 6q25-26 and found Lp(a) levels also to be significantly associated with a SNP near the APOA5-APOA4-APOC3-APOA1 gene cluster on chromosome 11q23 linked in the Amish to the APOC3 R19X null mutation. On 6q locus, we detected associations of Lp(a)-cholesterol with 118 common variants (P = 5 × 10(-8) to 3.91 × 10(-19)) spanning a â¼5.3 Mb region that included the LPA gene. To further elucidate variation within LPA, we sequenced LPA and identified two variants most strongly associated with Lp(a)-cholesterol, rs3798220 (P = 1.07 × 10(-14)) and rs10455872 (P = 1.85 × 10(-12)). We also measured copy numbers of kringle IV-2 (KIV-2) in LPA using qPCR. KIV-2 numbers were significantly associated with Lp(a)-cholesterol (P = 2.28 × 10(-9)). Conditional analyses revealed that rs3798220 and rs10455872 were associated with Lp(a)-cholesterol levels independent of each other and KIV-2 copy number. Furthermore, we determined for the first time that levels of LPA mRNA were higher in the carriers than non-carriers of rs10455872 (P = 0.0001) and were not different between carriers and non-carriers of rs3798220. Protein levels of apo(a) were higher in the carriers than non-carriers of both rs10455872 and rs3798220. In summary, we identified multiple independent genetic determinants for Lp(a)-cholesterol. These findings provide new insights into Lp(a) regulation.
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Aterosclerosis/genética , Colesterol/metabolismo , Lipoproteína(a)/genética , Adulto , Anciano , Aterosclerosis/metabolismo , Cromosomas Humanos Par 6/genética , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Kringles , Lipoproteína(a)/química , Lipoproteína(a)/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
The glucocorticoid-induced leucine zipper (GILZ), a primary target of glucocorticoids, is expressed in human adipocytes, but its importance in adipocyte function is unknown. Because TNFα is increased in obese adipose tissue and antagonizes a number of glucocorticoid actions, we investigated the interplay of these pathways. GILZ knockdown increased and GILZ overexpression decreased interleukin-6 (IL-6) and leptin mRNA and protein secretion. GILZ knockdown increased the magnitude of the glucocorticoid effect on leptin secretion, but did not affect the glucocorticoid suppression of IL-6. Although GILZ silencing decreased adiponectin mRNA levels, it did not affect the amount of adiponectin secreted. GILZ negatively modulated pro-inflammatory signaling pathways, blocking basal and TNFα-stimulated (1 h) p65 nuclear factor κB nuclear translocation and transcriptional activity by binding to p65 in the cytoplasm. GILZ silencing increased basal ERK1/2 and JNK phosphorylation, and decreased MAPK phosphatase-1 protein levels. Longer term TNFα (4 h or 24 h) treatment decreased GILZ expression in human adipocytes. Furthermore, adipose tissue GILZ mRNA levels were reduced in proportion to the degree of obesity and expression of inflammatory markers. Overall, these results suggest that GILZ antagonizes the pro-inflammatory effects of TNFα in human adipocytes, and its downregulation in obesity may contribute to adipose inflammation and dysregulated adipokine production, and thereby systemic metabolism.
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Inflamación/genética , Obesidad/genética , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/metabolismo , Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Adulto , Biopsia , Fosfatasa 1 de Especificidad Dual/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , Leptina/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Obesidad/metabolismo , Obesidad/patología , ARN Mensajero/biosíntesis , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factores de Transcripción/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Alanine transaminase (ALT) plays an important role in gluconeogenesis by converting alanine into pyruvate for glucose production. Early studies have shown that ALT activities are upregulated in gluconeogenic conditions and may be implicated in the development of diabetes. ALT consists of two isoforms, ALT1 and ALT2, with distinctive subcellular and tissue distributions. Whether and how they are regulated are largely unknown. METHODS: By using Western blotting analysis, we measured hepatic ALT isoforms at the protein level in obese and diabetic animals and in Fao hepatoma cells treated with dexamethasone and insulin. In addition, we measured glucose output in Fao cells over-expressing ALT1 and ALT2. RESULTS: Both ALT isoforms in the liver were increased in diabetic Goto-Kakizaki rats and during fasting. However, in ob/ob mice, only ALT2, but not ALT1, protein levels were elevated, and the increase of ALT2 was correlated with that of ALT activity. We further demonstrated that, in vitro, both ALT1 and ALT2 were induced by glucocorticoid dexamethasone, but suppressed by insulin in Fao cells. Finally, we showed that the over-expression of ALT1 and ALT2 in Fao cells directly increased glucose output. CONCLUSIONS: We have shown the similarity and difference in the regulation of ALT isoforms in gluconeogenic conditions at the protein level, supporting that ALT isoenzymes play an important role in glucose metabolism and may be implicated the development of insulin resistance and diabetes.
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Alanina Transaminasa/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Inducción Enzimática , Gluconeogénesis , Hígado/enzimología , Obesidad/enzimología , Alanina Transaminasa/antagonistas & inhibidores , Alanina Transaminasa/química , Alanina Transaminasa/genética , Animales , Línea Celular , Dexametasona/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Inducción Enzimática/efectos de los fármacos , Represión Enzimática/efectos de los fármacos , Glucocorticoides/farmacología , Gluconeogénesis/efectos de los fármacos , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Obesidad/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Intellectual disability is genetically heterogeneous, and it is likely that many of the responsible genes have not yet been identified. We describe three siblings with isolated, severe developmental encephalopathy. After extensive uninformative genetic and metabolic testing, whole exome sequencing identified a homozygous novel variant in glutamic pyruvate transaminase 2 (GPT2) or alanine transaminase 2 (ALT2), c.459 C > G p.Ser153Arg that segregated with developmental encephalopathy in the family. This variant was predicted to be damaging by all in silico prediction algorithms. GPT2 is the gene encoding ALT2 which is responsible for the reversible transamination of alanine and 2-oxoglutarate to form pyruvate and glutamate. GPT2 is expressed in brain and is in the pathway to generate glutamate, an excitatory neurotransmitter. Functional assays of recombinant wild-type and mutant ALT2 proteins demonstrated the p.Ser153Arg mutation resulted in a severe loss of enzymatic function. We suggest that recessively inherited loss of function GPT2 mutations are a novel cause of intellectual disability.
Asunto(s)
Encefalopatías/genética , Discapacidad Intelectual/genética , Mutación Missense , Transaminasas/genética , Adolescente , Alanina Transaminasa/genética , Encefalopatías/congénito , Preescolar , Consanguinidad , Femenino , Humanos , Masculino , Linaje , HermanosRESUMEN
OBJECTIVE: Obesity-associated non-alcoholic fatty liver disease (NAFLD) may cause liver dysfunction and failure. In a previously reported genome-wide association meta-analysis, single nucleotide polymorphisms (SNPs) near PNPLA3, NCAN, GCKR, LYPLAL1 and PPP1R3B were associated with NAFLD and with distinctive serum lipid profiles. The present study examined the relevance of these variants to NAFLD in extreme obesity. METHODS: In 1,092 bariatric surgery patients, the candidate SNPs were genotyped and association analyses with liver histology and serum lipids were performed. RESULTS: We replicated the association of hepatosteatosis with PNPLA3 rs738409[G] and with NCAN rs2228603[T]. We also replicated the association of rs2228603[T] with hepatic inflammation and fibrosis. rs2228603[T] was associated with lower serum low-density lipoprotein, total cholesterol and triglycerides. After stratification by the presence or absence of NAFLD, these associations were present predominantly in the subgroup with NAFLD. CONCLUSION: NCAN rs2228603[T] is a risk factor for liver inflammation and fibrosis, suggesting that this locus is responsible for progression from steatosis to steatohepatitis. In this bariatric cohort, rs2228603[T] was associated with low serum lipids only in patients with NAFLD. This supports a NAFLD model in which the liver may sequester triglycerides as a result of either increased triglyceride uptake and/or decreased lipolysis.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Hígado Graso/genética , Lectinas Tipo C/genética , Cirrosis Hepática/genética , Proteínas del Tejido Nervioso/genética , Obesidad Mórbida/genética , Adulto , Colesterol/sangre , Proteoglicanos Tipo Condroitín Sulfato/sangre , Hígado Graso/sangre , Femenino , Variación Genética , Genotipo , Humanos , Lectinas Tipo C/sangre , Cirrosis Hepática/sangre , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/sangre , Neurocano , Enfermedad del Hígado Graso no Alcohólico , Obesidad Mórbida/sangre , Polimorfismo de Nucleótido Simple , Triglicéridos/sangreRESUMEN
Alanine aminotransferase (ALT) is used as an indicator of hepatocellular injury. Since ALT consists of two isoenzymes, a better understanding of ALT isoenzyme biology in response to compounds that cause metabolic adaptive versus hepatotoxic responses will allow for a more accurate assessment of the significance of an ALT increase. The purpose of this study was to characterize the ALT isoenzyme response in mice treated with 25 or 75 mg/kg of dexamethasone, which is known to induce a progluconeogenic state, for 24 or 72 hr. Those mice treated with 75 mg/kg for 72 hr showed an increase in total liver ALT activity. Western blot showed that there was an increase in ALT2 at both doses and time points and there was a concurrent increase in ALT2 ribonucleic acid at 24 and 72 hr. The ALT isoenzyme response assessed by an activity assay showed an increase in ALT2. The increases in liver ALT were associated with an increase in liver glycogen and there was no hepatocellular necrosis. There was an increase in total serum ALT activity, although serum isoenzymes were not evaluated. Thus, the authors demonstrated that dexamethasone induced increases in hepatic and serum ALT, which reflect a hepatocellular progluconeogenic metabolic adaptive response.
Asunto(s)
Adaptación Biológica/efectos de los fármacos , Alanina Transaminasa/metabolismo , Dexametasona/toxicidad , Glucocorticoides/toxicidad , Isoenzimas/metabolismo , Hígado/efectos de los fármacos , Alanina Transaminasa/genética , Animales , Western Blotting , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucógeno/metabolismo , Isoenzimas/genética , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Serum alanine aminotransferase (ALT) activity is a widely-used surrogate marker for liver injury. However, mild elevation of serum ALT is frequently observed in apparently healthy individuals, making it sometimes challenging to interpret whether this laboratory abnormality is medically benign or serious. To obtain a better understanding of the factors influencing ALT levels, we examined the relation between ALT and a number of anthropometric and biochemistry measurements in humans. METHODS: We assessed the associations of ALT with hematocrit (HCT) in 1,200 apparently healthy adults from an Amish population. Multivariate analyses were carried out to determine whether observed associations were independent of other factors known to modulate ALT and HCT, including body mass index (BMI) and sex. The correlation detected in the Amish was then replicated in an independent population sample (N = 9,842) from the National Health and Nutrition Examination Survey (NHANES) III. RESULTS: ALT levels were positively correlated with HCT (r = 0.33, p < 0.0001) in both Amish and NHANES III. The magnitude of association was unchanged after adjustment for BMI, but was reduced by age/sex adjustment to r = 0.18 (p < 0.0001) and r = 0.17 (p < 0.0001) in the Amish and NHANES populations, respectively. HCT accounts for about 3% of the population variation in ALT, which is smaller than the contributions of gender and BMI, but larger than individual blood pressure and cholesterol components. CONCLUSIONS: We observed a correlation between ALT and HCT, suggesting that HCT may be a newly identified modulator of ALT in humans.
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Hematócrito , Adulto , Alanina Transaminasa/sangre , Amish , Presión Sanguínea , Índice de Masa Corporal , HDL-Colesterol , Femenino , Humanos , Masculino , Persona de Mediana Edad , Encuestas NutricionalesRESUMEN
Triple-negative breast cancer (TNBC) cells reprogram their metabolism to provide metabolic flexibility for tumor cell growth and survival in the tumor microenvironment. While our previous findings indicated that endothelial lipase (EL/LIPG) is a hallmark of TNBC, the precise mechanism through which LIPG instigates TNBC metabolism remains undefined. Here, we report that the expression of LIPG is associated with long non-coding RNA DANCR and positively correlates with gene signatures of mitochondrial metabolism-oxidative phosphorylation (OXPHOS). DANCR binds to LIPG, enabling tumor cells to maintain LIPG protein stability and OXPHOS. As one mechanism of LIPG in the regulation of tumor cell oxidative metabolism, LIPG mediates histone deacetylase 6 (HDAC6) and histone acetylation, which contribute to changes in IL-6 and fatty acid synthesis gene expression. Finally, aided by a relaxed docking approach, we discovered a new LIPG inhibitor, cynaroside, that effectively suppressed the enzyme activity and DANCR in TNBC cells. Treatment with cynaroside inhibited the OXPHOS phenotype of TNBC cells, which severely impaired tumor formation. Taken together, our study provides mechanistic insights into the LIPG modulation of mitochondrial metabolism in TNBC and a proof-of-concept that targeting LIPG is a promising new therapeutic strategy for the treatment of TNBC.
RESUMEN
We carried out a genome-wide association study of serum aspartate aminotransferase (AST) activity in 866 Amish participants of the Heredity and Phenotype Intervention Heart Study and identified significant association of AST activity with a cluster of single nucleotide polymorphisms located on chromosome 10q24.1 (peak association was rs17109512; P=2.80E-14), in the vicinity of GOT1, the gene encoding cytosolic AST (cAST). Sequencing of GOT1 revealed an in-frame deletion of three nucleic acids encoding asparagine at position 389 c.1165_1167delAAC (p.Asn389del) in the gene. Deletion carriers had significantly lower AST activity levels compared with homozygotes for the common allele (mean±s.d.: 10.0±2.8 versus 18.8±5.2 U l(-1); P=2.80E-14). Further genotyping of the deletion in other Amish samples (n=1932) identified an additional 20 carriers (minor allele frequency (MAF)=0.0052). The deletion was not detected in 647 outbred Caucasians. Asn at codon 389 is conserved among known mammalian cASTs. In vitro transient transfection of wild-type and mutant cAST indicated that mutant cAST protein was barely detectable in the cells. Furthermore, even after correction for cAST expression, mutant cAST had markedly diminished enzymatic activity. Remarkably, we did not find any association between the deletion and metabolic traits including serum fasting glucose or insulin, fasting and post-meal lipids, inflammatory markers, or sub-clinical markers of cardiovascular disease. In conclusion, we discovered a rare in-frame deletion in GOT1 gene, which inactivates cAST enzyme in the Old Order Amish. This finding will help us to understand structure and function of the enzyme and would be useful for predicting serum AST levels.
Asunto(s)
Aspartato Aminotransferasa Citoplasmática/sangre , Aspartato Aminotransferasa Citoplasmática/genética , Estudio de Asociación del Genoma Completo , Polimorfismo de Nucleótido Simple , Adulto , Secuencia de Aminoácidos , Amish/genética , Secuencia de Bases , Femenino , Genotipo , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Alineación de SecuenciaRESUMEN
BACKGROUND: Alterations of endocannabinoid system in adipose tissue play an important role in lipid regulation and metabolic dysfunction associated with obesity. The purpose of this study was to determine whether gene expression levels of cannabinoid type 1 receptor (CB1) and fatty acid amide hydrolase (FAAH) are different in subcutaneous abdominal and gluteal adipose tissue, and whether hypocaloric diet and aerobic exercise influence subcutaneous adipose tissue CB1 and FAAH gene expression in obese women. METHODS: Thirty overweight or obese, middle-aged women (BMI = 34.3 ± 0.8 kg/m2, age = 59 ± 1 years) underwent one of three 20-week weight loss interventions: caloric restriction only (CR, N = 9), caloric restriction plus moderate-intensity aerobic exercise (CRM, 45-50% HRR, N = 13), or caloric restriction plus vigorous-intensity aerobic exercise (CRV, 70-75% HRR, N = 8). Subcutaneous abdominal and gluteal adipose tissue samples were collected before and after the interventions to measure CB1 and FAAH gene expression. RESULTS: At baseline, FAAH gene expression was higher in abdominal, compared to gluteal adipose tissue (2.08 ± 0.11 vs. 1.78 ± 0.10, expressed as target gene/ß-actin mRNA ratio × 10-3, P < 0.05). Compared to pre-intervention, CR did not change abdominal, but decreased gluteal CB1 (Δ = -0.82 ± 0.25, P < 0.05) and FAAH (Δ = -0.49 ± 0.14, P < 0.05) gene expression. CRM or CRV alone did not change adipose tissue CB1 and FAAH gene expression. However, combined CRM and CRV (CRM+CRV) decreased abdominal adipose tissue FAAH gene expression (Δ = -0.37 ± 0.18, P < 0.05). The changes in gluteal CB1 and abdominal FAAH gene expression levels in the CR alone and the CRM+CRV group were different (P < 0.05) or tended to be different (P = 0.10). CONCLUSIONS: There are depot differences in subcutaneous adipose tissue endocannabinoid system gene expression in obese individuals. Aerobic exercise training may preferentially modulate abdominal adipose tissue endocannabinoid-related gene expression during dietary weight loss.
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Amidohidrolasas/metabolismo , Ejercicio Físico , Regulación de la Expresión Génica , Sobrepeso/terapia , Receptor Cannabinoide CB1/metabolismo , Grasa Subcutánea Abdominal/metabolismo , Grasa Subcutánea/metabolismo , Anciano , Amidohidrolasas/genética , Índice de Masa Corporal , Nalgas , Moduladores de Receptores de Cannabinoides/fisiología , Estudios de Cohortes , Dieta Reductora , Endocannabinoides , Femenino , Humanos , Persona de Mediana Edad , Obesidad/dietoterapia , Obesidad/metabolismo , Obesidad/terapia , Sobrepeso/dietoterapia , Sobrepeso/metabolismo , Esfuerzo Físico , ARN Mensajero/metabolismo , Receptor Cannabinoide CB1/genética , Pérdida de PesoRESUMEN
Peroxisome proliferator-activated receptor-γ (PPARγ) is the master regulator of adipogenesis, but knowledge about how PPARγ is regulated at the protein level is very limited. We aimed to identify PPARγ-interacting proteins which modulate PPARγ's protein levels and transactivating activities in human adipocytes. We expressed Flag-tagged PPARγ in human preadipocytes as bait to capture PPARγ-associated proteins, followed by mass spectroscopy and proteomics analysis, which identified serine/threonine kinase 38 (STK38) as a major PPARγ-associated protein. Protein pulldown studies confirmed this protein-protein interaction in transfected cells, and reporter assays demonstrated that STK38 enhanced PPARγ's transactivating activities without requiring STK38's kinase activity. In cell-based assays, STK38 increased PPARγ protein stability, extending PPARγ's half-life from ~1.08 to 1.95 h. Notably, in human preadipocytes, the overexpression of STK38 enhanced adipogenesis, whereas knockdown impaired the process in a PPARγ-dependent manner. Thus, we discovered that STK38 is a novel PPARγ-cofactor promoting adipogenesis, likely through stabilization of PPARγ.
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Adipogénesis , PPAR gamma , Proteínas Serina-Treonina Quinasas , Adipocitos/metabolismo , Humanos , PPAR gamma/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
UNLABELLED: The elevation of serum alanine aminotransferase (ALT) is regarded as an indicator of liver damage based on the presumption that ALT protein is specifically and abundantly expressed in the liver. However, ALT elevation is also observed in non-liver injury conditions (for example, muscle injury) and in apparently healthy people. Conversely, serum ALT activity is normal in many patients with confirmed liver diseases (for example, cirrhosis and hepatitis C infection). To improve the diagnostic value of the ALT assay and to understand the molecular basis for serum ALT changes in various pathophysiological conditions, we have cloned rat ALT isoenzyme ALT1 and ALT2 complementary DNAs (cDNAs), examined their tissue expressions at the messenger RNA and protein levels, and determined ALT1 and ALT 2 serum levels in response to liver damage in rodents. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis shows that ALT1 messenger RNA is widely distributed and mainly expressed in intestine, liver, fat tissues, colon, muscle, and heart, in the order of high to low expression level, whereas ALT2 gene expression is more restricted, mainly in liver, muscle, brain, and white adipose tissue. The tissue distribution pattern of ALT1 and ALT2 proteins largely agrees with their messenger RNA expression. Interestingly, hepatic ALT2 protein is approximately four times higher in male rats than in female rats. In addition, ALT isoenzymes distribute differentially at the subcellular level in that ALT1 is a cytoplasmic protein and ALT2 a mitochondrial protein, supporting bioinformatic prediction of mitochondrial localization of ALT2. CONCLUSION: Using animal models of hepatoxicity induced by carbon tetrachloride and acetaminophen, we found that both serum ALT1 and ALT2 protein levels were significantly elevated and correlated with ALT activity, providing, for the first time, the molecular basis for the elevated total serum ALT activity.
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Alanina Transaminasa/genética , Regulación Enzimológica de la Expresión Génica , Isoenzimas/genética , Hígado/patología , Alanina Transaminasa/sangre , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , Humanos , Isoenzimas/sangre , Hígado/enzimología , Masculino , Ratones , Mitocondrias Hepáticas/enzimología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , RatasRESUMEN
Activation of the apelin receptor, or APJ, by apelin is considered a therapeutic avenue for cardiovascular disease, including heart failure. Recently, a novel endogenous ligand for APJ named Elabela (ELA) has been discovered and is known to possess anti-heart failure activity in animal models. However, the short in vivo half-life of ELA constrains its clinical potential. To extend its half-life in vivo, we attempted to make IgG-Fc-ELA fusion proteins. We found that Fc-ELA-32 fusion proteins are cleaved during protein production, whereas Fc-ELA-21 fusion proteins are expressed intact, so we focused our studies on the latter. The Fc-ELA-21 fusion protein retained its functionality in vitro and had a half-life of approximately 44â¯h in circulation in mice after subcutaneous injection. Daily injection of the fusion protein in MI rats for 4â¯weeks significantly mitigated heart dysfunction with respect to hemodynamics. At the cellular and tissue levels, treatment of Fc-ELA-21 fusion protein significantly increased angiogenesis, promoted cardiomyocyte proliferation and reduced apoptosis and heart fibrosis near the infarct area. In comparison, ELA-21 had a half-life of 13â¯min and showed no significant cardioprotective activities. These data suggest that Fc-ELA-21 may be a potential therapeutic for heart failure.
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Infarto del Miocardio/sangre , Infarto del Miocardio/tratamiento farmacológico , Hormonas Peptídicas/sangre , Hormonas Peptídicas/uso terapéutico , Proteínas Recombinantes de Fusión/sangre , Proteínas Recombinantes de Fusión/uso terapéutico , Secuencia de Aminoácidos , Animales , Células HEK293 , Semivida , Humanos , Masculino , Hormonas Peptídicas/administración & dosificación , Hormonas Peptídicas/genética , Ratas , Ratas Sprague-DawleyRESUMEN
Alanine aminotransferase (ALT) is a key enzyme for gluconeogenesis as well as a widely used serum marker for liver injury. We have identified two ALT isoenzymes, ALT1 and ALT2, which are encoded by separate genes. In this study, we described the expression, purification and initial characterization of human ALT1 and ALT2 proteins in High-five insect cells. Human ALT1 and ALT2 were expressed as His-tagged fusion proteins by recombinant baculovirus in insect cells and purified into homogeneity in one step by using immobilized Ni2+-affinity chromatography. Tag-free ALT1 and ALT2 were obtained by cleavage of enterokinase digestion and used for initial characterization of the enzymes. The specific ALT activity of purified fusion or His-tag-removed ALT1 was about 15-fold higher than that of ALT2 and their enzymatic activities decreased quickly at 37 degrees C and -20 degrees C, but were well preserved at -80 degrees C. Nevertheless, the ALT1 and ALT2 activities remained stable in a buffer containing 25% glycerol. The pH profile was similar between hALT1 and hALT2 in that both enzymes remained fully active between pH 6.5 and 8.0. The purified ALT recombinant proteins can not only be used as a reference protein standard for the ALT assay in clinical chemistry, but also will be useful for understanding the biochemical and biological significance of the isoenzymes and for developing ALT isoform-specific assays for clinical or preclinical diagnostic use.
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Alanina Transaminasa/genética , Alanina Transaminasa/aislamiento & purificación , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Alanina Transaminasa/química , Animales , Secuencia de Bases , Western Blotting , Cartilla de ADN , Vectores Genéticos , Humanos , Concentración de Iones de Hidrógeno , Isoenzimas/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Spodoptera , TemperaturaRESUMEN
Apelin is a peptide hormone with anti-oxidative and anti-inflammatory activities and is proposed to be a potential therapeutic for many disease conditions, including sepsis. However, short in vivo half-life of the apelin peptide would limit its potential clinical applications. This study aims to investigate the effects of Fc-apelin, a novel long-acting apelin fusion protein, on lipopolysaccharide (LPS)-induced liver injury. Liver injury was induced by systemic injection of LPS in mice. Hepatoprotective activities of Fc-apelin against inflammation were evaluated in LPS mice and/or hepatoma Huh-7 cells with respect to serum ALT, apoptosis, oxidative stress, macrophage infiltration and gene expression. We found that LPS induced systemic inflammation and liver damage. Co-administration of Fc-apelin significantly attenuated serum ALT elevation, diminished LPS-induced apoptosis and ROS production in the liver and in Huh-7 cells, mitigated hepatic macrophage infiltration, and reduced TNFα and IL-6 gene expression. Collectively, Fc-apelin fusion protein exerts protective effects against LPS-induced liver damage and may serve as a potential therapeutic for endotoxin-induced liver injury.
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Apelina/farmacología , Hígado/lesiones , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Alanina Transaminasa/sangre , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Interleucina-6/sangre , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos , Hígado/efectos de los fármacos , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Sustancias Protectoras/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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RESUMEN
BACKGROUND: Obesity is associated with low-grade chronic inflammation, and serum markers of inflammation are independent risk factors for cardiovascular disease (CVD). However, the molecular and cellular mechanisms that link obesity to chronic inflammation and CVD are poorly understood. METHODS AND FINDINGS: Acute-phase serum amyloid A (A-SAA) mRNA levels, and A-SAA adipose secretion and serum levels were measured in obese and nonobese individuals, obese participants who underwent weight-loss, and persons treated with the insulin sensitizer rosiglitazone. Inflammation-eliciting activity of A-SAA was investigated in human adipose stromal vascular cells, coronary vascular endothelial cells and a murine monocyte cell line. We demonstrate that A-SAA was highly and selectively expressed in human adipocytes. Moreover, A-SAA mRNA levels and A-SAA secretion from adipose tissue were significantly correlated with body mass index (r = 0.47; p = 0.028 and r = 0.80; p = 0.0002, respectively). Serum A-SAA levels decreased significantly after weight loss in obese participants (p = 0.006), as well as in those treated with rosiglitazone (p = 0.033). The magnitude of the improvement in insulin sensitivity after weight loss was significantly correlated with decreases in serum A-SAA (r = -0.74; p = 0.034). SAA treatment of vascular endothelial cells and monocytes markedly increased the production of inflammatory cytokines, e.g., interleukin (IL)-6, IL-8, tumor necrosis factor alpha, and monocyte chemoattractant protein-1. In addition, SAA increased basal lipolysis in adipose tissue culture by 47%. CONCLUSIONS: A-SAA is a proinflammatory and lipolytic adipokine in humans. The increased expression of A-SAA by adipocytes in obesity suggests that it may play a critical role in local and systemic inflammation and free fatty acid production and could be a direct link between obesity and its comorbidities, such as insulin resistance and atherosclerosis. Accordingly, improvements in systemic inflammation and insulin resistance with weight loss and rosiglitazone therapy may in part be mediated by decreases in adipocyte A-SAA production.