RESUMEN
BACKGROUND: Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is higher in men than in women. Hormonal and genetic causes may account for the sex differences in MASLD. Current human in vitro liver models do not sufficiently take the influence of biological sex and sex hormones into consideration. METHODS: Primary human hepatocytes (PHHs) were isolated from liver specimen of female and male donors and cultured with sex hormones (17ß-estradiol, testosterone and progesterone) for up to 72 h. mRNA expression levels of 8 hepatic lipid metabolism genes were analyzed by RT-qPCR. Sex hormones and their metabolites were determined in cell culture supernatants by LC-MS analyses. RESULTS: A sex-specific expression was observed for LDLR (low density lipoprotein receptor) with higher mRNA levels in male than female PHHs. All three sex hormones were metabolized by PHHs and the effects of hormones on gene expression levels varied depending on hepatocyte sex. Only in female PHHs, 17ß-estradiol treatment affected expression levels of PPARA (peroxisome proliferator-activated receptor alpha), LIPC (hepatic lipase) and APOL2 (apolipoprotein L2). Further changes in mRNA levels of female PHHs were observed for ABCA1 (ATP-binding cassette, sub-family A, member 1) after testosterone and for ABCA1, APOA5 (apolipoprotein A-V) and PPARA after progesterone treatment. Only the male PHHs showed changing mRNA levels for LDLR after 17ß-estradiol and for APOA5 after testosterone treatment. CONCLUSIONS: Male and female PHHs showed differences in their expression levels of hepatic lipid metabolism genes and their responsiveness towards sex hormones. Thus, cellular sex should be considered, especially when investigating the pathophysiological mechanisms of MASLD.
Asunto(s)
Hormonas Esteroides Gonadales , Hepatocitos , Metabolismo de los Lípidos , Humanos , Masculino , Femenino , Hepatocitos/metabolismo , Hepatocitos/efectos de los fármacos , Metabolismo de los Lípidos/genética , Metabolismo de los Lípidos/efectos de los fármacos , Hormonas Esteroides Gonadales/farmacología , Hormonas Esteroides Gonadales/metabolismo , Células Cultivadas , Persona de Mediana Edad , Testosterona/farmacología , Testosterona/metabolismo , Estradiol/farmacología , Adulto , Progesterona/farmacología , Progesterona/metabolismo , Factores SexualesRESUMEN
The pyrrolysyl-tRNA synthetase/tRNAPyl pair is the most versatile and widespread system for the incorporation of non-canonical amino acids (ncAAs) into proteins in mammalian cells. However, low yields of ncAA incorporation severely limit its applicability to relevant biological targets. Here, we generate two tRNAPyl variants that significantly boost the performance of the pyrrolysine system. Compared to the original tRNAPyl, the engineered tRNAs feature a canonical hinge between D- and T-loop, show higher intracellular concentrations and bear partially distinct post-transcriptional modifications. Using the new tRNAs, we demonstrate efficient ncAA incorporation into a G-protein coupled receptor (GPCR) and simultaneous ncAA incorporation at two GPCR sites. Moreover, by incorporating last-generation ncAAs for bioorthogonal chemistry, we achieve GPCR labeling with small organic fluorophores on the live cell and visualize stimulus-induced GPCR internalization. Such a robust system for incorporation of single or multiple ncAAs will facilitate the application of a wide pool of chemical tools for structural and functional studies of challenging biological targets in live mammalian cells.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Lisina/análogos & derivados , Ingeniería de Proteínas/métodos , Aminoacil-ARN de Transferencia/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Secuencia de Bases , Codón de Terminación , Escherichia coli/genética , Genes Reporteros , Células HEK293 , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Lisina/genética , Lisina/metabolismo , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , Aminoacil-ARN de Transferencia/síntesis química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Receptores de Hormona Liberadora de Corticotropina/química , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
Overweight has become a major health care problem in Western societies and is accompanied by an increasing incidence and prevalence of non-alcoholic fatty liver disease (NAFLD). The progression from NAFLD to non-alcoholic steatohepatitis (NASH) marks a crucial tipping point in the progression of severe and irreversible liver diseases. This study aims to gain further insight into the molecular processes leading to the evolution from steatosis to steatohepatitis. Steatosis was induced in cultures of primary human hepatocytes by continuous five-day exposure to free fatty acids (FFAs). The kinetics of lipid accumulation, lipotoxicity, and oxidative stress were measured. Additionally, ER stress was evaluated by analyzing the protein expression profiles of its key players: PERK, IRE1a, and ATF6a. Our data revealed that hepatocytes are capable of storing enormous amounts of lipids without showing signs of lipotoxicity. Prolonged lipid accumulation did not create an imbalance in hepatocyte redox homeostasis or a reduction in antioxidative capacity. However, we observed an FFA-dependent increase in ER stress, revealing thresholds for triggering the activation of pathways associated with lipid stress, inhibition of protein translation, and apoptosis. Our study clearly showed that even severe lipid accumulation can be attenuated by cellular defenses, but regenerative capacities may be reduced.
Asunto(s)
Estrés del Retículo Endoplásmico , Ácidos Grasos no Esterificados/metabolismo , Hepatocitos/metabolismo , Estrés Oxidativo , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Metabolismo de los Lípidos , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Granulocyte colony-stimulating factor (GCSF) showed robust neuroprotective and neuroregenerative properties after stroke in rodents but failed to meet study end points in patients. Because immunologic side effects of GCSF may have escaped preclinical testing because of nonallometric dose translation, we hypothesized those as possible reasons. METHODS: Stroke was induced in C57BL/6 mice by 45-minute filament middle cerebral artery occlusion. GCSF was administered at 50 and 832.5 µg/kg body weight. Treatment was controlled by vehicle injection, sham surgery, and naive animals. Immune cell counts were assessed in blood, spleen, and brain by multidimensional flow cytometry 1 day after stroke. RESULTS: High-dose GCSF significantly altered myeloid and T-cell subpopulations in blood and spleen and caused a tremendous increase of monocytes/macrophages infiltrating the ischemic brain. CONCLUSIONS: Dose-dependent immunomodulation superimposes central nervous system-specific effects of GCSF after stroke. Adaption of dose or treatment time may overcome this drawback.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Inmunomodulación , Infarto de la Arteria Cerebral Media/patología , Inflamación/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Nervios Periféricos/inmunología , Nervios Periféricos/patología , Bazo/citología , Bazo/patología , Accidente Cerebrovascular/complicaciones , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
Metabolic alterations in hepatocellular carcinoma (HCC) are fundamental for the development of diagnostic screening and therapeutic intervention since energy metabolism plays a central role in differentiated hepatocytes. In HCC research, hepatoma cell lines (HCLs) like HepG2 and Huh7 cells are still the gold standard. In this study, we characterized the metabolic profiles of primary human hepatoma cells (PHCs), HCLs and primary human hepatocytes (PHHs) to determine their differentiation states. PHCs and PHHs (HCC-PHHs) were isolated from surgical specimens of HCC patients and their energy metabolism was compared to PHHs from non-HCC patients and the HepG2 and Huh7 cells at different levels (transcript, protein, function). Our analyses showed successful isolation of PHCs with a purity of 50-73% (CK18+). The transcript data revealed that changes in mRNA expression levels had already occurred in HCC-PHHs. While many genes were overexpressed in PHCs and HCC-PHHs, the changes were mostly not translated to the protein level. Downregulated metabolic key players of PHCs revealed a correlation with malign transformation and were predominantly pronounced in multilocular HCC. Therefore, HCLs failed to reflect these expression patterns of PHCs at the transcript and protein levels. The metabolic characteristics of PHCs are closer to those of HCC-PHHs than to HCLs. This should be taken into account for future optimized tumor metabolism research.
RESUMEN
Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue's energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.