Generation of new hepatocyte-like in vitro models better resembling human lipid metabolism.
- 作者列表："Pramfalk C","Jakobsson T","Verzijl CRC","Minniti ME","Obensa C","Ripamonti F","Olin M","Pedrelli M","Eriksson M","Parini P
:In contrast to human hepatocytes in vivo, which solely express acyl-coenzyme A:cholesterol acyltransferase (ACAT) 2, both ACAT1 and ACAT2 (encoded by SOAT1 and SOAT2) are expressed in primary human hepatocytes and in human hepatoma cell lines. Here, we aimed to create hepatocyte-like cells expressing the ACAT2, but not the ACAT1, protein to generate a model that - at least in this regard - resembles the human condition in vivo and to assess the effects on lipid metabolism. Using the Clustered Regularly Interspaced Short Palindromic Repeats technology, we knocked out SOAT1 in HepG2 and Huh7.5 cells. The wild type and SOAT2-only-cells were cultured with fetal bovine or human serum and the effects on lipoprotein and lipid metabolism were studied. In SOAT2-only-HepG2 cells, increased levels of cholesterol, triglycerides, apolipoprotein B and lipoprotein(a) in the cell media were detected; this was likely dependent of the increased expression of key genes involved in lipid metabolism (e.g. MTP, APOB, HMGCR, LDLR, ACACA, and DGAT2). Opposite effects were observed in SOAT2-only-Huh7.5 cells. Our study shows that the expression of SOAT1 in hepatocyte-like cells contributes to the distorted phenotype observed in HepG2 and Huh7.5 cells. As not only parameters of lipoprotein and lipid metabolism but also some markers of differentiation/maturation increase in the SOAT2-only-HepG2 cells cultured with HS, this cellular model represent an improved model for studies of lipid metabolism.
: 与体内仅表达酰基辅酶a 的人肝细胞相反: 胆固醇酰基转移酶 (ACAT) 2，ACAT1 和 ACAT2 (由 SOAT1 和 SOAT2 编码) 在人原代肝细胞和人肝癌细胞系中表达。在这里，我们旨在创造表达 ACAT2 而不是 ACAT1 的肝细胞样细胞, 蛋白质，以生成一个模型，至少在这方面-类似于人体条件在体内，并评估对脂质代谢的影响。使用成簇的规则间隔短回文重复技术，我们在 HepG2 和 Huh7.5 细胞中敲除 SOAT1。用胎牛或人血清培养野生型和 SOAT2-only-cells，研究其对脂蛋白和脂代谢的影响。在 SOAT2-only-HepG2 细胞中，检测到细胞培养基中的胆固醇，甘油三酯，载脂蛋白 B 和脂蛋白 (a) 水平增加; 这可能依赖于参与脂质代谢的关键基因的表达增加 (e。 g. MTP 、 APOB 、 HMGCR 、 LDLR 、 ACACA 和 DGAT2)。在 SOAT2-only-Huh7.5 细胞中观察到相反的作用。我们的研究表明，SOAT1 在肝细胞样细胞中的表达有助于在 HepG2 和 Huh7.5 细胞中观察到的扭曲表型。由于 HS 培养的 SOAT2-only-HepG2 细胞不仅具有脂蛋白和脂质代谢的参数，而且具有一些分化/成熟的标志物，因此该细胞模型为脂质代谢的研究提供了一种改进的模型。
METHODS:BACKGROUND:Given the importance of habitual dietary protein intake, distribution patterns and dietary sources in the aetiology of age-related declines of muscle mass and function, the present study examined these factors as a function of sex and age in Irish adults aged 18-90 years comprising The National Adult Nutrition Survey (NANS). METHODS:In total, 1051 (males, n = 523; females, n = 528) undertook a 4-day semi-weighed food diary. Total, body mass relative intake and percentage contribution to total energy intake of dietary protein were determined in addition to protein distribution scores (PDS), as well as the contribution of food groups, animal- and plant-based foods to total protein intake. RESULTS:Total and relative protein intake [mean (SD)] were highest in those aged 18-35 years [96 (3) g day , 1.32 (0.40) g kg day ], with lower protein intakes with increasing age (i.e. in adults aged ≥65 years [82 (22) g, 1.15 (0.34) g kg day , P < 0.001 for both]. Differences in protein intake between age groups were more pronounced in males compared to females. Protein distribution followed a skewed pattern for all age groups [breakfast, 15 (10) g; lunch, 30 (15) g; dinner, 44 (17) g]. Animal-based foods were the dominant protein source within the diet [63% (11%) versus 37% (11%) plant protein, P < 0.001]. CONCLUSIONS:Protein intake and the number of meals reaching the purported threshold for maximising post-prandial anabolism were highest in young adults, and lower with increasing age. For main meals, breakfast provided the lowest quantity of protein across all age categories and may represent an opportunity for improving protein distribution, whereas, in older adults, increasing the number of meals reaching the anabolic threshold regardless of distribution pattern may be more appropriate.
METHODS:BACKGROUND:Low cardiorespiratory fitness (CRF) increases risk of all-cause mortality and cardiovascular events. Periodic CRF assessment can have an important preventive function. OBJECTIVE:To develop a protocol-free method to estimate CRF in daily life based on heart rate (HR) and body acceleration measurements. METHODS:Acceleration and HR data were collected from 37 subjects (M=49%) while performing a standardized laboratory activity protocol (sitting, walking, running, cycling) and during a 5-days free-living monitoring period. CRF was determined by oxygen uptake (VO2max) during maximal exercise testing. A doubly-labeled water validated equation was used to predict total energy expenditure (TEE) from acceleration data. A fitness index was defined as the ratio between TEE and HR (TEE-pulse). Activity recognition techniques were used to process acceleration features and classify sedentary, ambulatory and other activity types. Regression equations based on TEE-pulse data from each activity type were developed to predict VO2max. RESULTS:TEE-pulse measured within each activity type of the laboratory protocol was highly correlated to VO2max (r from 0.74 to 0.91). Averaging the outcome of each activity-type specific equation based on TEE-pulse from the laboratory data led to accurate estimates of VO2max (RMSE: 300.0 mlO2/min or 10%). The difference between laboratory and free-living determined TEE-pulse was 3.7 ± 11% (r =0.85). The prediction method preserved the prediction accuracy when applied to free-living data (RMSE: 367 mlO2/min or 12%). CONCLUSIONS:Measurements of body acceleration and HR can be used to predict VO2max in daily life. Activity-specific prediction equations are needed to achieve highly accurate estimates of CRF.
METHODS:OBJECTIVE:Postprandial dyslipidemia is a common feature of insulin resistant states and contributes to increased cardiovascular disease risk. Recently, bile acids have been recognized beyond their emulsification properties as important signaling molecules that promote energy expenditure, improve insulin sensitivity, and lower fasting lipemia. While bile acid receptors have become novel pharmaceutical targets, their effects on postprandial lipid metabolism remain unclear. Here we investigated the potential role of bile acids in regulation of postprandial chylomicron production and triglyceride excursion. Approach and Results: Healthy C57BL/6 mice were given an intraduodenal infusion of taurocholic acid (TA) under fat-loaded conditions and circulating lipids were measured. Targeting of bile acid receptors was achieved with GW4064, a synthetic agonist to the farnesoid X receptor (FXR), and with deoxycholic acid (DCA), an activator of the Takeda G-protein-coupled receptor 5. TA, GW4064, and DCA treatments all lowered postprandial lipemia. FXR agonism also reduced intestinal triglyceride content and activity of microsomal triglyceride transfer protein, involved in chylomicron assembly. Importantly, TA effects (but not DCA) were largely lost in FXR knockout mice. These bile acid effects are reminiscent of the anti-diabetic hormone glucagon-like peptide-1 (GLP-1). While the GLP-1 receptor agonist exendin-4 retained its ability to acutely lower postprandial lipemia during bile acid sequestration and FXR deficiency, it did raise hepatic expression of the rate limiting enzyme for bile acid synthesis. CONCLUSIONS:Bile acid signaling may be an important mechanism of controlling dietary lipid absorption and bile acid receptors may constitute novel targets for the treatment of postprandial dyslipidemia.