Dataset Information

Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity.

ABSTRACT: Free fatty acids are essentially involved in the pathogenesis of chronic diseases such as diabetes mellitus, non-alcoholic fatty liver disease, and cardiovascular disease. They promote mitochondrial dysfunction, oxidative stress, respiratory chain uncoupling, and endoplasmic reticulum stress and modulate stress-sensitive pathways. These detrimental biological effects summarized as lipotoxicity mainly depend on fatty acid carbon chain length, degree of unsaturation, concentration, and treatment time. Preparation of fatty acid solutions involves dissolving and complexing. Solvent toxicity and concentration, amount of bovine serum albumin (BSA), and ratio of albumin to fatty acids can vary significantly between equal concentrations, mediating considerable harmful effects and/or interference with certain assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Herein, we studied the impact of commonly used solvents ethanol and dimethyl sulfoxide and varying concentrations of BSA directly and in solution with oleic acid on MTT to formazan conversion, adenosine triphosphate level, and insulin content and secretion of murine β-cell line MIN6 employing different treatment duration. Our data show that experimental outcomes and assay readouts can be significantly affected by mere preparation of fatty acid solutions and should thus be carefully considered and described in detail to ensure comparability and distinct evaluation of data.

SUBMITTER: Romer A

PROVIDER: S-EPMC8754478 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Icosabutate is a structurally engineered eicosapentaenoic acid derivative under development for nonalcoholic steatohepatitis (NASH). In this study, we investigated the absorption and distribution properties of icosabutate in relation to liver targeting and used rodents to evaluate the effects of icosabutate on glucose metabolism, insulin resistance, as well as hepatic steatosis, inflammation, lipotoxicity, and fibrosis. The absorption, tissue distribution, and excretion of icosabutate was investigated in rats along with its effects in mouse models of insulin resistance (ob/ob) and metabolic inflammation/NASH (high-fat/cholesterol-fed APOE*3Leiden.CETP mice) and efficacy was compared with synthetic peroxisome proliferator-activated receptor α (PPAR-α) (fenofibrate) and/or PPAR-γ/(α) (pioglitazone and rosiglitazone) agonists. Icosabutate was absorbed almost entirely through the portal vein, resulting in rapid hepatic accumulation. Icosabutate demonstrated potent insulin-sensitizing effects in ob/ob mice, and unlike fenofibrate or pioglitazone, it significantly reduced plasma alanine aminotransferase. In high-fat/cholesterol-fed APOE*3Leiden.CETP mice, icosabutate, but not rosiglitazone, reduced microvesicular steatosis and hepatocellular hypertrophy. Although both rosiglitazone and icosabutate reduced hepatic inflammation, only icosabutate elicited antifibrotic effects in association with decreased hepatic concentrations of multiple lipotoxic lipid species and an oxidative stress marker. Hepatic gene-expression analysis confirmed the changes in lipid metabolism, inflammatory and fibrogenic response, and energy metabolism, and revealed the involved upstream regulators. In conclusion, icosabutate selectively targets the liver through the portal vein and demonstrates broad beneficial effects following insulin sensitivity, hepatic microvesicular steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis. Icosabutate therefore offers a promising approach to the treatment of both dysregulated glucose/lipid metabolism and inflammatory disorders of the liver, including NASH.

Project description:Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate-choline or the phosphatidylethanolamine N-methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet-induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity-induced hepatic steatosis, male mice fed a 60% high-fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter-like 1 (CTL1) in the liver of mice fed a high-fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity-induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis.

Dataset Information

Preparation of fatty acid solutions exerts significant impact on experimental outcomes in cell culture models of lipotoxicity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets