Abstract
Ectopic lipid accumulation in skeletal muscle and liver drives the pathogenesis of diabetes mellitus type 2 (DMT2). Mild hyperbilirubinaemia has been repeatedly suggested to play a role in the prevention of DMT2 and is known for its capacity to shape an improved lipid phenotype in humans and in animals. To date, the effect of bilirubin on lipid accumulation in tissues that are prone to ectopic lipid deposition is unclear. Therefore, we analyzed the effect of bilirubin on lipid accumulation in skeletal muscle and liver cell lines. C2C12 skeletal mouse muscle and HepG2 human liver cells were treated with physiological concentrations of free fatty acids (FFA) (0.5 mM and 1 mM) and unconjugated bilirubin (UCB) (17.1 and 55 µM). The intracellular presence of UCB upon exogenous UCB administration was confirmed by HPLC and the lipid accumulation was assessed by using Nile red. Exposure of both cell lines to UCB significantly reduced lipid accumulation by up to 23% (p ≤ 0.001) in HepG2 and by up to 17% (p ≤ 0.01) in C2C12 cells at 0.5 and 5 h under hypoglycaemic conditions. Simultaneously, UCB slightly increased FFA uptake in HepG2 cells after 0.5 and 5 h and in C2C12 cells after 12 h as confirmed by gas chromatographic analyses of the remaining FFA content in the incubation media. The effects of UCB on lipid accumulation and uptake were abolished in the presence of higher glucose concentrations. Monitoring the uptake of a radiolabeled glucose analogue [18F]FDG: (2-deoxy-2-[18F]fluoro-D-glucose) into both cell types further indicated higher glucose consumption in the presence of UCB. In conclusion, our findings show that UCB considerably decreases lipid accumulation in skeletal muscle and liver cells within a short incubation time of max. 5 h which suggests that mildly elevated bilirubin levels could lower ectopic lipid deposition, a major key element in the pathogenesis of DMT2.
Highlights
The increase in diabetes mellitus type 2 (DMT2) is emerging with 463 million cases worldwide, a prevalence that tripled over the last 20 years and is projected to rise to 700 million cases in 2045 (International Diabetes Federation, 2019)
Subjects with Gilberts syndrome (GS), an existing human model with constant mildly increased unconjugated blood bilirubin (UCB) levels have repeatedly been linked to a reduced fat mass, BMI and blood lipids (Wallner et al, 2013) and exhibited a health beneficial glucose metabolic phenotype including a reduction in fasting glucose, insulin, C-peptide and insulin resistance (Mölzer et al, 2016; Khoei et al, 2018)
Cells were treated with 0.5 mM or 1 mM BSA-coupled free fatty acids (FFA) (Svedberg et al, 1990) using palmitic acid (PA, P9767, Sigma-Aldrich, US), oleic acid (OA, O7501, Sigma-Aldrich, US) and linoleic acid (LA, L8134, Sigma-Aldrich, US) at a ratio of 2:1:2 which is consistent with the physiological FFA concentration and composition in human blood (Hodson et al, 2008)
Summary
The increase in diabetes mellitus type 2 (DMT2) is emerging with 463 million cases worldwide, a prevalence that tripled over the last 20 years and is projected to rise to 700 million cases in 2045 (International Diabetes Federation, 2019). Subjects with Gilberts syndrome (GS), an existing human model with constant mildly increased unconjugated blood bilirubin (UCB) levels have repeatedly been linked to a reduced fat mass, BMI and blood lipids (Wallner et al, 2013) and exhibited a health beneficial glucose metabolic phenotype including a reduction in fasting glucose, insulin, C-peptide and insulin resistance (Mölzer et al, 2016; Khoei et al, 2018). This might suggest that mild hyperbilirubinaemia reduces the risk of DMT2 by protecting from disadvantageous, diabetes related changes in lipid metabolism. This study aims for the first time to investigate the effect of UCB on intracellular lipid accumulation in C2C12 skeletal mouse muscle cells and HepG2 hepatoblastomaderived cells
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