title: Altered mitochondrial function and cholesterol synthesis influences protein synthesis in extended HepG2 spheroid cultures.
abstract: Cultures of hepatocytes and HepG2 cells provide useful in vitro models of liver specific function. In this study, we investigated metabolic and biosynthetic function in 3-D HepG2 spheroid cultures, in particular to characterise changes on prolonged culture. We show that HepG2 cells cultured in spheroids demonstrate a reduction in mitochondrial membrane potential and respiration following 10 days of culture. This coincides with a modest reduction in glycolysis but an increase in glucose uptake where increased glycogen synthesis occurs at the expense of the intracellular ATP pool. Lowered biosynthesis coincides with and is linked to mitochondrial functional decline since low glucose-adapted spheroids, which exhibit extended mitochondrial function, have stable biosynthetic activity during extended culture although biosynthetic function is lower. This indicates that glucose is required for biosynthetic output but sustained mitochondrial function is required for the maintenance of biosynthetic function. Furthermore, we show that cholesterol synthesis is markedly increased in spheroids cf. monolayer culture and that inhibition of cholesterol synthesis by lovastatin extends mitochondrial and biosynthetic function. Therefore, increased cholesterol synthesis and/or its derivatives contributes to mitochondrial functional decline in extended HepG2 spheroid cultures.
authors.affiliation: Department of Medicine, Royal Free Campus, Royal Free and UCL Medical School, Rowland Hill Street, Hampstead, London, NW3 2PF, United Kingdom. l.damelin@rfc.ucl.ac.uk
authors.forename: Leonard H
authors.initials: LH
authors.lastname: Damelin
authors.forename: Sam
authors.initials: S
authors.lastname: Coward
authors.forename: Sarah F
authors.initials: SF
authors.lastname: Choudhury
authors.forename: Sherri-Ann
authors.initials: SA
authors.lastname: Chalmers
authors.forename: I Jane
authors.initials: IJ
authors.lastname: Cox
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Robertson
authors.forename: Gilbert
authors.initials: G
authors.lastname: Revial
authors.forename: Marianne
authors.initials: M
authors.lastname: Miles
authors.forename: Rosemary
authors.initials: R
authors.lastname: Tootle
authors.forename: Humphrey J F
authors.initials: HJ
authors.lastname: Hodgson
authors.forename: Clare
authors.initials: C
authors.lastname: Selden
mesh_terms: D000418:Albumins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D019069:Cell Respiration / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000096:biosynthesis*; D004777:Environment; D005947:Glucose / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D008148:Lovastatin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008863:Microspheres; D008930:Mitochondria, Liver / Q000187:drug effects / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D000418:Albumins; D024101:Mitochondrial Proteins; D002784:Cholesterol; D008148:Lovastatin; D005947:Glucose; D010100:Oxygen