title: Role of adipocyte-derived lipoprotein lipase in adipocyte hypertrophy.
abstract: BACKGROUND
  A major portion of available fatty acids for adipocyte uptake is derived from lipoprotein lipase (LPL)-mediated hydrolysis of circulating lipoprotein particles. In vivo studies aimed at identifying the precise role of adipocyte-derived LPL in fat storage function of adipose tissue have been unable to provide conclusive evidence due to compensatory mechanisms that activate endogenous fatty acid synthesis. To address this gap in knowledge, we have measured the effect of reducing adipocyte LPL expression on intracellular lipid accumulation using a well-established cultured model of adipocyte differentiation.
  
  
  METHODS
  siRNA specific for mouse LPL was transfected into 3T3-L1 adipocytes. Expression of LPL was measured by quantitative real-time PCR and cell surface-associated LPL enzymatic activity was measured by colorimetric detection following substrate (p-nitrophenyl butyrate) hydrolysis. Apolipoprotein CII and CIII expression ratios were also measured by qRT-PCR. Intracellular lipid accumulation was quantified by Nile Red staining.
  
  
  RESULTS
  During differentiation of 3T3-L1 pre-adipocytes, LPL mRNA expression increases 6-fold resulting in a 2-fold increase in cell surface-associated LPL enzymatic activity. Parallel to this increase in LPL expression, we found that intracellular lipids increased ~10-fold demonstrating a direct correlation between adipocyte-derived LPL expression and lipid storage. We next reduced LPL expression in adipocytes using siRNA transfections to directly quantify the contributions of adipocyte-derived LPL to lipid storage, This treatment reduced LPL mRNA expression and cell surface-associated LPL enzymatic activity to ~50% of non-treated controls while intracellular lipid levels were reduced by 80%. Exogenous addition of purified LPL (to restore extracellular lipolytic activity) or palmitate (as a source of free fatty acids) to siRNA-treated cells restored intracellular lipid levels to those measured for non-treated controls. We also found that adipocytes express apolipoprotein CII and CIII and, in addition, the apoCII/apoCIII ratio remains largely unchanged in cells with reduced LPL expression.
  
  
  CONCLUSIONS
  We provide evidence that adipocyte-derived LPL is required for efficient fatty acid uptake and storage, and that adipocytes express their own source of apoCII and apoCIII for regulating extracellular LPL activity. These findings demonstrate that adipocytes are capable of producing the necessary enzymatic components and co-factors for efficient lipid storage independent of vascular sources.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of New Mexico, School of Medicine, MSC08 4670, 1 University of New Mexico, Albuquerque, New Mexico, 87131, USA. rorlando@salud.unm.edu.
authors.forename: Amanda M
authors.initials: AM
authors.lastname: Gonzales
authors.forename: Robert A
authors.initials: RA
authors.lastname: Orlando