3T3-L1 adipocytes express two isoforms of phospholipase D in distinct subcellular compartments

Phospholipase D has been implicated as an important enzyme in a range of cellular responses, including regulated secretion and the formation of secretory vesicles, cell proliferation and control of cell morphology. As insulin treatment of adipocytes has been shown to stimulate a phosphatidylcholine-specific phospholipase D and also modulates membrane trafficking, we wished to determine which isoform(s) of phospholipase D were present within adipocytes, to identify their subcellular distribution, and examine how this distribution may change in response to insulin. Using RT-PCR, 3T3-L1 adipocytes were found to express two isoforms of phospholipase D, specifically PLD1b and PLD2a. Using isoform-specific antibodies, PLD1 and PLD2 were found to be present predominantly in intracellular membranes, unlike the situation reported in other cells. Detailed analysis of the intracellular localisation of PLD1 and PLD2 revealed that these isoforms are differentially localised within adipocytes, implying functionally distinct roles for PLD activity in distinct subcellular compartments.