We are interested in studying how organisms control and regulate lipid metabolism as it represents a complex process whose alterations affect the functioning of various tissues and organs, and the physiology and behavior of the entire organism. C. elegans is an attractive animal model for this study as it allows for the systematic identification of genes coding for the components of fatty acid metabolism. b-oxidation of fatty acids, which occurrs in mitochondria, plays a central role in lipid metabolism and carnitine is a key molecule required for the transfer of fatty acids across the mitochondrial membrane. Two mammalian genes involved in carnitine biosynthesis have been identified, one, BBOX1, maps in the 11p14 region and one, BBOX2, maps in the Xq28 region. Both genes seemingly code for gamma-butyrobetaine hydroxylases . Systematic search of the C. elegans DNA sequence databases has revealed two predicted homologs of the human BBOX genes. We have named D2089.5,
gbh-1 and M05D6.7,
gbh-2 for gamma butyrobetaine hydroxilases 1 and 2. They appear to be orthologues of the human genes in that
gbh-1 aligns best with hBBOX1 while
gbh-2 aligns best with hBBOX2. We analyzed the expression pattern of
gbh-2 , using 2.7 kb upstream of the ATG to drive expression of GFP with and without a nuclear localization signal. The most prominent GFP expression was in intestinal cells while a weaker signal could be detected in head and body muscles. Expression begins very early in embryogenesis, is restricted to 8 intestinal cells and does not appear to change substantially during development. A similar construct for
gbh-1 showed an expression pattern identical to
gbh-2 in larval stages and in adults but different in the embryo where expression is not restricted to the gut, but appears more generalized. We have used RNA interference to reduce expression of
gbh-1 and
gbh-2 and to study the resulting phentoype(s). The main defects we have observed are: i.The presence of bubble-shaped fatty acid accumulation in the pseudocaelomic cavity of RNAi treated worms. The fat droplets are more easily visible after sexual maturity has been reached. Fat appears to accumulate not only in the extracellular space but also inside the cells of the intestine. We have used the dye Nile Red to confirm the presence of fat droplets in the intestinal and hypodermal cells of living C. elegans . ii. Many RNAi hermaphrodite worms have twisted gonads. In some worms the distal arm of the gonad is longer and misplaced to the ventral side. iii. Interfered worms have reduced fertility and often the germ line cells appear degenerated or abnormal. Interference with
gbh-1 and
gbh-2 probably results in reduced synthesis of carnitine. Since carnitine is necessary for fatty acid utilization it is conceivable that RNA interfered worms under utilize lipids as energy sources and accumulate fatty acids. The abnormal gonad morphogenesis and the reduced fertility of
gbh-1 and
gbh-2 interfered worms are probably secondary to the alteration in fatty acid metabolism. It is interesting that also in mammals, carnitine deficiency, due either to inefficient transport or to reduced biosynthesis, results in extensive fat accumulation not only in the cytoplasm of cells but also in the extracellular space and that it is associated with a variety of pathological conditions.