Characterization of a novel 34 kDa protein from adipocytes

Adipose tissue, which plays a fundamental role in vertebrate energy balance and overall body homeostasis, produces and secretes peptides, proteins, lipids and other components and acts as a paracrine/endocrine organ. In addition to its central role in energy metabolism, adipose tissue is also able to store considerable amounts of unesterified cholesterol (FC), more than any other peripheral cell. Fundamental processes governing the development of adipose tissue and of its most abundant cell type, the adipocytes, are currently a central focus of biochemical and medical research. Despite the well-established finding that obesity represents an independent risk factor for atherosclerosis and related diseases, information on the nature of adipocyte triglyceride droplets (TGD) is still scarce. It is generally accepted that the hydrophobic triglyceride clusters are surrounded by a phospholipid monolayer, and by protein constituents (cage proteins), but only few of them have been characterized so far. In search of novel proteins involved in the biogenesis and metabolism of adipocyte TGD, we partially purified from a TGD subfraction of differentiated adipocytes a previously undescribed 34-kDa protein constituent of the TGD surface layer. N-terminal peptide sequences from the undigested as well as from four internal tryptic protein fragments were obtained, and an anti-peptide antibody has been produced. The proposed project focuses on the genetic characterization, expression, and intracellular localization of the novel 34-kDa protein. Specific aims of the study: 1) cDNA cloning to elucidate the primary structure of the 34-kDa protein. 2) Expression of recombinant protein in prokaryotic and eukaryotic cells, and antibody production. 3) To analyze the tissue-specific expression of the protein. 4) To investigate the regulation of expression of the 34-kDa protein by hormones. 5) To explore the the intracellular distribution and potential function by over-expression and by expression of antisense cDNA to hinder the expression of the 34-kDa protein in adipocytes. The presence of the investigated protein in triglyceride droplets suggests a potential function in triglyceride droplet biogenesis and/or lipid mobilization. Both potential functions are especially interesting with regard to the elucidation of the structural organization of triglyceride droplets in physiological and diseased states.

The biogenesis of intracellular lipid dropets as storage compartments for triglycerides (energy store) as well as for cholesteryl esters (supply of unesterified cholesterol for membrane biosynthesis) in not fully understood. Informations on the mechanisms involved in the synthesis of lipid droplets are scarce. Moreover, only few components (proteins and lipids) of the monolayer shell of lipid droplets have been characterized so far. Our studies relate to the biogenesis and nature of the monolayer shell of adipocyte lipid droplets. We demonstrate in adipocytes the expression of the enzyme PEMT (phosphatidyl-ethanolamine methyltransferase), the presence of substantial PEMT activity, and the implication of the enzyme in the synthesis of a specific glycerophospholipid subclass containing mono-unsaturated fatty acids which is involved in the biogenesis of lipid droplets. Furthermore, we show that the morphology and the basal lipolysis of adipocytes is substantially modified, when a choline analogue, dimethylaminoethanol (DMAE), but not choline, is used as precursor for phospholipid synthesis. Upon biogenic incorporation of DMAE into the corresponding phospholipid, the enzyme PEMT converts it to phosphatidylcholine. As a consequence of DMAE treatment, adipocyte lipid droplets shrink, and the basal lipolysis is substantially increased. Fundamental cellular functions (such as uptake of glukose), however, remain unaltered. Our results imply an important function of the enzyme PEMT for the biogenesis of intracellular lipid droplets.