cerulenin and stearic-acid

Although increased bone marrow fat in age-related bone loss has been associated with lower trabecular mass, the underlying mechanism responsible remains unknown. We hypothesized that marrow adipocytes exert a lipotoxic effect on osteoblast function and survival through the reversible biosynthesis of fatty acids (FA) into the bone marrow microenvironment. We have used a two-chamber system to co-culture normal human osteoblasts (NHOst) with differentiating pre-adipocytes in the absence or presence of an inhibitor of FA synthase (cerulenin) and separated by an insert that allowed unidirectional trafficking of soluble factors only and prevented direct cell-cell contact. Supernatants were assayed for the presence of FA using mass spectophotometry. After 3 weeks in co-culture, NHOst showed significantly lower levels of differentiation and function based on lower mineralization and expression of alkaline phosphatase, osterix, osteocalcin and Runx2. In addition, NHOst survival was affected by the presence of adipocytes as determined by MTS-formazan and TUNEL assays as well as higher activation of caspases 3/7. These toxic effects were inhibited by addition of cerulenin. Furthermore, culture of NHOst with either adipocyte-conditioned media alone in the absence of adipocytes themselves or with the addition of the most predominant FA (stearate or palmitate) produced similar toxic results. Finally, Runx2 nuclear binding was affected by addition of either adipocyte conditioned media or FA into the osteogenic media. We conclude that the presence of FA within the marrow milieu can contribute to the age-related changes in bone mass and can be prevented by the inhibition of FA synthase.

Topics: Adipocytes; Adult; Apoptosis; Calcification, Physiologic; Cell Differentiation; Cell Nucleus; Cell Proliferation; Cell Survival; Cerulenin; Coculture Techniques; Core Binding Factor Alpha 1 Subunit; Fatty Acid Synthases; Fatty Acids; Female; Humans; Lipids; Male; Osteoblasts; Palmitic Acid; Protein Binding; Stearic Acids; Transcription, Genetic; Young Adult

Paramecium requires oleate for growth. The phospholipids of the ciliate contain high concentrations of palmitate and 18- and 20-carbon unsaturated fatty acids. We previously showed that radiolabeled oleate is desaturated and elongated to provide these 18- and 20-carbon unsaturated acids. We now report on saturated fatty acid (SFA) metabolism in Paramecium. Radiolabeled palmitate and stearate were incorporated directly into cellular phospholipids with little or no desaturation and/or elongation. Radiolabeled acetate, malonate, pyruvate, citrate, or glucose added to cultures were not incorporated into cellular phospholipid fatty acids indicating that these exogenously supplied putative precursors were not utilized for fatty acid synthesis by Paramecium. Radiolabel from octanoate or hexanoate appeared in fatty acyl groups of phospholipids, possibly by partial beta-oxidation and reincorporation of the label. Under oleate-free conditions in which cultures do not grow, radiolabel from these shorter chain SFA were beta-oxidized and preferentially used for the formation of arachidonate, the major end-product of fatty acid synthesis in Paramecium. Cerulenin inhibited culture growth apparently by inhibiting de novo fatty acid synthesis. Cerulenin-treated cells did not incorporate radioactivity from [1-14C]octanoate into esterified palmitate. However, total saponifiable phospholipid fatty acids, including SFA, per cell increased under these conditions.

Topics: Acetates; Acetic Acid; Animals; Cerulenin; Culture Media; Fatty Acids; Fatty Acids, Unsaturated; Oleic Acid; Oleic Acids; Palmitic Acid; Palmitic Acids; Paramecium; Phospholipids; Stearic Acids

Cerulenin and dodecanoic acid prevented the synthesis and secretion of glucosyltransferase in non-proliferating cell suspensions of Streptococcus salivarius ATCC 25975 under conditions that also inhibited the incorporation of radioactively labelled acetate into the cell. In the presence of Tween 80, acetate incorporation was not markedly affected by cerulenin despite the fact that glucosyltransferase secretion was still inhibited. Cerulenin and dodecanoic acid were found to prevent the incorporation of radioactively labelled acetate by affecting the uptake of acetate by the cell. In the case of cerulenin, the inhibition of uptake of acetate by the cell was partially relieved by the addition of Tween 80. These and other observations strongly suggested that cerulenin inhibited glucosyltransferase secretion and acetate incorporation by perturbing the membrane, rather than by directly inhibiting lipid synthesis.

Topics: Acetates; Antifungal Agents; Cell Membrane; Cerulenin; Deoxyglucose; Fatty Acids; Glucosyltransferases; Lauric Acids; Polysorbates; Stearic Acids; Streptococcus