Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells
Introduction
Lipotoxicity refers to the inhibitory effects of lipid on tissue function. Lipotoxicity has been best characterized in metabolic tissues wherein excess lipid inhibits insulin signaling, leading to dysregulated metabolism [1]. However, the toxic effects of lipid include decreased cell viability and differentiation. Recently, bone has been identified as an important site of lipotoxicity, and it has been suggested that lipotoxicity may be a key mechanism in age-related osteoporosis [2], [3]. Published results show that lipids cause increased osteoblast apoptosis and inhibit the differentiation of osteoblasts [4], [5]. The mechanisms of lipotoxicity in bone are, however, incompletely understood. In bone marrow it is believed that with age, there is a switch from osteoblastogenesis to adipogenesis [2]; and that fatty acids, released from the adipocytes, act in a paracrine manner to inhibit osteoblast function [4], [5], [6], [7]. In calvarial cells, on the other hand, there is evidence that osteoblasts co-express adipogenic and osteogenic markers in response to PPARγ agonists [8].
Regardless of the precise change in cellularity, it is evident that lipotoxicity involves the dysregulation of free fatty acid metabolism leading to inhibitory effects on cells [1]. In soft tissues, increased de novo fatty acid biosynthesis and/or ineffective fatty acid oxidation leads to metabolism of fatty acids through alternative pathways resulting in the production of toxic compounds such as ceramides [1]. A key regulator of the balance of fatty acid metabolism is the enzyme, acetyl CoA carboxylase (ACC), which catalyzes the conversion of acetyl CoA to malonyl CoA. Malonyl CoA in turn acts as a switch by providing the substrate for fatty acid biosynthesis and by acting as an allosteric inhibitor of fatty acid oxidation. Thus, acetyl CoA carboxylase inhibition may attenuate lipotoxicity by inhibiting fatty acid biosynthesis and by promoting fatty acid oxidation [9]. Previously we found that palmitate inhibited the expression of two osteogenic markers in FRC cells and that this effect was blocked by the ACC inhibitor TOFA [10]. In the present study we have further tested the hypothesis that stimulation of fatty acid oxidation may attenuate the lipotoxic effects of palmitate on fetal rat calvarial (FRC) cell.
Section snippets
Materials
Fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Woodland, CA). Alpha MEM (αMEM), Hanks’ Balanced Salt Solution (HBSS), penicillin–streptomycin stock, and trypsin–EDTA were purchased from Gibco/Invitrogen (Carlsbad, CA). Recombinant human BMP-7 was provided by Stryker Biotech (Hopkinton, MA) and dissolved in 47.5% ethanol/0.01% trifluoroacetic acid. TOFA was purchased from Cayman Chemical (Ann Arbor, MI) and Cell Proliferation (MTT) kit was from Promega.
Fetal rat calvarial cell culture
Animals were purchased
Results
In order to evaluate the influence of lipid on osteoblast function, we incubated fetal rat calvarial (FRC) cells with palmitate, the primary product of de novo lipogenesis and a major component of triacylglycerol. We then evaluated effects of palmitate on FRC cell proliferation and osteogenic differentiation. As shown in Fig. 1A, palmitate did not inhibit cell proliferation to a significant extent but inhibited mineralized bone nodule formation in FRC cultures as revealed by Alizarin Staining (
Discussion
Several published studies have modeled the effect of fatty acids released from adipocytes in the bone marrow environment on osteoblast function in endochondral bone formation [6], [17], [18]. Our previously published [10] and current studies examined the effect of palmitate on calvarial cells, which are involved in intramembranous bone formation. Our results show that palmitate does not affect osteoblast cell proliferation but inhibits the osteogenic differentiation function of fetal rat
Acknowledgment
The study was supported by NIA/NIH grant R21AG040612.
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