Lipid bodies (lipid droplets) are emerging as dynamic organelles involved in lipid metabolism and inflammation. Increased lipid body numbers have been described in tumor cells; however, its functional significance in cancer has never been addressed. Here, we showed increased number of lipid bodies in tumor tissues from patients with adenocarcinoma of colon submitted to surgical resection when compared with an adjacent normal tissue. Accordingly, increased numbers of lipid bodies were observed in human colon adenocarcinoma cell lines and in a H-rasV12-transformed intestinal epithelial cell line (IEC-6 H-rasV12) compared with nontransformed IEC-6 cells. The functions of lipid bodies in eicosanoid synthesis in cancer cells were investigated. CACO-2 cells have increased expression of cyclooxygenase-2 (COX-2) when compared with IEC-6 cells. We showed by immunolocalization that, in addition to perinuclear stain, COX-2 and prostaglandin E (PGE) synthase present punctate cytoplasmic localizations that were concordant with adipose differentiation-related protein-labeled lipid bodies. The colocalization of COX-2 at lipid bodies was confirmed by immunoblot of subcellular fractionated cells. Direct localization of PGE 2 at its synthesis locale showed that lipid bodies are sources of eicosanoids in the transformed colon cancer cells. Treatment with either aspirin or the fatty acid synthase inhibitor C75 significantly reduced the number of lipid bodies and PGE 2 production in CACO-2 and in IEC-6 H-rasV12 cells with effects in cell proliferation. Together, our results showed that lipid bodies in colon cancer cells are dynamic and functional active organelles centrally involved in PGE 2 synthesis and may potentially have implications in the pathogenesis of adenocarcinoma of colon. [Cancer Res 2008;68(6):1732-40]
Macrophages have important roles in both lipid metabolism and inflammation and are central to immunity to intracellular pathogens. Foam-like, lipid-laden macrophages are present during the course of mycobacterial infection and have recently been implicated in mycobacterial pathogenesis. In this study, we analyzed the molecular mechanisms underlying the formation of macrophage lipid bodies (lipid droplets) during Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection, focusing on the role of the lipid-activated nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). We found that BCG infection induced increased expression of PPARγ that paralleled the augmented lipid body formation and PGE2 synthesis in mouse peritoneal macrophages. BCG-induced PPARγ expression and lipid body formation were diminished in macrophages from TLR2-deficient mice, suggesting a key role for TLR2. The function of PPARγ in modulating BCG infection was demonstrated by the capacity of the PPARγ agonist BRL49653 to potentiate lipid body formation and PGE2 production; furthermore, pretreatment with the PPARγ antagonist GW9662 inhibited BCG-induced lipid body formation and PGE2 production. BCG-induced MIP-1α, IL12p70, TNF-α, and IL6 production was not inhibited by GW9662 treatment. Nonpathogenic Mycobacterium smegmatis failed to induce PPARγ expression or lipid body formation. Moreover, inhibition of PPARγ by GW9662 enhanced the mycobacterial killing capacity of macrophages. Our findings show that PPARγ is involved in lipid body biogenesis, unravels a cross-talk between the innate immune receptor TLR2 and the lipid-activated nuclear receptor PPARγ that coordinates lipid metabolism and inflammation in BCG-infected macrophages, thereby potentially affecting mycobacterial pathogenesis.
Lipid bodies (also known as lipid droplets) are emerging as inflammatory organelles with roles in the innate immune response to infections and inflammatory processes. In this study, we identified MCP-1 as a key endogenous mediator of lipid body biogenesis in infection-driven inflammatory disorders and we described the cellular mechanisms and signaling pathways involved in the ability of MCP-1 to regulate the biogenesis and leukotriene B4 (LTB4) synthetic function of lipid bodies. In vivo assays in MCP-1−/− mice revealed that endogenous MCP-1 produced during polymicrobial infection or LPS-driven inflammatory responses has a critical role on the activation of lipid body-assembling machinery, as well as on empowering enzymatically these newly formed lipid bodies with LTB4 synthetic function within macrophages. MCP-1 triggered directly the rapid biogenesis of distinctive LTB4-synthesizing lipid bodies via CCR2-driven ERK- and PI3K-dependent intracellular signaling in in vitro-stimulated macrophages. Disturbance of microtubule organization by microtubule-active drugs demonstrated that MCP-1-induced lipid body biogenesis also signals through a pathway dependent on microtubular dynamics. Besides biogenic process, microtubules control LTB4-synthesizing function of MCP-1-elicited lipid bodies, in part by regulating the compartmentalization of key proteins, as adipose differentiation-related protein and 5-lipoxygenase. Therefore, infection-elicited MCP-1, besides its known CCR2-driven chemotactic function, appears as a key activator of lipid body biogenic and functional machineries, signaling through a microtubule-dependent manner.
Leptin is an adipocyte-derived hormone/cytokine that links nutritional status with neuroendocrine and immune functions. Lipid bodies (lipid droplets) are emerging as dynamic organelles with roles in lipid metabolism and inflammation. Here we investigated the roles of leptin in signaling pathways involved in cytoplasmic lipid body biogenesis and leukotriene B 4 synthesis in macrophages. Our results demonstrated that leptin directly activated macrophages and induced the formation of adipose differentiation-related protein-enriched lipid bodies. Newly formed lipid bodies were sites of 5-lipoxygenase localization and correlated with an enhanced capacity of leukotriene B 4 production. We demonstrated that leptin-induced macrophage activation was dependent on phosphatidylinositol 3-kinase (PI3K) activity, since the lipid body formation was inhibited by LY294002 and was absent in the PI3K knock-out mice. Leptin induces phosphorylation of p70 S6K and 4EBP1 key downstream signaling intermediates of the mammalian target of rapamycin (mTOR) pathway in a rapamycin-sensitive mechanism. The mTOR inhibitor, rapamycin, inhibited leptin-induced lipid body formation, both in vivo and in vitro. In addition, rapamycin inhibited leptin-induced adipose differentiation-related protein accumulation in macrophages and lipid bodydependent leukotriene synthesis, demonstrating a key role for mTOR in lipid body biogenesis and function. Our results establish PI3K/mTOR as an important signaling pathway for leptin-induced cytoplasmic lipid body biogenesis and adipose differentiation-related protein accumulation. Furthermore, we demonstrate a previously unrecognized link between intracellular (mTOR) and systemic (leptin) nutrient sensors in macrophage lipid metabolism. Leptin-induced increased formation of cytoplasmic lipid bodies and enhanced inflammatory mediator production in macrophages may have implications for obesity-related cardiovascular diseases.
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.
Lipid droplets (lipid bodies, LDs) are dynamic organelles that have important roles in regulating lipid metabolism, energy homeostasis, cell signaling, membrane trafficking, and inflammation. LD biogenesis, composition, and functions are highly regulated and may vary according to the stimuli, cell type, activation state, and inflammatory environment. Increased cytoplasmic LDs are frequently observed in leukocytes and other cells in a number of infectious diseases. Accumulating evidence reveals LDs participation in fundamental mechanisms of host-pathogen interactions, including cell signaling and immunity. LDs are sources of eicosanoid production, and may participate in different aspects of innate signaling and antigen presentation. In addition, intracellular pathogens evolved mechanisms to subvert host metabolism and may use host LDs, as ways of immune evasion and nutrients source. Here, we review mechanisms of LDs biogenesis and their contributions to the infection progress, and discuss the latest discoveries on mechanisms and pathways involving LDs roles as regulators of the immune response to protozoan infection.
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