Docosahexaenoic acid impairs the maturation of very low density lipoproteins in rat hepatic cells.

One mechanism of the lipid-lowering effects of the fish oil n-3 fatty acids [e.g., docosahexaenoic acid (DHA)] in cell and animal models is induced hepatic apolipoprotein B100 (apoB) presecretory degradation. This degradation occurs post-endoplasmic reticulum, but whether DHA induces it before or after intracellular VLDL formation remains unanswered. We found in McA-RH7777 rat hepatic cells that DHA and oleic acid (OA) treatments allowed formation of pre-VLDL particles and their transport to the Golgi, but, in contrast to OA, with DHA pre-VLDL particles failed to quantitatively assemble into fully lipidated (mature) VLDL. This failure required lipid peroxidation and was accompanied by the formation of apoB aggregates (known to be degraded by autophagy). Preventing the exit of proteins from the Golgi blocked the aggregation of apoB but did not restore VLDL maturation, indicating that failure to fully lipidate apoB preceded its aggregation. ApoB autophagic degradation did not appear to require an intermediate step of cytosolic aggresome formation. Taken with other examples in the literature, the results of this study suggest that pre-VLDL particles that are competent to escape endoplasmic reticulum quality control mechanisms but fail to mature in the Golgi remain subject to quality control surveillance late in the secretory pathway.

Cell-free supernatants from probiotic Lactobacillus casei and Lactobacillus rhamnosus GG decrease colon cancer cell invasion in vitro.

Probiotics have been shown to have a preventative role in colorectal carcinogenesis but research concerning their prophylactic potential in the later stages of colorectal cancer, specifically metastasis is limited. This study explored the potential of cell-free supernatants (CFS) from 2 probiotic Lactobacillus sp., Lactobacillus casei and Lactobacillus rhamnosus GG, to inhibit colon cancer cell invasion by influencing matrix metalloproteinase-9 (MMP-9) activity and levels of the tight junction protein zona occludens-1 (ZO-1) in cultured metastatic human colorectal carcinoma cells. HCT-116 cells were treated with CFS from L. casei, L. rhamnosus, or Bacteroides thetaiotaomicron (a gut commensal); or with uninoculated bacterial growth media. Treatment with CFS from both Lactobacillus sp. decreased colorectal cell invasion but treatment with CFS from B. thetaiotaomicron did not. CFS from both Lactobacillus sp. decreased MMP-9 and increased ZO-1 protein levels. L. rhamnosus CFS also lowered MMP-9 activity. To begin elucidating the secreted bacterial factor conveying these responses, Lactobacillus sp. CFS were fractionated into defined molecular weight ranges and cell invasion assessed. Fractionation revealed that the inhibitory activity was contained primarily in the >100 kDa and 50-100 kDa fractions, suggesting the inhibitory compound may be a macromolecule such as a protein, nucleic acid, or a polysaccharide.

Bioassays of quorum sensing compounds using Agrobacterium tumefaciens and Chromobacterium violaceum.

In most bacteria, a global level of regulation exists involving intercellular communication via the production and response to cell density-dependent signal molecules. This cell density-dependent regulation has been termed quorum sensing (QS). QS is a global regulator, which has been associated with a number of important features in bacteria including virulence regulation and biofilm formation. Consequently, there is considerable interest in understanding, detecting, and inhibiting QS. Acyl homoserine lactones (acyl HSLs) are used as extracellular QS signals by a variety of Gram-negative bacteria. Chromobacterium violaceum, a Gram-negative bacterium commonly found in soil and water, produces the characteristic purple pigment violacein, the production of which is regulated by acyl HSL-mediated QS. Based on this readily observed pigmentation phenotype, C. violaceum strains can be used to detect various aspects of acyl HSL-mediated QS activity. In another commonly used bioassay organism, Agrobacterium tumefaciens, QS can be detected by the use of a reporter gene such as lacZ. Here, we describe several commonly used approaches incorporating C. violaceum and A. tumefaciens that can be used to detect acyl HSLs and QS inhibition.

Presecretory oxidation, aggregation, and autophagic destruction of apoprotein-B: a pathway for late-stage quality control.

Hepatic secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins, is regulated through posttranslational degradation. We reported a degradation pathway, post-ER pre secretory proteolysis (PERPP), that is increased by reactive oxygen species (ROS) generated within hepatocytes from dietary polyunsaturated fatty acids (PUFA). We now report the molecular processes by which PUFA-derived ROS regulate PERPP of apoB. ApoB exits the ER; undergoes limited oxidant-dependent aggregation; and then, upon exit from the Golgi, becomes extensively oxidized and converted into large aggregates. The aggregates slowly degrade by an autophagic process. None of the oxidized, aggregated material leaves cells, thereby preventing export of apoB-lipoproteins containing potentially toxic lipid peroxides. In summary, apoB secretory control via PERPP/autophagosomes is likely a key component of normal and pathologic regulation of plasma apoB levels, as well as a means for remarkably late-stage quality control of a secreted protein.

Differences in cell morphology, lipid and apo B secretory capacity in caco-2 cells following long term treatment with saturated and monounsaturated fatty acids.

The suitability of the caco-2 cell line as a model for studying the long term impact of dietary fatty acids on intestinal lipid handling and chylomicron production was examined. Chronic supplementation of caco-2 cells with palmitic acid (PA) resulted in a lower triacylglycerol secretion than oleic acid (OA). This was coupled with a detrimental effect of PA, but not OA, on transepithelial electrical resistance (TER) measurements, suggesting a loss of structural integrity across the cell monolayer. Addition of OA reversed the adverse effects of PA and stearic acid on TER and increased the ability of cells to synthesise and accumulate lipid, but did not normalise the secretion of lipids by caco-2 cells. Increasing amounts of OA and decreasing amounts of PA in the incubation media markedly improved the ability of cells to synthesise apolipoprotein B and secrete lipids. Real time RT-PCR revealed a down regulation of genes involved in lipoprotein synthesis following PA than OA. Electron microscopy showed adverse effects of PA on cellular morphology consistent with immature enterocytes such as stunted microvilli and poor tight junction formation. In conclusion, previously reported differences in lipoprotein secretion by caco-2 cells supplemented with saturated fatty acids (SFA) and OA may partly reflect early cytotoxic effects of SFA on cellular integrity and function.

Saturated fat-induced changes in Sf 60-400 particle composition reduces uptake of LDL by HepG2 cells.

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.

Prebiotics and synbiotics: towards the next generation.

Recent research in the area of prebiotic oligosaccharides and synbiotic combinations with probiotics is leading towards a more targeted development of functional food ingredients. Improved molecular techniques for analysis of the gut microflora, new manufacturing biotechnologies, and increased understanding of the metabolism of oligosaccharides by probiotics are facilitating development. Such developments are leading us to the time when we will be able to rationally develop prebiotics and synbiotics for specific functional properties and health outcomes.