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BackgroundObesity-related diseases, including type 2 diabetes and cardiovascular disease, have reached epidemic proportions in industrialized nations, and dietary interventions for their prevention are therefore important. Resistant starches (RS) improve insulin sensitivity in clinical trials, but the mechanisms underlying this health benefit remain poorly understood. Because RS fermentation by the gut microbiota results in the formation of physiologically active metabolites, we chose to specifically determine the role of the gut microbiota in mediating the metabolic benefits of RS. To achieve this goal, we determined the effects of RS when added to a Western diet on host metabolism in mice with and without a microbiota.ResultsRS feeding of conventionalized mice improved insulin sensitivity and redressed some of the Western diet-induced changes in microbiome composition. However, parallel experiments in germ-free littermates revealed that RS-mediated improvements in insulin levels also occurred in the absence of a microbiota. RS reduced gene expression of adipose tissue macrophage markers and altered cecal concentrations of several bile acids in both germ-free and conventionalized mice; these effects were strongly correlated with the metabolic benefits, providing a potential microbiota-independent mechanism to explain the physiological effects of RS.ConclusionsThis study demonstrated that some metabolic benefits exerted by dietary RS, especially improvements in insulin levels, occur independently of the microbiota and could involve alterations in the bile acid cycle and adipose tissue immune modulation. This work also sets a precedent for future mechanistic studies aimed at establishing the causative role of the gut microbiota in mediating the benefits of bioactive compounds and functional foods.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0230-5) contains supplementary material, which is available to authorized users.

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BackgroundOxysterols are cholesterol derivatives that have been suggested to play a role in inflammatory diseases such as obesity, atherosclerosis, or neuroinflammatory diseases. However, the effect of neuroinflammation on oxysterol levels has only been partially studied so far.MethodsWe used an HPLC-MS method to quantify over ten oxysterols both in in vitro and in vivo models of neuroinflammation. In the same models, we used RT-qPCR to analyze the expression of the enzymes responsible for oxysterol metabolism. Using the BV2 microglial cell line, we explored the effect of lipopolysaccharide (LPS)-induced (M1-type) and IL-4-induced (M2-type) cell activation on oxysterol levels. We also used LPS-activated co-cultures of mouse primary microglia and astrocytes. In vivo, we induced a neuroinflammation by administering LPS to mice. Finally, we used a mouse model of multiple sclerosis, namely the experimental autoimmune encephalomyelitis (EAE) model, that is characterized by demyelination and neuroinflammation.ResultsIn vitro, we found that LPS activation induces profound alterations in oxysterol levels. Interestingly, we could discriminate between control and LPS-activated cells based on the changes in oxysterol levels both in BV2 cells and in the primary co-culture of glial cells. In vivo, the changes in oxysterol levels were less marked than in vitro. However, we found in both models increased levels of the GPR183 agonist 7α,25-dihydroxycholesterol. Furthermore, we studied in vitro the effect of 14 oxysterols on the mRNA expression of inflammatory markers in LPS-activated co-culture of microglia and astrocytes. We found that several oxysterols decreased the LPS-induced expression of pro-inflammatory markers.ConclusionsThese data demonstrate that inflammation profoundly affects oxysterol levels and that oxysterols can modulate glial cell activation. This further supports the interest of a large screening of oxysterol levels when studying the interplay between neuroinflammation and bioactive lipids.Electronic supplementary materialThe online version of this article (10.1186/s12974-018-1114-8) contains supplementary material, which is available to authorized users.

Oxysterols are bioactive lipids derived from cholesterol that are linked to inflammatory processes. Because obesity and metabolic syndrome are characterized by inflammation and altered cholesterol metabolism, we sought to investigate the variations of oxysterol levels and their metabolic pathways induced by obesity in the liver, hypothalamus, adipose tissue and plasma. To this end, we used diet-induced and genetic (ob/ob and db/db) models of obesity. Among the oxysterols measured, we found that 4β-oxysterol levels were consistently decreased in the high-fat diet study, at different time-points, and in the ob/ob model. Overall, we did not find any correlation between cytochromes mRNA expression and variations of oxysterol levels. We also measured the levels of hepatic primary bile acids, in these three models and found similar profiles between HFD and ob/ob mice. However, although they are downstream metabolites of oxysterols, the variations in bile acid levels did not reflect the variations of their precursors. Our data show that, when considering oxysterol metabolism, the high-fat diet and ob/ob models are more closely related when compared to the db/db model. However, we were able to discriminate between lean and obese phenotypes based on liver oxysterol (4β-hydroxycholesterol, 27- hydroxycholesterol, 7-hydroxycholestenone) levels and enzyme (CYP3A11, CYP27A1, CYP7A1) expression.

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Oxysterols, ceramides, and endocannabinoids are three families of bioactive lipids suggested to be involved in obesity and metabolic syndrome. To facilitate the quantification of these potentially interconnected lipids, we have developed and validated a liquid chromatography coupled to mass spectrometry method allowing for their simultaneous quantification from tissues. Sample purification is of great importance when quantifying oxysterols due to the potential artifactual conversion of cholesterol into oxysterols. Therefore, we developed a novel solid-phase extraction procedure and demonstrated that it allowed for good recoveries of the three families of analytes without artifactual oxidation of cholesterol. The oxysterols, ceramides, and endocannabinoids and their respective internal standards were chromatographically separated by HPLC and ionized using the atmospheric pressure chemical ionization (APCI) source of an LTQ-orbitrap mass spectrometer. The repeatability and bias were within the acceptance limits for all 23 lipids of interest. The sensitivity (limit of detection (LOD) and limit of quantification (LOQ)) and specificity of the method allowed us to quantify all the analytes in the liver and adipose tissue of control and high-fat diet-fed C57BL/6 mice. We found that 16 weeks of high-fat diet strongly impacted the hepatic levels of several oxysterols, ceramides, and endocannabinoids. A partial least-squares discriminant analysis (PLS-DA) based on the variations of the hepatic levels of these 23 bioactive lipids allowed differentiating the lean mice from the obese mice.

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