Colitis Alters Oxysterol Metabolism and is Affected by 4ß-Hydroxycholesterol Administration.

BACKGROUND AND AIMS: Inflammatory bowel diseases [IBD] represent a challenging health issue with a complex aetiology involving genetic and environmental parameters. Although our understanding of the pathophysiology of IBD has improved, much remains to be explored. In this context, bioactive lipids, more specifically oxysterols, i.e. oxygenated derivatives of cholesterol, represent an interesting avenue to investigate. Indeed, oxysterols or their receptors are involved in inflammation and immune regulation. Therefore, we set out to study the oxysterome in IBD. METHODS: We used both high-performance liquid chromatograph/mass spectroscopy and molecular biology tools to quantify oxysterol levels and the expression of their metabolic enzymes in several models of murine colitis [both acute and chronic], as well as in colon biopsies from patients with Crohn's disease and ulcerative colitis. RESULTS: We found that the oxysterome is altered in IBD, in both acute and chronic murine models as well as in human IBD. Two of the oxysterols quantified, 4ß-hydroxycholesterol and 25-hydroxycholesterol, were consistently altered in all our models and therefore could be of interest in this context. Hence, we administered them to mice with colitis. While 25-hydroxycholesterol had no effect, 4ß-hydroxycholesterol worsened colon inflammation. CONCLUSIONS: Our study addresses the potential involvement of oxysterols in colitis and clearly points towards an active role as well as a clinical relevance for these bioactive lipids.

Post-operative pain in mice is prolonged by diet-induced obesity and rescued by dietary intervention.

The prevalence of obesity has increased at an alarming rate during past decades. Obesity is associated with pathophysiological disorders that can evolve and increase the risk of heart disease, diabetes and hypertension. While the impact of diabetes on post-operative recovery is now known, the consequences of obesity on post-operative pain remain much less explored. Here, we show that obesity affects post-operative pain resolution and leads to a chronic pain state in mice. Several mechanisms were identified as implicated in the prolonged post-operative pain. Indeed, we found that following a hind paw incision, high fat diet prolonged glial cell activation in the spinal cord. It also altered the expression of neurotrophins and increased inflammatory and endoplasmic reticulum stress markers in both central and peripheral nervous systems. Moreover, we show that a dietary intervention, leading to weight reduction and decreased inflammation, was able to restore normal pain sensitivity in mice suffering from chronic pain for more than 10 weeks. In conclusion, our data demonstrate that obesity is responsible for pain chronicization. This is clearly of importance in a clinical post-operative setting.

Oxysterol levels and metabolism in the course of neuroinflammation: insights from in vitro and in vivo models.

BACKGROUND: Oxysterols 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. METHODS: We 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. RESULTS: In 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. CONCLUSIONS: These 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.

Resistant starch can improve insulin sensitivity independently of the gut microbiota.

BACKGROUND: Obesity-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. RESULTS: RS 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. CONCLUSIONS: This 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.

Oxysterols: From cholesterol metabolites to key mediators.

Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4ß-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.

Oxysterols in Metabolic Syndrome: From Bystander Molecules to Bioactive Lipids.

Oxysterols are cholesterol metabolites now considered bona fide bioactive lipids. Recent studies have identified new receptors for oxysterols involved in immune and inflammatory processes, hence reviving their appeal. Through multiple receptors, oxysterols are involved in numerous metabolic and inflammatory processes, thus emerging as key mediators in metabolic syndrome. This syndrome is characterized by complex interactions between inflammation and a dysregulated metabolism. Presently, the use of synthetic ligands and genetic models has facilitated a better understanding of the roles of oxysterols in metabolism, but also raised interesting questions. We discuss recent findings on the absolute levels of oxysterols in tissues, their newly identified targets, and the mechanistic studies emphasizing their importance in metabolic disease, as there is a pressing need to further comprehend these intriguing bioactive lipids.

Obesity is associated with changes in oxysterol metabolism and levels in mice liver, hypothalamus, adipose tissue and plasma.

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 4ß-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 (4ß-hydroxycholesterol, 27- hydroxycholesterol, 7-hydroxycholestenone) levels and enzyme (CYP3A11, CYP27A1, CYP7A1) expression.

Development and validation of an HPLC-MS method for the simultaneous quantification of key oxysterols, endocannabinoids, and ceramides: variations in metabolic syndrome.

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.