Prebiotic fibre mixtures counteract the manifestation of gut microbial dysbiosis induced by the chemotherapeutic 5-Fluorouracil (5-FU) in a validated in vitro model of the colon.

BACKGROUND: 5-Fluorouracil (5-FU) is used as an antineoplastic agent in distinct cancer types. Increasing evidence suggests that the gut microbiota might modulate 5-FU efficacy and toxicity, potentially affecting the patient's prognosis. The current experimental study investigated 5-FU-induced microbiota alterations, as well as the potential of prebiotic fibre mixtures (M1-M4) to counteract these shifts. METHODS: A pooled microbial consortium was derived from ten healthy donors, inoculated in an in vitro model of the colon, and treated with 5-FU, with or without prebiotic fibre mixtures for 72 h. Four different prebiotic fibre mixtures were tested: M1 containing short-chain galacto-oligosaccharides (sc GOS), long-chain fructo-oligosaccharides (lcFOS), and low viscosity pectin (lvPect), M2 consisting of arabinoxylan, beta-glucan, pectin, and resistant starch, M3 which was a mixture of scGOS and lcFOS, and M4 containing arabinoxylan, beta-glucan, pectin, resistant starch, and inulin. RESULTS: We identified 5-FU-induced changes in gut microbiota composition, but not in microbial diversity. Administration of prebiotic fibre mixtures during 5-FU influenced gut microbiota composition and taxa abundance. Amongst others, prebiotic fibre mixtures successfully stimulated potentially beneficial bacteria (Bifidobacterium, Lactobacillus, Anaerostipes, Weissella, Olsenella, Senegalimassilia) and suppressed the growth of potentially pathogenic bacteria (Klebsiella, Enterobacter) in the presence of 5-FU. The short-chain fatty acid (SCFA) acetate increased slightly during 5-FU, but even more during 5-FU with prebiotic fibre mixtures, while propionate was lower due to 5-FU with or without prebiotic fibre mixtures, compared to control. The SCFA butyrate and valerate did not show differences among all conditions. The branched-chain fatty acids (BCFA) iso-butyrate and iso-valerate were higher in 5-FU, but lower in 5-FU + prebiotics, compared to control. CONCLUSIONS: These data suggest that prebiotic fibre mixtures represent a promising strategy to modulate 5-FU-induced microbial dysbiosis towards a more favourable microbiota, thereby possibly improving 5-FU efficacy and reducing toxicity, which should be evaluated further in clinical studies.

Sex-opposed inflammatory effects of 27-hydroxycholesterol are mediated via differences in estrogen signaling.

Despite the increased awareness of differences in the inflammatory response between men and women, only limited research has focused on the biological factors underlying these sex differences. The cholesterol derivative 27-hydroxycholesterol (27HC) has been shown to have opposite inflammatory effects in independent experiments using mouse models of atherosclerosis and non-alcoholic steatohepatitis (NASH), pathologies characterized by cholesterol-induced inflammation. As the sex of mice in these in vivo models differed, we hypothesized that 27HC exerts opposite inflammatory effects in males compared to females. To explore whether the sex-opposed inflammatory effects of 27HC translated to humans, plasma 27HC levels were measured and correlated with hepatic inflammatory parameters in obese individuals. To investigate whether 27HC exerts sex-opposed effects on inflammation, we injected 27HC into female and male Niemann-Pick disease type C1 mice (Npc1nih ) that were used as an extreme model of cholesterol-induced inflammation. Finally, the involvement of estrogen signaling in this mechanism was studied in bone marrow-derived macrophages (BMDMs) that were treated with 27HC and 17ß-estradiol (E2). Plasma 27HC levels showed opposite correlations with hepatic inflammatory indicators between female and male obese individuals. Likewise, hepatic 27HC levels oppositely correlated between female and male Npc1nih mice. Twenty-seven hydroxycholesterol injections reduced hepatic inflammation in female Npc1nih mice in contrast to male Npc1nih mice, which showed increased hepatic inflammation after 27HC injections. Furthermore, 27HC administration also oppositely affected inflammation in female and male BMDMs cultured in E2-enriched medium. Remarkably, female BMDMs showed higher ERα expression compared to male BMDMs. Our findings identify that the sex-opposed inflammatory effects of 27HC are E2-dependent and are potentially related to differences in ERα expression between females and males. Hence, the individual's sex needs to be taken into account when 27HC is employed as a therapeutic tool as well as in macrophage estrogen research in general. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

No influence of sarcopenia on survival of ovarian cancer patients in a prospective validation study.

OBJECTIVE: Decrease in skeletal muscle index (SMI) during neoadjuvant chemotherapy (NACT) has been associated with worse outcome in patients with advanced ovarian cancer. To validate these findings, we tested if a decrease in SMI was a prognostic factor for a homogenous cohort of patients who received NACT in the randomized phase 3 OVHIPEC-trial. METHODS: CT-scans were performed at baseline and after two cycles of neoadjuvant chemotherapy in stage III ovarian cancer patients. The SMI (skeletal muscle area in cm2 divided by body surface area in m2) was calculated using SliceOMatic software. The difference in SMI between both CT-scans (ΔSMI) was calculated. Cox-regression analyses were performed to analyze the independent effect of a difference in SMI (ΔSMI) on outcome. Log-rank tests were performed to plot recurrence-free (RFS) and overall survival (OS). The mean number of adverse events per patient were compared between groups using t-tests. RESULTS: Paired CT-scans were available for 212 out of 245 patients (87%). Thirty-four of 74 patients (58%) in the group with a decrease in ΔSMI and 73 of 138 of the patients (53%) in the group with stable/increase in ΔSMI had died. Median RFS and OS did not differ significantly (p = 0.297 and p = 0.764) between groups. Patients with a decrease in SMI experienced more pre-operative adverse events, and more grade 3-4 adverse events. CONCLUSION: Decreased SMI during neoadjuvant chemotherapy was not associated with worse outcome in patients with stage III ovarian cancer included in the OVHIPEC-trial. However, a strong association between decreasing SMI and adverse events was found.

The endothelial function biomarker soluble E-selectin is associated with nonalcoholic fatty liver disease.

BACKGROUND & AIMS: Plasma soluble E-selectin (sE-selectin) is a frequently used biomarker of systemic endothelial dysfunction. The present study explored the relationship between nonalcoholic fatty liver disease (NAFLD) and plasma sE-selectin levels. METHODS: Expression of E-selectin in liver, visceral adipose tissue (VAT) and muscle was studied in relation to plasma sE-selectin in severely obese individuals (n = 74). The course of hepatic E-selectin expression in relation to hepatic steatosis and inflammation was examined in C57BL/6J LDLR-/- mice on a Western-type diet. The relationship between biomarkers of NAFLD, that is, plasma aminotransferase (ALT) and NAFLD susceptibility genes (rs738409 [PNPLA3] and rs1260326 [GCKR]), and plasma sE-selectin was studied in the combined CODAM (n = 571) and Hoorn (n = 694) studies. RESULTS: E-selectin expression in liver, not VAT or muscle, was associated with plasma sE-selectin in severely obese individuals (ß = 0.26; 95% CI: 0.05-0.47). NAFLD severity was associated with hepatic E-selectin expression (P = .02) and plasma sE-selectin (P = .003). LDLR-/- mice on a Western-type diet displayed increased hepatic E-selectin expression that followed the same course as hepatic inflammation, but not steatosis. In the CODAM study, plasma ALT was associated with plasma sE-selectin, independent of potential confounders (ß = 0.25; 95% CI: 0.16-0.34). Both rs738409 and rs1260326 were associated with higher plasma sE-selectin in the combined CODAM and Hoorn studies (P = .01 and P = .004 respectively). CONCLUSIONS: NAFLD and related markers are associated with higher expression of hepatic E-selectin and higher levels of plasma sE-selectin. Further studies are required to investigate the role of E-selectin in the pathogenesis of NAFLD and the applicability of sE-selectin as a plasma biomarker of NAFLD/NASH.

Low thoracic muscle radiation attenuation is associated with postoperative pneumonia following partial hepatectomy for colorectal metastasis.

BACKGROUND: Low skeletal muscle radiation attenuation (SM-RA) is indicative of myosteatosis and diminished muscle function. It is predictive of poor outcome following oncological surgery in several cancer types. Postoperative pneumonia is a known risk factor for increased postoperative mortality. We hypothesized that low SM-RA of the respiratory muscles at the 4th thoracic-vertebra (T4) is associated with postoperative pneumonia following liver surgery. METHODS: Postoperative pneumonia was identified using prospective infection control data. Computed tomography body composition analysis was performed at the L3-and T4 level to determine SM-RA. Body composition variables were corrected for confounders and related to postoperative pneumonia and admission time by multivariable logistic regression. RESULTS: Body composition analysis of 180 patients was performed. Twenty-one patients developed postoperative pneumonia (11.6%). Multivariable analysis showed that low T4 SM-RA as well as low L3 SM-RA were significantly associated with postoperative pneumonia (OR 3.65, 95% CI 1.41-9.49, p < 0.01) and (OR 3.22, 95% CI 1.20-8.61, p = 0.02, respectively). CONCLUSION: Low SM-RA at either the L3-or T4-level is associated with a higher risk of postoperative pneumonia following CLRM resection.

The Clinical Link between Human Intestinal Microbiota and Systemic Cancer Therapy.

Clinical interest in the human intestinal microbiota has increased considerably. However, an overview of clinical studies investigating the link between the human intestinal microbiota and systemic cancer therapy is lacking. This systematic review summarizes all clinical studies describing the association between baseline intestinal microbiota and systemic cancer therapy outcome as well as therapy-related changes in intestinal microbiota composition. A systematic literature search was performed and provided 23 articles. There were strong indications for a close association between the intestinal microbiota and outcome of immunotherapy. Furthermore, the development of chemotherapy-induced infectious complications seemed to be associated with the baseline microbiota profile. Both chemotherapy and immunotherapy induced drastic changes in gut microbiota composition with possible consequences for treatment efficacy. Evidence in the field of hormonal therapy was very limited. Large heterogeneity concerning study design, study population, and methods used for analysis limited comparability and generalization of results. For the future, longitudinal studies investigating the predictive ability of baseline intestinal microbiota concerning treatment outcome and complications as well as the potential use of microbiota-modulating strategies in cancer patients are required. More knowledge in this field is likely to be of clinical benefit since modulation of the microbiota might support cancer therapy in the future.

Adipose tissue macrophages induce hepatic neutrophil recruitment and macrophage accumulation in mice.

OBJECTIVE: Obesity is a risk factor for non-alcoholic steatohepatitis (NASH). This risk has been attributed to visceral adipose tissue (vAT) expansion associated with increased proinflammatory mediators. Accumulation of CD11c+ proinflammatory adipose tissue macrophages (ATM) is an important driver of vAT inflammation. We investigated the role of ATMs in hepatic inflammation during NASH development. DESIGN: vAT isolated from lean, obese or ATM-depleted (using clodronate liposomes) obese mice was transplanted to lean ldlr-/- acceptor mice. Systemic and hepatic inflammation was assessed either after 2 weeks on standard chow or after 8 weeks on high cholesterol diet (HCD) to induce NASH. RESULTS: Transplanting donor vAT from obese mice increased HCD-induced hepatic macrophage content compared with lean-transplanted mice, worsening liver damage. ATM depletion prior to vAT transplantation reduced this increased hepatic macrophage accumulation. On chow, vAT transplantation induced a more pronounced increase in circulating and hepatic neutrophil numbers in obese-transplanted than lean-transplanted mice, while ATM depletion prior to vAT transplantation reversed this effect. Microarray analysis of fluorescence-activated cell sorting of CD11c+ and CD11c- macrophages isolated from donor adipose tissue showed that obesity resulted in enhanced expression of neutrophil chemotaxis genes specifically in CD11c+ ATMs. Involvement of the neutrophil chemotaxis proteins, CXCL14 and CXCL16, was confirmed by culturing vAT. In humans, CD11c expression in vAT of obese individuals correlated with vAT expression of neutrophil chemotactic genes and with hepatic expression of neutrophil and macrophage marker genes. CONCLUSION: ATMs from obese vAT induce hepatic macrophage accumulation during NASH development, possibly by enhancing neutrophil recruitment.

A novel human cell culture model to study visceral smooth muscle phenotypic modulation in health and disease.

Adaptation of the smooth muscle cell (SMC) phenotype is essential for homeostasis and is often involved in pathologies of visceral organs (e.g., uterus, bladder, gastrointestinal tract). In vitro studies of the behavior of visceral SMCs under (patho)-physiological conditions are hampered by a spontaneous, uncontrolled phenotypic modulation of visceral SMCs under regular tissue culture conditions. We aimed to develop a new visceral SMC culture model that allows controlled phenotypic modulation. Human uterine SMCs [ULTR and telomerase-immortalized human myometrial cells (hTERT-HM)] were grown to confluency and kept for up to 6 days on regular tissue culture surfaces or basement membrane (BM) matrix-coated surfaces in the presence of 0-10% serum. mRNA and protein expression and localization of SMC-specific phenotype markers and their transcriptional regulators were investigated by quantitative PCR, Western blotting, and immunofluorescence. Maintaining visceral SMCs confluent for 6 days increased α-smooth muscle actin (1.9-fold) and smooth muscle protein 22-α (3.1-fold), whereas smooth muscle myosin heavy chain was only slightly upregulated (1.3-fold). Culturing on a BM matrix-coated surface further increased these proteins and also markedly promoted mRNA expression of γ-smooth muscle actin (15.0-fold), smoothelin (3.5-fold), h-caldesmon (5.2-fold), serum response factor (7.6-fold), and myocardin (8.1-fold). Whereas additional serum deprivation only minimally affected contractile markers, platelet-derived growth factor-BB and transforming growth factor ß1 consistently reduced versus increased their expression. In conclusion, we present a simple and reproducible visceral SMC culture system that allows controlled phenotypic modulation toward both the synthetic and the contractile phenotype. This may greatly facilitate the identification of factors that drive visceral SMC phenotypic changes in health and disease.

Mitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression.

Nonalcoholic fatty liver disease (NAFLD) comprises fat-accumulating conditions within hepatocytes that can cause severe liver damage and metabolic comorbidities. Studies suggest that mitochondrial dysfunction contributes to its development and progression and that the hepatic lipidome changes extensively in obesity and in NAFLD. To gain insight into the relationship between lipid metabolism and disease progression through different stages of NAFLD, we performed lipidomic analysis of plasma and liver biopsy samples from obese patients with nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) and from those without NAFLD. Congruent with earlier studies, hepatic lipid levels overall increased with NAFLD. Lipid species that differed with NAFLD severity were related to mitochondrial dysfunction; specifically, hepatic cardiolipin and ubiquinone accumulated in NAFL, and levels of acylcarnitine increased with NASH. We propose that increased levels of cardiolipin and ubiquinone may help to preserve mitochondrial function in early NAFLD, but that mitochondrial function eventually fails with progression to NASH, leading to increased acylcarnitine. We also found a negative association between hepatic odd-chain phosphatidylcholine and NAFLD, which may result from mitochondrial dysfunction-related impairment of branched-chain amino acid catabolism. Overall, these data suggest a close link between accumulation of specific hepatic lipid species, mitochondrial dysfunction, and the progression of NAFLD.

Spatial Systems Lipidomics Reveals Nonalcoholic Fatty Liver Disease Heterogeneity.

Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery ( n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component-linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.

A liver-specific long noncoding RNA with a role in cell viability is elevated in human nonalcoholic steatohepatitis.

Hepatocyte apoptosis in nonalcoholic steatohepatitis (NASH) can lead to fibrosis and cirrhosis, which permanently damage the liver. Understanding the regulation of hepatocyte apoptosis is therefore important to identify therapeutic targets that may prevent the progression of NASH to fibrosis. Recently, increasing evidence has shown that long noncoding (lnc) RNAs are involved in various biological processes and that their dysregulation underlies a number of complex human diseases. By performing gene expression profiling of 4,383 lncRNAs in 82 liver samples from individuals with NASH (n = 48), simple steatosis but no NASH (n = 11), and healthy controls (n = 23), we discovered a liver-specific lncRNA (RP11-484N16.1) on chromosome 18 that showed significantly elevated expression in the liver tissue of NASH patients. This lncRNA, which we named lnc18q22.2 based on its chromosomal location, correlated with NASH grade (r = 0.51, P = 8.11 × 10-7 ), lobular inflammation (r = 0.49, P = 2.35 × 10-6 ), and nonalcoholic fatty liver disease activity score (r = 0.48, P = 4.69 × 10-6 ). The association of lnc18q22.2 to liver steatosis and steatohepatitis was replicated in 44 independent liver biopsies (r = 0.47, P = 0.0013). We provided a genetic structure of lnc18q22.2 showing an extended exon 2 in liver. Knockdown of lnc18q22.2 in four different hepatocyte cell lines resulted in severe phenotypes ranging from reduced cell growth to lethality. This observation was consistent with pathway analyses of genes coexpressed with lnc18q22.2 in human liver or affected by lnc18q22.2 knockdown. CONCLUSION: We identified an lncRNA that can play an important regulatory role in liver function and provide new insights into the regulation of hepatocyte viability in NASH. (Hepatology 2017;66:794-808).

Myosteatosis predicts survival after surgery for periampullary cancer: a novel method using MRI.

BACKGROUND: Myosteatosis, characterized by inter- and intramyocellular fat deposition, is strongly related to poor overall survival after surgery for periampullary cancer. It is commonly assessed by calculating the muscle radiation attenuation on computed tomography (CT) scans. However, since magnetic resonance imaging (MRI) is replacing CT in routine diagnostic work-up, developing methods based on MRI is important. We developed a new method using MRI-muscle signal intensity to assess myosteatosis and compared it with CT-muscle radiation attenuation. METHODS: Patients were selected from a prospective cohort of 236 surgical patients with periampullary cancer. The MRI-muscle signal intensity and CT-muscle radiation attenuation were assessed at the level of the third lumbar vertebra and related to survival. RESULTS: Forty-seven patients were included in the study. Inter-observer variability for MRI assessment was low (R2 = 0.94). MRI-muscle signal intensity was associated with short survival: median survival 9.8 (95%-CI: 1.5-18.1) vs. 18.2 (95%-CI: 10.7-25.8) months for high vs. low intensity, respectively (p = 0.038). Similar results were found for CT-muscle radiation attenuation (low vs. high radiation attenuation: 10.8 (95%-CI: 8.5-13.1) vs. 15.9 (95%-CI: 10.2-21.7) months, respectively; p = 0.046). MRI-signal intensity correlated negatively with CT-radiation attenuation (r=-0.614, p < 0.001). CONCLUSIONS: Myosteatosis may be adequately assessed using either MRI-muscle signal intensity or CT-muscle radiation attenuation.

Impact of chemotherapy-associated liver injury on tumour regression grade and survival in patients with colorectal liver metastases.

BACKGROUND: An inverse relation between chemotherapy-associated liver injury (CALI) and tumour response to chemotherapy has been reported. The aim was to validate these findings, and further investigate the impact of CALI on survival in patients who underwent partial hepatectomy for colorectal liver metastases (CRLM). METHODS: Patients who received neoadjuvant chemotherapy and underwent partial hepatectomy for CRLM between 2008 and 2014 were included. Liver and tumour specimens were histologically examined for CALI (steatosis, steatohepatitis, sinusoidal dilatation [SD], nodular regeneration) and tumour regression grade (TRG). TRG 1-2 was defined as complete tumour response. RESULTS: 166 consecutive patients were included with a median survival of 30 and 44 months for recurrence-free and overall survival, respectively. Grade 2-3 SD was found in 44 (27%) and TRG 1-2 was observed in 33 (20%) patients. Of studied CALI, only grade 2-3 SD was associated with increased TRG 3-5 (odds ratio 3.99, 95% CI 1.17-13.65, p = 0.027). CALI was not significantly related to survival. TRG 1-2 was associated with prolonged recurrence-free (hazard ratio 0.47, 95% CI 0.25-0.89, p = 0.020) and overall survival (hazard ratio 0.35, 95% CI 0.18-0.68, p = 0.002). CONCLUSION: CALI was not directly related to survival. CALI was, however, associated with diminished complete tumour response, and diminished complete tumour response, in turn, was associated with decreased survival.

Human splanchnic amino-acid metabolism.

Plasma levels of several amino acids are correlated with metabolic dysregulation in obesity and type 2 diabetes. To increase our understanding of human amino-acid metabolism, we aimed to determine splanchnic interorgan amino-acid handling. Twenty patients planned to undergo a pylorus preserving pancreatico-duodenectomy were included in this study. Blood was sampled from the portal vein, hepatic vein, superior mesenteric vein, inferior mesenteric vein, splenic vein, renal vein, and the radial artery during surgery. The difference between arterial and venous concentrations of 21 amino acids was determined using liquid chromatography as a measure of amino-acid metabolism across a given organ. Whereas glutamine was significantly taken up by the small intestine (121.0 ± 23.8 µmol/L; P < 0.0001), citrulline was released (-36.1 ± 4.6 µmol/L; P < 0.0001). This, however, was not seen for the colon. Interestingly, the liver showed a small, but a significant uptake of citrulline from the circulation (4.8 ± 1.6 µmol/L; P = 0.0138) next to many other amino acids. The kidneys showed a marked release of serine and alanine into the circulation (-58.0 ± 4.4 µmol/L and -61.8 ± 5.2 µmol/L, P < 0.0001), and a smaller, but statistically significant release of tyrosine (-12.0 ± 1.3 µmol/L, P < 0.0001). The spleen only released taurine (-9.6 ± 3.3 µmol/L; P = 0.0078). Simultaneous blood sampling in different veins provides unique qualitative and quantitative information on integrative amino-acid physiology, and reveals that the well-known intestinal glutamine-citrulline pathway appears to be functional in the small intestine but not in the colon.

Plasma cholesteryl ester transfer protein is predominantly derived from Kupffer cells.

UNLABELLED: The role of Kupffer cells (KCs) in the pathophysiology of the liver has been firmly established. Nevertheless, KCs have been underexplored as a target for diagnosis and treatment of liver diseases owing to the lack of noninvasive diagnostic tests. We addressed the hypothesis that cholesteryl ester transfer protein (CETP) is mainly derived from KCs and may predict KC content. Microarray analysis of liver and adipose tissue biopsies, obtained from 93 obese subjects who underwent elective bariatric surgery, showed that expression of CETP is markedly higher in liver than adipose tissue. Hepatic expression of CETP correlated strongly with that of KC markers, and CETP messenger RNA and protein colocalized specifically with KCs in human liver sections. Hepatic KC content as well as hepatic CETP expression correlated strongly with plasma CETP concentration. Mechanistic and intervention studies on the role of KCs in determining the plasma CETP concentration were performed in a transgenic (Tg) mouse model expressing human CETP. Selective elimination of KCs from the liver in CETP Tg mice virtually abolished hepatic CETP expression and largely reduced plasma CETP concentration, consequently improving the lipoprotein profile. Conversely, augmentation of KCs after Bacille-Calemette-Guérin vaccination largely increased hepatic CETP expression and plasma CETP. Also, lipid-lowering drugs fenofibrate and niacin reduced liver KC content, accompanied by reduced plasma CETP concentration. CONCLUSIONS: Plasma CETP is predominantly derived from KCs, and plasma CETP level predicts hepatic KC content in humans.

Protein-Bound Plasma Nε-(Carboxymethyl)lysine Is Inversely Associated With Central Obesity and Inflammation and Significantly Explain a Part of the Central Obesity-Related Increase in Inflammation: The Hoorn and CODAM Studies.

OBJECTIVE: Adipose tissue inflammation contributes to the development of complications, such as insulin resistance and type 2 diabetes mellitus. We previously reported that plasma levels of N(ε)-(carboxymethyl)lysine (CML) were decreased in obese subjects resulting from CML accumulation in adipose tissue and that this CML accumulation plays an important role in adipose tissue inflammation. The objective of this study is to investigate associations between obesity (body mass index, waist circumference, and trunk fat mass), plasma CML (as an inversely correlated marker of CML accumulation in adipose tissue), and low-grade inflammation (LGI) in a large sample of individuals whose weight status ranged from normal to morbid obesity. APPROACH AND RESULTS: We studied 1270 individuals of the Cohort on Diabetes and Atherosclerosis Maastricht Study and Hoorn Study, in whom protein-bound CML levels were measured by UPLC-Tandem MS (ultra performance liquid chromatography-tandem mass spectrometry), and 6 inflammatory markers were measured with multiarrays. These inflammatory markers were compiled into an LGI score. Multiple linear regression, adjusted for covariates, showed that (1) waist circumference was inversely associated with protein-bound CML plasma levels (standardized regression coefficient [ß]=-0.357 [95% confidence interval: -0.414; -0.301]); (2) protein-bound CML was inversely associated with LGI score (ß=-0.073 [-0.130;-0.015]); and (3) the association between waist circumference and LGI (ß=0.262 [0.203;0.321]) was attenuated after adjustment for protein-bound CML plasma levels and other potential mediators (to ß=0.202 [0.138;0.266]), with CML explaining the greatest portion of the attenuation (≈12%). Further analysis with dual-energy X-ray absorptiometry measures of body composition confirmed a strong inverse association of fat mass preferentially accumulated in the trunk with protein-bound CML plasma levels, significantly explaining ≈21% of the trunk fat-LGI association. CONCLUSIONS: Obesity, in particular central obesity, is characterized by greater levels of LGI but by lower levels of circulating CML; the latter significantly explaining a portion of the positive association between central obesity and inflammation.

Navigating through metaproteomics data: a logbook of database searching.

Metaproteomic research involves various computational challenges during the identification of fragmentation spectra acquired from the proteome of a complex microbiome. These issues are manifold and range from the construction of customized sequence databases, the optimal setting of search parameters to limitations in the identification search algorithms themselves. In order to assess the importance of these individual factors, we studied the effect of strategies to combine different search algorithms, explored the influence of chosen database search settings, and investigated the impact of the size of the protein sequence database used for identification. Furthermore, we applied de novo sequencing as a complementary approach to classic database searching. All evaluations were performed on a human intestinal metaproteome dataset. Pyrococcus furiosus proteome data were used to contrast database searching of metaproteomic data to a classic proteomic experiment. Searching against subsets of metaproteome databases and the use of multiple search engines increased the number of identifications. The integration of P. furiosus sequences in a metaproteomic sequence database showcased the limitation of the target-decoy-controlled false discovery rate approach in combination with large sequence databases. The selection of varying search engine parameters and the application of de novo sequencing represented useful methods to increase the reliability of the results. Based on our findings, we provide recommendations for the data analysis that help researchers to establish or improve analysis workflows in metaproteomics.

Colonic metaproteomic signatures of active bacteria and the host in obesity.

Obesity is associated with the intestinal microbiota in humans but the underlying mechanisms are yet to be fully understood. Our previous phylogenetic study showed that the faecal microbiota profiles of nonobese versus obese and morbidly obese individuals differed. Here, we have extended this analysis with a characterization of the faecal metaproteome, in order to detect differences at a functional level. Proteins were extracted from crude faecal samples of 29 subjects, separated by 1D gel electrophoresis and characterized using RP LC-MS/MS. The peptide data were analyzed in database searches with two complementary algorithms, OMSSA and X!Tandem, to increase the number of identifications. Evolutionary genealogy of genes: nonsupervised orthologous groups (EggNOG) database searches resulted in the functional annotation of over 90% of the identified microbial and human proteins. Based on both bacterial and human proteins, a clear clustering of obese and nonobese samples was obtained that exceeded the phylogenetic separation in dimension. Moreover, integration of the metaproteomics and phylogenetic datasets revealed notably that the phylum Bacteroidetes was metabolically more active in the obese than nonobese subjects. Finally, significant correlations between clinical measurements and bacterial gene functions were identified. This study emphasizes the importance of integrating data of the host and microbiota to understand their interactions.

Nε-(carboxymethyl)lysine-receptor for advanced glycation end product axis is a key modulator of obesity-induced dysregulation of adipokine expression and insulin resistance.

OBJECTIVE: Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N(ε)-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML-RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. APPROACH AND RESULTS: In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance. CONCLUSIONS: RAGE-mediated CML accumulation in adipose tissue and the activation of the CML-RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.

Unraveling the regulatory mechanisms underlying tissue-dependent genetic variation of gene expression.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3' and 5' untranslated regions (P = 1.84×10(-5) and 4.7×10(-4), respectively) and SNPs that are synonymous-coding (P = 9.9×10(-4)). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10(-10)). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.