This protocol outlines a translational lipidomic approach to discover lipid biomarkers that could predict morphometric body and histological organ measurements (e.g., weight and adiposity gains) during specific stages of life (e.g., early life). We describe procedures ranging from animal experimentation and histological analyses to downstream analytical steps through lipid profiling, both in mice and humans. This protocol represents a reliable and versatile approach to translate and validate candidate lipid biomarkers from animal models to a human cohort.

SCOPE:Milk fat globule membrane (MFGM) is an essential component of milk. Bovine MFGM (bMFGM) has been shown to support cognitive development and increase relative concentrations of serum phospholipids. This study investigates bioavailability of bMFGM components after oral administration in two preclinical models to explore whether dietary bMFGM induces parallel changes to plasma and brain lipidomes.METHODS AND RESULTS:Transgenic APOE*3.Leiden mice (n = 18 per group) and Sprague-Dawley rats (n = 12 per group) are fed bMFGM-enriched (MFGM+) or Control diet, followed by phospholipid profile-determination in plasma, hippocampus, and prefrontal cortex tissue by targeted mass spectrometry. Multivariate analysis of lipidomic profiles demonstrates a separation between MFGM+ and Control plasma across rodents. In plasma, sphingomyelins contributed the most to the separation of lipid patterns among both models, where three sphingomyelins (d18:1/14:0, d18:1/23:0, d18:1/23:1[9Z]) are consistently higher in the circulation of MFGM+ groups. A similar trend is observed in rat prefrontal cortex, although no significant separation of the brain lipidome is demonstrated.CONCLUSION:bMFGM-enriched diet alters plasma phospholipid composition in rodents, predominantly increasing sphingomyelin levels in the systemic circulation with similar, but non-significant, trends in central brain regions. These changes may contribute to the beneficial effects of bMFGM on neurodevelopment during early life.

The development of obesity is characterized by the metabolic overload of tissues and subsequent organ inflammation. The health effects of krill oil (KrO) on obesity-associated inflammation remain largely elusive, because long-term treatments with KrO have not been performed to date. Therefore, we examined the putative health effects of 28 weeks of 3% (w/w) KrO supplementation to an obesogenic diet (HFD) with fat derived mostly from lard. The HFD with KrO was compared to an HFD control group to evaluate the effects on fatty acid composition and associated inflammation in epididymal white adipose tissue (eWAT) and the liver during obesity development. KrO treatment increased the concentrations of EPA and DHA and associated oxylipins, including 18-HEPE, RvE2 and 14-HDHA in eWAT and the liver. Simultaneously, KrO decreased arachidonic acid concentrations and arachidonic-acid-derived oxylipins (e.g., HETEs, PGD2, PGE2, PGF2α, TXB2). In eWAT, KrO activated regulators of adipogenesis (e.g., PPARγ, CEBPα, KLF15, STAT5A), induced a shift towards smaller adipocytes and increased the total adipocyte numbers indicative for hyperplasia. KrO reduced crown-like structures in eWAT, and suppressed HFD-stimulated inflammatory pathways including TNFα and CCL2/MCP-1 signaling. The observed eWAT changes were accompanied by reduced plasma leptin and increased plasma adiponectin levels over time, and improved insulin resistance (HOMA-IR). In the liver, KrO suppressed inflammatory signaling pathways, including those controlled by IL-1β and M-CSF, without affecting liver histology. Furthermore, KrO deactivated hepatic REL-A/p65-NF-κB signaling, consistent with increased PPARα protein expression and a trend towards an increase in IkBα. In conclusion, long-term KrO treatment increased several anti-inflammatory PUFAs and oxylipins in WAT and the liver. These changes were accompanied by beneficial effects on general metabolism and inflammatory tone at the tissue level. The stimulation of adipogenesis by KrO allows for safe fat storage and may, together with more direct PPAR-mediated anti-inflammatory mechanisms, attenuate inflammation.

The majority of intestinal in vitro screening models use cell lines that do not reflect the complexity of the human intestinal tract and hence often fail to accurately predict intestinal drug absorption. Tissue explants have intact intestinal architecture and cell type diversity, but show short viability in static conditions. Here, we present a medium throughput microphysiological system, Intestinal Explant Barrier Chip (IEBC), that creates a dynamic microfluidic microenvironment and prolongs tissue viability. Using a snap fit mechanism, we successfully incorporated human and porcine colon tissue explants and studied tissue functionality, integrity and viability for 24 hours. With a proper distinction of transcellular over paracellular transport (ratio >2), tissue functionality was good at early and late timepoints. Low leakage of FITC-dextran and preserved intracellular lactate dehydrogenase levels indicate maintained tissue integrity and viability, respectively. From a selection of low to high permeability drugs, 6 out of 7 properly ranked according to their fraction absorbed. In conclusion, the IEBC is a novel screening platform benefitting from the complexity of tissue explants and the flow in microfluidic chips.

Objective: Obesity is one of the greatest risk factors for osteoarthritis (OA) and evidence is accumulating that inflammatory mediators and innate immunity play an important role. The infrapatellar fat pad (IPFP) could be a potential local source of inflammatory mediators in the knee. Here, we combine surgical joint damage with high-fat feeding in mice to investigate inflammatory responses in the IPFP during OA development. Design: Mice (n = 30) received either a low-fat diet (LFD), high-fat diet (HFD) for 18 weeks or switched diets (LFD > HFD) after 10 weeks. OA was induced by surgical destabilization of the medial meniscus (DMM), contralateral knees served as sham controls. An additional HFD-only group (n = 15) received no DMM. Results: The most pronounced inflammation, characterized by macrophage crown-like structures (CLS), was found in HFD + DMM mice, CLS increased compared to HFD only (mean difference = 7.26, 95%CI [1.52-13.0]) and LFD + DMM (mean difference = 6.35, 95%CI [0.53-12.18). The M1 macrophage marker iNOS increased by DMM (ratio = 2.48, 95%CI [1.37-4.50]), while no change in M2 macrophage marker CD206 was observed. Fibrosis was minimal by HFD alone, but in combination with DMM it increased with 23.45% (95%CI [13.67-33.24]). Conclusions: These findings indicate that a high-fat diet alone does not trigger inflammation or fibrosis in the infrapatellar fat pad, but in combination with an extra damage trigger, like DMM, induces inflammation and fibrosis in the infrapatellar fat pad. These data suggest that HFD provides a priming effect on the infrapatellar fat pad and that combined actions bring the joint in a metabolic state of progressive OA. (C) 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

•Mechanisms of cardiovascular disease are complex and multifactorial, driven by genetic variants.•Attractive drug targets require a substantial body of mechanistic and clinical evidence.•Drug target discovery in CVD is only possible through multidisciplinary collaboration.•To maximise impact, collaboration between independent research consortia is advised.

Research consortia in Europe often compete with each other for skills, human and technical resources and, eventually, recognition of the scientific impact of their work. In response to the same EU Horizon2020 call, we received funding for our research project proposals to identify and validate novel drug targets for cardiovascular disease treatment. Each consortium followed a unique and independent research strategy. However, as coordinators of these consortia we envisioned we could increase impact, outcomes and efficiency by intensifying our interaction. At an agreed stage during our projects we chose to share our knowledge, vision and ideas. In this paper we present what we learned, in the hope that future consortia will see the benefits of this approach.

Purpose: Obesity is one of the highest risk factors for osteoarthritis (OA) and evidence is rising that inflammatory mediators play an important role in obesity-related OA. A potential local source of inflammatory mediators in the knee is the infrapatellar fat pad (IPFP) or Hoffa's fat pad, which is located within the articular capsule of the joint. Next to adipocytes, an important cell type in this fat tissue capable of producing large amounts of signaling factors, are macrophages. In the healthy situation macrophages in fat tissue consist mainly of anti-inflammatory CD206-positive M2 type macrophages. However, in obesity, fat depots throughout the body show a switch to a pro-inflammatory M1 macrophage phenotype that produces inducible nitric oxide synthase (iNOS). Adipose inflammation in obesity is reflected by an increase in adipocyte size and an increase in crown-like structures (CLS), which consist of macrophages surrounding dying adipocytes. We wondered whether an increase in inflammation is also present in the IPFP during a high-fat diet and whether this could play a role in the development of OA. Methods: Mice (C57BL/6J) were randomized into groups (n=10/group) receiving either a low-fat diet (LFD+DMM) or a high-fat diet (HFD+DMM) for a total of 18 weeks, with surgical destabilization of the medial meniscus (DMM) of the right knee at 10 weeks. The left knee served as an internal sham control. A last group of mice with an C57BL/6J background (n=15) received only a HFD for 38 weeks, without DMM surgery. All knees were processed for histology to determine the OARSI score and number of CLS. Number and size of adipocytes were determined using the ImageJ Adiposoft plug-in. Macrophage polarization was examined using a triple immunohistochemical staining against F4/80 (general pan macrophage), CD206 (M2) and iNOS (M1). Individual markers were analyzed based on images of unmixed color spectra for each chromogen, in order to determine the positive area of each marker and its co-localization with other markers. Results: The average OA score of DMM-knees in the LFD group was 13.8 vs. 19.4 in the HFD group, all sham operated knees showed minimal OA development, which was comparable to the HFD only group with an OA score of 4.3. We did not see an increase in CLS in the IPFP as a result of the high-fat diet alone. Only when OA was induced by DMM surgery in HFD-fed mice, an increase in CLS (p=0.005) was observed. In the LFD+DMM group an average of 2.5 CLS/1000 adipocytes was observed compared to 8.8 CLS/1000 adipocytes in the HFD+DMM group (Figure 1A). Remarkably, the contralateral joint in the HFD+DMM group also showed a slight increase in the amount of CLS (5.2/1000 adipocytes). Average adipocyte size in HFD+DMM group was comparable to HFD group but significantly greater compared to LFD+DMM controls (p<0.001). The pan macrophage marker F4/80 showed a positive surface area of 0.76% in the IPFP of the HFD only group of (Figure 1B). This percentage was increased to 0.97% in the HFD+DMM group and 1.00% in the LFD+DMM group, although not significantly. When we evaluated colocalization of F4/80 with macrophage phenotype markers iNOS (M1) and CD206 (M2) we observed no change in CD206-positive area between groups (Figure 1C). However, iNOS positivity within the total F4/80 area increased significantly, from 9.6% in the HFD group to 15.7% (p=0.04) and 14.8% (p=0.07) in the LFD+DMM and HFD+DMM groups, respectively (Figure 1D). Conclusions: We found that DMM-induced OA on top of high-fat feeding increased IPFP inflammation, marked by an increase in CLS. In contrast to what was expected long-term HFD feeding alone did not induce IPFP inflammation. Interestingly, when DMM was applied, the contralateral (sham) joint also showed increased signs of IPFP inflammation. In addition, we observed an increase in the pan macrophage marker F4/80 in groups that underwent DMM surgery, compared to the HFD only group. This was accompanied by a minor shift in macrophage phenotype, suggesting an inflow of M1 macrophages to the IPFP, while resident M2 macrophages are unchanged. Altogether, these findings indicate that a high-fat diet alone does not trigger inflammation in the IPFP as it does in other adipose depots in the body, but that an extra trigger, like DMM is needed before the IPFP shows any signs of inflammation.

Obesity characterized by adiposity and ectopic fat accumulation is associated with the development of non-alcoholic fatty liver disease (NAFLD). Treatments that stimulate lipid utilization may prevent the development of obesity and comorbidities. This study evaluated the potential anti-obesogenic hepatoprotective effects of combined treatment with L-carnitine and nicotinamide riboside, i.e., components that can enhance fatty acid transfer across the inner mitochondrial membrane and increase nicotinamide adenine nucleotide (NAD+) levels, which are necessary for β-oxidation and the TCA cycle, respectively. Ldlr −/−.Leiden mice were treated with high-fat diet (HFD) supplemented with L-carnitine (LC; 0.4% w/w), nicotinamide riboside (NR; 0.3% w/w) or both (COMBI) for 21 weeks. L-carnitine plasma levels were reduced by HFD and normalized by LC. NR supplementation raised its plasma metabolite levels demonstrating effective delivery. Although food intake and ambulatory activity were comparable in all groups, COMBI treatment significantly attenuated HFD-induced body weight gain, fat mass gain (−17%) and hepatic steatosis (−22%). Also, NR and COMBI reduced hepatic 4-hydroxynonenal adducts. Upstream-regulator gene analysis demonstrated that COMBI reversed detrimental effects of HFD on liver metabolism pathways and associated regulators, e.g., ACOX, SCAP, SREBF, PPARGC1B, and INSR. Combination treatment with LC and NR exerts protective effects on metabolic pathways and constitutes a new approach to attenuate HFD-induced obesity and NAFLD.

Background In this study we investigated the contribution of high-fat diet-induced metabolic overload to osteoarthritis (OA) progression originally caused by mild mechanical trauma to the mouse knee joint. We hypothesized that metabolic stress would induce a proinflammatory environment by altering systemic lipid levels and immune cell populations. Methods Twelve-week-old male C57BL/6J mice (n=20) were given a low-fat diet (LFD, 10%kcal from fat) or high-fat diet (HFD, 45%kcal from fat) for 18 weeks. OA was initiated by transecting the medial meniscotibial ligament of the right knee joint at t=10 weeks. OA severity and changes in M1/M2 polarization of synovial macrophage populations were determined in serial coronal FFPE-mounted sections. Eicosanoid levels and monocyte populations were evaluated before and after ligament transection. Results Diet-induced metabolic stress assessed by body weight, systemic cholesterol levels, and insulin resistance index was significantly higher in HFD mice. This group also showed increased cartilage damage, synovitis, and osteophyte formation compared with LFD controls. High-fat feeding elevated systemic arachidonic acid levels, which mainly resulted in increased levels of its cytochrome P450-catalysed diol metabolites 5,6-dihydroxyeicosatrienoic acid (DHET) and 8,9-DHET. High-fat feeding also triggered an increase in pro-inflammatory intermediate CD43++Ly6Cint monocytes after ligament resection. Ligament resection in addition to high-fat feeding induced increased expression of the activation marker CD11c selectively on non-classical CD43++Ly6Clow monocytes. No significant changes in synovial macrophage polarization were observed. Conclusions Metabolic stress resulted in a proinflammatory environment and aggravated injury-induced OA progression. Our results suggest that a CYP450-focused eicosanoid metabolism and activated circulating monocytes may be drivers of this metabolic stress-induced OA progression, contributing to the mechanistic understanding and potentially serving as future diagnostic and prognostic biomarkers for metabolic OA.

Objective: Human cohort studies have demonstrated a role for systemic metabolic dysfunction in osteoarthritis (OA) pathogenesis in obese patients. To explore the mechanisms underlying this metabolic phenotype of OA, we examined cartilage degradation in the knees of mice from different genetic backgrounds in which a metabolic phenotype was established by various dietary approaches. Design: Wild-type C57BL/6J mice and genetically modified mice (hCRP, LDLr-/-. Leiden and ApoE*3Leiden. CETP mice) based on C57BL/6J background were used to investigate the contribution of inflammation and altered lipoprotein handling on diet-induced cartilage degradation. High-caloric diets of different macronutrient composition (i.e., high-carbohydrate or high-fat) were given in regimens of varying duration to induce a metabolic phenotype with aggravated cartilage degradation relative to controls. Results: Metabolic phenotypes were confirmed in all studies as mice developed obesity, hypercholesteremia, glucose intolerance and/or insulin resistance. Aggravated cartilage degradation was only observed in two out of the twelve experimental setups, specifically in long-term studies in male hCRP and female ApoE*3Leiden. CETP mice. C57BL/6J and LDLr-/-. Leiden mice did not develop HFD-induced OA under the conditions studied. Osteophyte formation and synovitis scores showed variable results between studies, but also between strains and gender. Conclusions: Long-term feeding of high-caloric diets consistently induced a metabolic phenotype in various C57BL/6J (-based) mouse strains. In contrast, the induction of articular cartilage degradation proved variable, which suggests that an additional trigger might be necessary to accelerate diet-induced OA progression. Gender and genetic modifications that result in a humanized pro-inflammatory state (human CRP) or lipoprotein metabolism (human-E3L. CETP) were identified as important contributing factors. (c) 2017 Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International.

Objective: C-reactive protein (CRP) levels can be elevated in osteoarthritis (OA) patients. In addition to indicating systemic inflammation, it is suggested that CRP itself can play a role in OA development. Obesity and metabolic syndrome are important risk factors for OA and also induce elevated CRP levels. Here we evaluated in a human CRP (hCRP)-transgenic mouse model whether CRP itself contributes to the development of ` metabolic' OA. Design: Metabolic OA was induced by feeding 12-week-old hCRP-transgenic males (hCRP-tg, n = 30) and wild-type littermates (n = 15) a 45 kcal% high-fat diet (HFD) for 38 weeks. Cartilage degradation, osteophytes and synovitis were graded on Safranin O-stained histological knee joint sections. Inflammatory status was assessed by plasma lipid profiling, flow cytometric analyses of blood immune cell populations and immunohistochemical staining of synovial macrophage subsets. Results: Male hCRP-tg mice showed aggravated OA severity and increased osteophytosis compared with their wild-type littermates. Both classical and non-classical monocytes showed increased expression of CCR2 and CD86 in hCRP-tg males. HFD-induced effects were evident for nearly all lipids measured and indicated a similar low-grade systemic inflammation for both genotypes. Synovitis scores and synovial macrophage subsets were similar in the two groups. Conclusions: Human CRP expression in a background of HFD-induced metabolic dysfunction resulted in the aggravation of OA through increased cartilage degeneration and osteophytosis. Increased recruitment of classical and non-classical monocytes might be a mechanism of action through which CRP is involved in aggravating this process. These findings suggest interventions selectively directed against CRP activity could ameliorate metabolic OA development. (c) 2018 The Authors. Published by Elsevier Ltd on behalf of Osteoarthritis Research Society International. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

BACKGROUND & AIMS: The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. METHODS: A time-course study in low-density lipoprotein-receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. RESULTS: High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. CONCLUSIONS: An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre) clinical experimental time frames.

Introduction: To support the validation of novel molecular drug targets in cardiovascular disease (CVD), molecular imaging and staining technologies are essential tools to provide documentation for...

Context: Metabolic dysregulation underlies key metabolic risk factors—obesity, dyslipidemia, and dysglycemia. Objective: To uncover mechanistic links between metabolomic dysregulation and metabolic risk by testing metabolite associations with risk factors cross-sectionally and with risk factor changes over time. Design: Cross-sectional—discovery samples (n = 650; age, 36–69 years) from the Framingham Heart Study (FHS) and replication samples (n = 670; age, 61–76 years) from the BioImage Study, both following a factorial design sampled from high vs low strata of body mass index, lipids, and glucose. Longitudinal—FHS participants (n = 554) with 5–7 years of follow-up for risk factor changes. Setting: Observational studies. Participants: Cross-sectional samples with or without obesity, dysglycemia, and dyslipidemia, excluding prevalent cardiovascular disease and diabetes or dyslipidemia treatment. Age- and sex-matched by group. Interventions: None. Main Outcome Measure(s): Gas chromatography-mass spectrometry detected 119 plasma metabolites. Cross-sectional associations with obesity, dyslipidemia, and dysglycemia were tested in discovery, with external replication of 37 metabolites. Single- and multi-metabolite markers were tested for association with longitudinal changes in risk factors. Results: Cross-sectional metabolite associations were identified with obesity (n = 26), dyslipidemia (n = 21), and dysglycemia (n = 11) in discovery. Glutamic acid, lactic acid, and sitosterol associated with all three risk factors in meta-analysis (P < 4.5 × 10−4). Metabolites associated with longitudinal risk factor changes were enriched for bioactive lipids. Multi-metabolite panels explained 2.5–15.3% of longitudinal changes in metabolic traits. Conclusions: Cross-sectional results implicated dysregulated glutamate cycling and amino acid metabolism in metabolic risk. Certain bioactive lipids were associated with risk factors cross-sectionally and over time, suggesting their upstream role in risk factor progression. Functional studies are needed to validate findings and facilitate translation into treatments or preventive measures.

Consumption of starchy products with slowly digestible starch is implicated with a lower risk of metabolic diseases. Previously, it has been shown that pasta consumption resulted in a lower rate of appearance of exogenous glucose (RaE) and lower glucose clearance rate quantified with a dual isotope technique, which was in accordance with a lower insulin and GIP response after pasta intake. To get more insight in the acute metabolic consequences of the intake of products with differential rate of appearance of starch‐derived glucose, plasma metabolic profiles in 9 healthy men were analyzed after consumption of wheat bread and pasta, both enriched with wheat‐bran.Differences in postprandial responses of 134 plasma metabolites were analyzed using a GC‐MS ‐based metabolomic approach. Six samples were analyzed per subject; one baseline sample and 30, 60, 90, 120 and 180 min after consumption. A two‐way ANOVA was used to analyze the effect of time, treatment, and their interaction. Additionally, pathway analysis was performed.The observed differences occurred primarily in pathways related to protein and energy metabolism. Profiles of various amino acids had similar postprandial patterns and showed lower and attenuated responses after pasta, which could be explained by a slower absorption (parallel to the lower RaE) as well as the lower insulin response after pasta. Metabolites of glucose metabolism showed similar profiles as RaE, whereas metabolites of lipid metabolism were suppressed for a longer time after pasta intake. Remarkable higher plasma concentrations of arabinose (A) and xylose (X) after consumption of pasta were found. A and X are derived from arabinoxylans (AX), which are important components of wheat bran. The higher bioavailability of A and X coincided with a lower rate of appearance of glucose and amino acids. This is possibly due to higher viscosity of AX in the small intestine after pasta consumption, which is hindering absorption. The lower rate of glucose appearance is thus apparently not caused by slow starch digestion, but by slow absorption of starch‐derived glucose. As both food products contained the same amount of AX, different methods of processing may explain the difference in viscosity. It is speculated that the higher bioavailability of A and X after pasta is due to higher degradation of AX by small intestinal microbiota, facilitated by higher viscosity of AX after pasta. These results suggest that also wheat bran, depending of processing, may increase viscosity of the meal bolus in the small intestine and interfere with macronutrient absorption, thereby influencing postprandial glucose and insulin response.Support or Funding InformationThe project was supported by UMCG, TNO, TIFN and ZON‐MW