Johan Gerrits

Interplay between metabolic identities in the intestinal crypt supports stem cell function

The small intestinal epithelium self-renews every four or five days. Intestinal stem cells (Lgr5+ crypt base columnar cells (CBCs)) sustain this renewal and reside between terminally differentiated Paneth cells at the bottom of the intestinal crypt. Whereas the signalling requirements for maintaining stem cell function and crypt homeostasis have been well studied, little is known about how metabolism contributes to epithelial homeostasis. Here we show that freshly isolated Lgr5+ CBCs and Paneth cells from the mouse small intestine display different metabolic programs. Compared to Paneth cells, Lgr5+ CBCs display high mitochondrial activity. Inhibition of mitochondrial activity in Lgr5+ CBCs or inhibition of glycolysis in Paneth cells strongly affects stem cell function, as indicated by impaired organoid formation. In addition, Paneth cells support stem cell function by providing lactate to sustain the enhanced mitochondrial oxidative phosphorylation in the Lgr5+ CBCs. Mechanistically, we show that oxidative phosphorylation stimulates p38 MAPK activation by mitochondrial reactive oxygen species signalling, thereby establishing the mature crypt phenotype. Together, our results reveal a critical role for the metabolic identity of Lgr5+ CBCs and Paneth cells in supporting optimal stem cell function, and we identify mitochondria and reactive oxygen species signalling as a driving force of cellular differentiation.

Increased Plasma Levels of Chemoresistance-Inducing Fatty Acid 16:4(n-3) After Consumption of Fish and Fish Oil

IMPORTANCE Our research group previously identified specific endogenous platinum-induced fatty acids (PIFAs) that, in picomolar quantities, activate splenic macrophages leading to resistance to chemotherapy in mouse models. Fish oil was shown to contain the PIFA 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) and when administered to mice neutralized chemotherapy activity. OBJECTIVE Because patients with cancer frequently use fish oil supplements, we set out to determine exposure to 16:4(n-3) after intake of fish or fish oil. DESIGN, SETTING, AND PARTICIPANTS (1) In November 2011, 400 patients with cancer undergoing treatment at the University Medical Center Utrecht were surveyed to determine their use of fish oil supplements; 118 patients responded to the questionnaire (30%); (2) pharmacokinetic analysis of the 16:4(n-3) content of 6 fish oils and 4 fishes was carried out; (3) from April through November 2012, a healthy volunteer study was performed to determine 16:4(n-3) plasma levels after intake of 3 different brands of fish oil or 4 different fish species. Thirty healthy volunteers were randomly selected for the fish oil study; 20 were randomly selected for the fish study. These studies were supported by preclinical tumor experiments in mice to determine chemoresistance conducted between September 2011 and December 2012. MAIN OUTCOMES AND MEASURES (1) Rate of use of fish oil supplements among patients undergoing cancer treatment at our institution; (2) levels of 16:4(n-3) present in 3 brands of fish oil and 4 species of fish; and (3) plasma levels of 16:4(n-3) present in healthy volunteers after consuming fish oil or fish. RESULTS Eleven percent of respondents reported using omega-3 supplements. All fish oils tested contained relevant amounts of 16:4(n-3), from 0.2 to 5.7 µM. Mouse experiments showed that addition of 1 µL of fish oil to cisplatin was sufficient to induce chemoresistance, treatment having no impact on the growth rate of tumors compared with vehicle-treated controls (estimated tumor volume difference, 44.1 mm3; P > .99). When the recommended daily amount of 10 mL of fish oil was administered to healthy volunteers, rises in plasma 16:4(n-3) levels were observed, reaching up to 20 times the baseline levels. Herring and mackerel contained high levels of 16:4(n-3) in contrast to salmon and tuna. Consumption of fish with high levels of 16:4(n-3) also resulted in elevated plasma levels of 16:4(n-3). CONCLUSIONS AND RELEVANCE All tested fish oils and herring and mackerel fishes contained relevant levels of fatty acid 16:4(n-3), a fatty acid with chemotherapy-negating effects in preclinical models. After ingestion of these fish oils or fishes, 16:4(n-3) was rapidly taken up in the plasma of human volunteers. Until further data become available, fish oil and fish containing high levels of 16:4(n-3) may best be avoided on the days surrounding chemotherapy.

FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression

FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α‐ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2‐hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion.

CSF d-serine concentrations are similar in Alzheimer's disease, other dementias, and elderly controls

Cerebrospinal fluid (CSF) levels of d-serine were recently reported as a potential new biomarker for Alzheimers disease (AD), showing a perfect distinction between AD patients and healthy controls. In this study, we aimed to confirm these results and extend these previous findings to dementia with Lewy bodies and frontotemporal dementia. d-Serine levels in CSF of 29 AD patients, 8 dementia with Lewy bodies patients, 14 frontotemporal dementia patients, and 28 nondemented controls were measured using ultra-high-performance liquid chromatography-tandem mass spectrometry. In contrast to previous findings, in our study CSF d-serine levels were only slightly increased in AD patients compared with controls. CSF d-serine in AD did not differ from other dementias and was also not correlated to mini-mental state examination-scores. Owing to the large overlap of d-serine levels, we conclude that CSF d-serine is neither a suitable biomarker for AD nor for cognitive decline.

Development and validation of a quantitative LC–tandem MS assay for hexadeca-4,7,10,13-tetraenoic acid in human and mouse plasma

Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid-liquid extraction, solid phase clean-up and LC-MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1-100 nmol/L (r(2)>0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98-106% and 100-108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at -80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance.

Vitamin B6 is essential for serine de novo biosynthesis

Pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation.

Abstract B32: FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression

FOXO transcription factors are considered bona fide tumor suppressors, however recent studies showed FOXOs are also required for tumor survival. Here we identify FOXOs as transcriptional regulators of IDH1. FOXOs regulate IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise regulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation likely by repressing differentiation. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion. Furthermore these data suggest a role for FOXOs in maintaining the de-differentiated state by regulating α-ketoglutarate and/or 2-hydroxyglutarate levels. Indeed, in models of differentiation (e.g. 3T3-L1 differentiation towards adipocytes) loss of FOXO, as well as loss of IDH1 expression repress differentiation and we are currently further exploring the molecular underpinnings. Note: This abstract was not presented at the conference. Citation Format: Paraskevi Charitou, Maria Rodriguez-Colman, Johan Gerrits, Nanda Verhoeven-Duif, Boudewijn Burgering. FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B32.

Abstract 840: High levels of hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)) in fish oil induce resistance to chemotherapy in vivo

Environment-mediated resistance to chemotherapy is emerging as a cause of treatment failure. We recently discovered that two fatty acids, 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), induced resistance to a broad spectrum of chemotherapeutics in mice. These fatty acids were produced by mesenchymal stem cells upon platinum stimulation, and were therefore called platinum-induced fatty acids (PIFAs; Roodhart et al, Cancer Cell 2011). Fish oil, a complex, non-standardized mixture of fatty acids, was also shown to contain high levels of these PIFAs. Fish oil supplements are commonly used by cancer patients due to their perceived positive health effects. First, to determine the percentage of cancer patients taking fish oil supplements, a questionnaire was handed out at the UMC Utrecht medical oncology department. In an interim analysis, 12% of responders (11/90) were found to use fish oil supplements, the majority of whom continued supplementation during chemotherapy (82%). Second, we analyzed 5 commercially available fish oils for 16:4(n-3) content by ultra-high pressure liquid chromatography coupled to Orbitrap mass spectrometry. Even though 16:4(n-3) values differed, all tested fish oils contained significant amounts of the resistance-inducing PIFA. Concentrations ranged between 0.1 - 6.5 μM 16:4(n-3). We previously determined that as little as 25 nM purified 16:4(n-3) was sufficient to induce chemoresistance in mice. Here, we show that fish oils abundant in 16:4(n-3) interfered with chemotherapy actions in BALB/c mice bearing subcutaneous C26 tumors. Fish oil alone, administered by oral gavage, did not alter tumor volume compared to untreated mice. However, a single oral administration of each of three different fish oils together with intraperitoneal cisplatin therapy induced potent chemoresistance (tumor volume on day 4 after fish oil and chemotherapy was 222%, 223% and 246% compared to tumor volume after cisplatin alone). These results were confirmed in LLC-bearing C57Bl/6 mice. Finally, the fish oil containing the highest 16:4(n-3) level (6.5 μM) was tested for dose dependency by oral administration of 100, 10, 1 and 0.1 αl. As little as 1 αl of this fish oil was sufficient to induce resistance to cisplatin. On day 4, tumor volumes were 118±44 mm3 for vehicle-treated mice, 41±16 mm3 for cisplatin-treated and 94±63 mm3 for mice treated with cisplatin + 1 αl of fish oil (p 0.02). Concluding, commercially available fish oils contained variable levels of resistance-inducing PIFA 16:4(n-3). In clinically relevant doses, fish oil induced resistance to chemotherapy in mice. This implicates that fish oil use can interfere with chemotherapy outcome. 12% of cancer patients use fish oil, urging the need for clinical studies to determine the effects of fish oil intake on PIFA concentrations in human plasma, and on response to chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 840. doi:1538-7445.AM2012-840