Petterson, Xuan-Mai

We show how switching from a regular diet to an HCHF increases cholesterol absorption in patients with normal weight and obesity. The decrease in cholesterol absorption from an HCHF to an LCLF, on the other hand, was only seen in normal-weight controls, underlining the importance of body weight in this regulation. In addition, changes in caloric and fat content had an immediate and direct effect on hepatic bile acid production.

Abstract Introduction: Altered cellular metabolism is a hallmark of every cancer cell. Aerobic glycolysis ("The Warburg Effect") is one of the earliest recognized metabolic abnormalities in cancer cells whereby extracellular glucose is preferentially metabolized and eventually processed to generate lactate and energy in the form of ATP before the former is released extracellularly, irrespective of oxygen availability. While extracellular lactate produced and released from cancer cells has traditionally been considered a waste metabolic by-product, recent understanding of cell metabolism suggests that it can also serve as a primary metabolic fuel for cancer cells via uptake by monocarboxylate transporters (MCTs). Our goal was to evaluate this "Reverse Warburg Effect" phenomenon in multiple myeloma (MM) cells and determine if it can be exploited for therapeutic purposes. Methods: All HMCLs, MM1S, RPMI-8226 and U266, were grown in RPMI-1640 cell culture medium containing 11 mM glucose and supplemented with 10% dialyzed fetal bovine serum (FBS) and 2 mM Glutamine. Primary MM cells were extracted using magnetic bead CD138 positive selection from MM patient bone marrow aspirates. For 13C-labeling experiments, HMCLs and primary MM cells were suspended in RPMI-1640 cell culture media containing 13C-labeled isotopes. Isotopomer analysis of glycolytic and tricarboxylic acid (TCA) cycle metabolites from HMCL and primary MM cell pellets was performed using Agilent Technologies 5975C gas chromatography-mass spectrometry. Small molecule inhibitors, AZD3965 and syrosingopine, were purchased from Selleck Chemicals and Sigma respectively. Cellular viability and proliferation were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide (MTT) and CCK-8 assays respectively. MCT-1 and MCT-4 antibodies for western blotting were utilized to evaluate their cell membrane expression on HMCLs. Results: The HMCLs, MM1S and RPMI-8226 as well as primary CD138+ cells from MM patient bone marrow were cultured in cell culture media containing physiological levels (1 mM) of U-13C-Lactate. The incorporation of extracellular 13C into the intracellular glycolytic and TCA cycle metabolite pool was observed (Fig 1) based on the expected isotopomeric patterns, demonstrating the Reverse Warburg Effect in MM cells. The relative contribution of carbon substrate by extracellular lactate compared to extracellular glucose was assessed in the following HMCLs: MM1S, RPMI-8226 and U266 cells by culturing in cell culture media containing 3-13C-Lactate and U-13C-Glucose. Extracellular lactate (yellow bar) contribution to the formation of TCA metabolites equaled that of glucose (red bar) based on the expected isotopomer patterns, suggesting the relative importance of extracellular lactate as an essential nutrient like glucose (Fig 2). Since MCT-1 and MCT-4 are key bidirectional cell membrane transporters of lactate in and out of cells, we explored the clinical significance of their gene expression level on clinical outcomes using the COMMPASS dataset provided by the Multiple Myeloma Research Foundation (MMRF). When MM patients were dichotomized at above or below the median of the expression levels of fragments per kilobase of transcript per million (FPKM), MCT-1 and MCT-4 overexpression conferred a worse progression free survival and overall survival (Fig 3). The MCT-1/MCT-4 protein expression was detectable across the various HMCLs: MM1S, U266 and RPMI-8226 (Fig 4). Inhibition of MCT-1 by specific inhibitor AZD3965 was able to reduce proliferation but not affect viability of HMCLs at 48 hours (Fig 5). However, dual inhibition of MCT-1/MCT-4 using syrosingopine was able to significantly reduce proliferation and decrease viability of HMCLs in a dose dependent fashion (Fig 6). Finally, dual inhibition of MCT-1/MCT-4 using syrosingopine reduced the utilization of extracellular lactate into the TCA cycle pool by HMCLs in media containing 3-13C-Lactate (Fig 7). Conclusion: Utilization of extracellular lactate via Reverse Warburg Effect phenomenon appears highly active in MM cells. Disrupting the utilization of extracellular lactate by dual inhibition of both MCT-1 and MCT-4 appears therapeutic. In the future, dual inhibition of MCT-1/MCT-4 in combination with other anti-MM therapies should be evaluated to determine synergistic therapeutic potential. Figure 1 Figure 1. Disclosures Kumar: Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Antengene: Consultancy, Honoraria; Beigene: Consultancy; Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Consultancy; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Roche-Genentech: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Research Funding.

ABSTRACT Background With pathway‐specific trials in PD associated with variants in the glucocerebrosidase gene (PD GBA ) under way, we need markers that confirm the impact of genetic variants in patient‐derived biofluids in order to allow patient stratification merely based on genetics and that might serve as biochemical read‐out for target engagement. Objective To explore GBA‐pathway‐specific biomarker profiles cross‐sectionally (TUEPAC‐MIGAP, PPMI) and longitudinally (PPMI). Methods We measured enzyme activity of the lysosomal glucocerebrosidase, CSF levels of glucosylceramides (upstream substrate of glucocerebrosidase), CSF levels of ceramides (downstream product of glucocerebrosidase), lactosylceramides, sphingosines, sphingomyelin (by‐products) and CSF levels of total α‐synuclein in PD GBA patients compared to PD GBA_wildtype patients. Results Cross‐sectionally in both cohorts and longitudinally in PPMI: (1) glucocerebrosidase activity was significantly lower in PD GBA compared to PD GBA_wildtype . (2) CSF levels of upstream substrates (glucosylceramides species) were higher in PD GBA compared to PD GBA_wildtype . (3) CSF levels of total α‐synuclein were lower in PD GBA compared to PD GBA_wildtype . All of these findings were most pronounced in PD GBA with severe mutations (PD GBA_severe ). Cross‐sectionally in TUEPAC‐MIGAP and longitudinally in PPMI, CSF levels of downstream‐products (ceramides) were higher in PD GBA_severe . Cross‐sectionally in TUEPAC‐MIGAP by‐products sphinganine and sphingosine‐1‐phosphate and longitudinally in PPMI species of by‐products lactosylceramides and sphingomyelin were higher in PD GBA_severe . Interpretation These findings confirm that GBA mutations have a relevant functional impact on biomarker profiles in patients. Bridging the gap between genetics and biochemical profiles now allows patient stratification for clinical trials merely based on mutation status. Importantly, all findings were most prominent in PD GBA with severe variants. © 2021 International Parkinson and Movement Disorder Society

Abstract BackgroundOverexpression of c-Myc is required for the progression of pre-malignant plasma cells in monoclonal gammopathy of undetermined significance (MGUS) to malignant plasma cells in multiple myeloma (MM). c-Myc also increases glutamine anaplerosis into the tricarboxylic acid (TCA) cycle within cancer cells. Whether increased glutamine anaplerosis is associated with the progression of pre-malignant to malignant plasma cells is unknown.MethodsHuman volunteers (N = 7) and patients with MGUS (N = 11) and MM (N = 12) were prospectively recruited to undergo an intravenous infusion of 13C-labelled glutamine followed by a bone marrow aspiration to obtain bone marrow cells and plasma.ResultsDespite notable heterogeneity, stable isotope resolved metabolomics (SIRM) revealed that the mean 13C-labelled glutamine anaplerosis into the TCA cycle was higher in malignant compared to pre-malignant bone marrow plasma cells relative to the remainder of their paired bone marrow mononuclear cells. RNA sequencing demonstrated a higher relative mRNA expression of c-Myc and glutamine transporters such as ASCT2 and SN2 in malignant compared to pre-malignant bone marrow plasma cells. Finally, higher quantitative levels of TCA cycle intermediates in the bone marrow plasma differentiated MM from MGUS patients.ConclusionMeasurement of the in vivo activity of glutamine anaplerosis into the TCA cycle provides novel insight into the metabolic changes associated with the transformation of pre-malignant plasma cells in MGUS to malignant plasma cells in MM.