Insulin regulation of lysine and α-aminoadipic acid dynamics and amino metabolites in insulin-resistant and control women.

Insulin is a key regulator of amino acids (AAs) metabolism. Many plasma AAs, including lysine and its metabolite, α-aminoadipic acid (α-AA), a predictor for developing diabetes, are elevated in insulin resistance. In 18 insulin-resistant (IR) over-weight women with polycystic ovary syndrome compared to 12 lean controls, high physiological insulin during a euglycemic clamp failed to normalize many elevated AA metabolites, including branched-chain and aromatic AA, alphaamino- butyric acid, and lysine, but normalized α-AA. To understand the underpinning of differential responses of lysine and its metabolic product α-AA to high physiological insulin in IR compared to controls, we developed a kinetic model utilizing [α-15N1] lysine and [13C1] α-AA as tracers and measured the two tracers simultaneously in α-AA by innovative mass spectrometry. High insulin increased lysine conversion to α-AA in IR and controls but failed to normalize plasma lysine concentrations in IR due to a decrease in lysine metabolic clearance rate (MCR). In contrast, despite higher conversion rates of lysine to α-AA by high insulin, α-AA concentration decreased in IR because of the sustained greater MCR of α-AA. The abnormal AAs and metabolites, even while on high physiological insulin, could potentially explain many functional derangements in IR.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia.

BACKGROUND & AIMS: The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS: The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS: In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS: Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

Effect of caloric intake and macronutrient composition on intestinal cholesterol absorption and bile acids in patients with obesity.

Obesity is associated with alterations in cholesterol and bile acid (BA) metabolism. However, the interaction among dietary intake, cholesterol absorption, and BA metabolism in patients with obesity remains unclear. We conducted a 4-wk nutritional intervention nonrandomized clinical trial with three different sequential diets for a week in the following order: regular diet (RD); high calorie, high-fat diet (HCHF), washout period on RD; and low-calorie, low-fat diet (LCLF). We provided participants with meal replacements during HCHF and LCLF diets. A total of 16 participants completed the study [n = 8 normal weight (NW); n = 8 with obesity (OB)]. Overall, there was a significant increase in intestinal cholesterol uptake when changing from RD to HCHF and a reduction in intestinal cholesterol uptake from HCHF to LCLF. When analyzing by BMI groups, these findings were similar in patients with NW (RD to HCHF: P < 0.007; HCHF to LCLF: P = 0.02); however, in patients with obesity, the change in intestinal cholesterol uptake was only observed when changing from RD to HCHF (P = 0.006). There was no correlation between cholesterol absorption and fecal bile acids or other markers of BA metabolism in all patients or the subgroups. Dietary caloric content had a significant effect on cholesterol absorption, however, this effect is blunted in patients with obesity. These data are consistent with the impaired effect of a low-fat diet on cholesterol absorption in obesity.NEW & NOTEWORTHY 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.

The Mutation Matters: CSF Profiles of GCase, Sphingolipids, α-Synuclein in PDGBA.

BACKGROUND: With pathway-specific trials in PD associated with variants in the glucocerebrosidase gene (PDGBA ) 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 PDGBA patients compared to PDGBA_wildtype patients. RESULTS: Cross-sectionally in both cohorts and longitudinally in PPMI: (1) glucocerebrosidase activity was significantly lower in PDGBA compared to PDGBA_wildtype . (2) CSF levels of upstream substrates (glucosylceramides species) were higher in PDGBA compared to PDGBA_wildtype . (3) CSF levels of total α-synuclein were lower in PDGBA compared to PDGBA_wildtype . All of these findings were most pronounced in PDGBA with severe mutations (PDGBA_severe ). Cross-sectionally in TUEPAC-MIGAP and longitudinally in PPMI, CSF levels of downstream-products (ceramides) were higher in PDGBA_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 PDGBA_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 PDGBA with severe variants. © 2021 International Parkinson and Movement Disorder Society.

Metabolomic and Lipidomic Profiling of Bone Marrow Plasma Differentiates Patients with Monoclonal Gammopathy of Undetermined Significance from Multiple Myeloma.

Oncogenic drivers of progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) such as c-MYC have downstream effects on intracellular metabolic pathways of clonal plasma cells (PCs). Thus, extracellular environments such as the bone marrow (BM) plasma likely have unique metabolite profiles that differ from patients with MGUS compared to MM. This study utilized an untargeted metabolite and targeted complex lipid profiling of BM plasma to identify significant differences in the relative metabolite levels between patients with MGUS and MM from an exploratory cohort. This was followed by verification of some of the metabolite differences of interest by targeted quantification of the metabolites using isotopic internal standards in the exploratory cohort as well as an independent validation cohort. Significant differences were noted in the amino acid profiles such as decreased branch chain amino acids (BCAAs) and increased catabolism of tryptophan to the active kynurenine metabolite 3-hydroxy-kynurenine between patients with MGUS and MM. A decrease in the total levels of complex lipids such as phosphatidylethanolamines (PE), lactosylceramides (LCER) and phosphatidylinositols (PI) were also detected in the BM plasma samples from MM compared to MGUS patients. Thus, metabolite and complex lipid profiling of the BM plasma identifies differences in levels of metabolites and lipids between patients with MGUS and MM. This may provide insight into the possible differences of the intracellular metabolic pathways of their clonal PCs.

Radiation Induced Metabolic Alterations Associate With Tumor Aggressiveness and Poor Outcome in Glioblastoma.

Glioblastoma (GBM) is uniformly fatal with a 1-year median survival, despite best available treatment, including radiotherapy (RT). Impacts of prior RT on tumor recurrence are poorly understood but may increase tumor aggressiveness. Metabolic changes have been investigated in radiation-induced brain injury; however, the tumor-promoting effect following prior radiation is lacking. Since RT is vital to GBM management, we quantified tumor-promoting effects of prior RT on patient-derived intracranial GBM xenografts and characterized metabolic alterations associated with the protumorigenic microenvironment. Human xenografts (GBM143) were implanted into nude mice 24 hrs following 20 Gy cranial radiation vs. sham animals. Tumors in pre-radiated mice were more proliferative and more infiltrative, yielding faster mortality (p < 0.0001). Histologic evaluation of tumor associated macrophage/microglia (TAMs) revealed cells with a more fully activated ameboid morphology in pre-radiated animals. Microdialyzates from radiated brain at the margin of tumor infiltration contralateral to the site of implantation were analyzed by unsupervised liquid chromatography-mass spectrometry (LC-MS). In pre-radiated animals, metabolites known to be associated with tumor progression (i.e., modified nucleotides and polyols) were identified. Whole-tissue metabolomic analysis of pre-radiated brain microenvironment for metabolic alterations in a separate cohort of nude mice using 1H-NMR revealed a significant decrease in levels of antioxidants (glutathione (GSH) and ascorbate (ASC)), NAD+, Tricarboxylic acid cycle (TCA) intermediates, and rise in energy carriers (ATP, GTP). GSH and ASC showed highest Variable Importance on Projection prediction (VIPpred) (1.65) in Orthogonal Partial least square Discriminant Analysis (OPLS-DA); Ascorbate catabolism was identified by GC-MS. To assess longevity of radiation effects, we compared survival with implantation occurring 2 months vs. 24 hrs following radiation, finding worse survival in animals implanted at 2 months. These radiation-induced alterations are consistent with a chronic disease-like microenvironment characterized by reduced levels of antioxidants and NAD+, and elevated extracellular ATP and GTP serving as chemoattractants, promoting cell motility and vesicular secretion with decreased levels of GSH and ASC exacerbating oxidative stress. Taken together, these data suggest IR induces tumor-permissive changes in the microenvironment with metabolomic alterations that may facilitate tumor aggressiveness with important implications for recurrent glioblastoma. Harnessing these metabolomic insights may provide opportunities to attenuate RT-associated aggressiveness of recurrent GBM.

Distinct Influence of Omega-3 Fatty Acids on the Plasma Metabolome of Healthy Older Adults.

Omega-3 polyunsaturated fatty acids (n3-PUFA) are well recognized for their potent triglyceride-lowering effects, but the potential influence of these bioactive lipids on other biological processes, particularly in the context of healthy aging, remains unknown. With the goal of gaining new insight into some less well-characterized biological effects of n3-PUFAs in healthy older adults, we performed metabolomics of fasting peripheral blood plasma collected from 12 young adults and 12 older adults before and after an open-label intervention of n3-PUFA (3.9 g/day, 2.7 g eicosapentaenoic [EPA], 1.2 g docosahexaenoic [DHA]). Proton nuclear magnetic resonance (1H-NMR) based lipoprotein subclass analysis revealed the expected reduction in total triglyceride (TG), but also demonstrated that n3-PUFA supplementation reduced very low-density lipoprotein (VLDL) particle number, modestly increased high-density lipoprotein (HDL) cholesterol, and shifted the composition of HDL subclasses. Further metabolite profiling by 1H-NMR and mass spectrometry revealed pronounced changes in phospholipids, cholesterol esters, diglycerides, and triglycerides following n3-PUFA supplementation. Furthermore, significant changes in hydroxyproline, kynurenine, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) following n3-PUFA supplementation provide further insight into some less well-recognized biological effects of n3-PUFA supplementation, including possible effects on protein metabolism, the kynurenine pathway, and glucose metabolism.

A novel natural tracer method to measure complex carbohydrate metabolism.

While the triple tracer isotope dilution method has enabled accurate estimation of carbohydrate turnover after a mixed meal, use of the simple carbohydrate glucose as the carbohydrate source limits its translational applicability to everyday meals that typically contain complex carbohydrates. Hence, utilizing the natural enrichment of [13C]polysaccharide in commercially available grains, we devised a novel tracer method to measure postprandial complex carbohydrate turnover and indices of insulin action and ß-cell function and compared the parameters to those obtained after a simple carbohydrate containing mixed meal. We studied healthy volunteers after either rice (n = 8) or sorghum (n = 8) and glucose (n = 16) containing mixed meals and modified the triple tracer technique to calculate carbohydrate turnover. All meals were matched for calories and macronutrient composition. Rates of meal glucose appearance (2,658 ± 736 vs. 4,487 ± 909 µM·kg-1·2 h-1), endogenous glucose production (-835 ± 283 vs. -1,123 ± 323 µM·kg-1·2 h-1) and glucose disappearance (1,829 ± 807 vs. 3,606 ± 839 µM·kg-1·2 h-1) differed (P < 0.01) between complex and simple carbohydrate containing meals, respectively. Interestingly, there were significant increase in indices of insulin sensitivity (32.5 ± 3.5 vs. 25.6 ± 3.2 10-5 (dl·kg-1·min-2)/pM, P = 0.006) and ß-cell responsivity (disposition index: 1,817 ± 234 vs. 1,236 ± 159 10-14 (dl·kg-1·min-2)/pM, P < 0.005) with complex than simple carbohydrate meals. We present a novel triple tracer approach to estimate postprandial turnover of complex carbohydrate containing mixed meals. We also report higher insulin sensitivity and ß-cell responsivity with complex than with simple carbohydrates in mixed meals of identical calorie and macronutrient compositions in healthy adults.

A comprehensive protocol for multiplatform metabolomics analysis in patient-derived skin fibroblasts.

INTRODUCTION: Patient-derived skin fibroblasts offer a unique translational model to study molecular mechanisms of multiple human diseases. Metabolomics profiling allows to track changes in a broad range of metabolites and interconnected metabolic pathways that could inform on molecular mechanisms involved in disease development and progression, and on the efficacy of therapeutic interventions. Therefore, it is important to establish standardized protocols for metabolomics analysis in human skin fibroblasts for rigorous and reliable metabolic assessment. OBJECTIVES: We aimed to develop an optimized protocol for concurrent measure of the concentration of amino acids, acylcarnitines, and components of the tricarboxylic acid (TCA) cycle in human skin fibroblasts using gas (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS). METHODS: The suitability of four different methods of cell harvesting on the recovery of amino acids, acylcarnitines, and TCA cycle metabolites was established using GC/MS and LC/MS analytical platforms. For each method, metabolite stability was determined after 48 h, 2 weeks and 1 month of storage at - 80 °C. RESULTS: Harvesting cells in 80% methanol solution allowed the best recovery and preservation of metabolites. Storage of samples in 80% methanol up to 1  month at - 80 °C did not significantly impact metabolite concentrations. CONCLUSION: We developed a robust workflow for metabolomics analysis in human skin fibroblasts suitable for a high-throughput multiplatform analysis. This method allows a direct side-by-side comparison of metabolic changes in samples collected at different time that could be used for studies in large patient cohorts.

Distinct microbes, metabolites, and ecologies define the microbiome in deficient and proficient mismatch repair colorectal cancers.

BACKGROUND: Links between colorectal cancer (CRC) and the gut microbiome have been established, but the specific microbial species and their role in carcinogenesis remain an active area of inquiry. Our understanding would be enhanced by better accounting for tumor subtype, microbial community interactions, metabolism, and ecology. METHODS: We collected paired colon tumor and normal-adjacent tissue and mucosa samples from 83 individuals who underwent partial or total colectomies for CRC. Mismatch repair (MMR) status was determined in each tumor sample and classified as either deficient MMR (dMMR) or proficient MMR (pMMR) tumor subtypes. Samples underwent 16S rRNA gene sequencing and a subset of samples from 50 individuals were submitted for targeted metabolomic analysis to quantify amino acids and short-chain fatty acids. A PERMANOVA was used to identify the biological variables that explained variance within the microbial communities. dMMR and pMMR microbial communities were then analyzed separately using a generalized linear mixed effects model that accounted for MMR status, sample location, intra-subject variability, and read depth. Genome-scale metabolic models were then used to generate microbial interaction networks for dMMR and pMMR microbial communities. We assessed global network properties as well as the metabolic influence of each microbe within the dMMR and pMMR networks. RESULTS: We demonstrate distinct roles for microbes in dMMR and pMMR CRC. Bacteroides fragilis and sulfidogenic Fusobacterium nucleatum were significantly enriched in dMMR CRC, but not pMMR CRC. These findings were further supported by metabolic modeling and metabolomics indicating suppression of B. fragilis in pMMR CRC and increased production of amino acid proxies for hydrogen sulfide in dMMR CRC. CONCLUSIONS: Integrating tumor biology and microbial ecology highlighted distinct microbial, metabolic, and ecological properties unique to dMMR and pMMR CRC. This approach could critically improve our ability to define, predict, prevent, and treat colorectal cancers.

Synthesis of multi-omic data and community metabolic models reveals insights into the role of hydrogen sulfide in colon cancer.

Multi-omic data and genome-scale microbial metabolic models have allowed us to examine microbial communities, community function, and interactions in ways that were not available to us historically. Now, one of our biggest challenges is determining how to integrate data and maximize data potential. Our study demonstrates one way in which to test a hypothesis by combining multi-omic data and community metabolic models. Specifically, we assess hydrogen sulfide production in colorectal cancer based on stool, mucosa, and tissue samples collected on and off the tumor site within the same individuals. 16S rRNA microbial community and abundance data were used to select and inform the metabolic models. We then used MICOM, an open source platform, to track the metabolic flux of hydrogen sulfide through a defined microbial community that either represented on-tumor or off-tumor sample communities. We also performed targeted and untargeted metabolomics, and used the former to quantitatively evaluate our model predictions. A deeper look at the models identified several unexpected but feasible reactions, microbes, and microbial interactions involved in hydrogen sulfide production for which our 16S and metabolomic data could not account. These results will guide future in vitro, in vivo, and in silico tests to establish why hydrogen sulfide production is increased in tumor tissue.

Glutamine-derived 2-hydroxyglutarate is associated with disease progression in plasma cell malignancies.

The production of the oncometabolite 2-hydroxyglutarate (2-HG) has been associated with c-MYC overexpression. c-MYC also regulates glutamine metabolism and drives progression of asymptomatic precursor plasma cell (PC) malignancies to symptomatic multiple myeloma (MM). However, the presence of 2-HG and its clinical significance in PC malignancies is unknown. By performing 13C stable isotope resolved metabolomics (SIRM) using U[13C6]Glucose and U[13C5]Glutamine in human myeloma cell lines (HMCLs), we show that 2-HG is produced in clonal PCs and is derived predominantly from glutamine anaplerosis into the TCA cycle. Furthermore, the 13C SIRM studies in HMCLs also demonstrate that glutamine is preferentially utilized by the TCA cycle compared with glucose. Finally, measuring the levels of 2-HG in the BM supernatant and peripheral blood plasma from patients with precursor PC malignancies such as smoldering MM (SMM) demonstrates that relatively elevated levels of 2-HG are associated with higher levels of c-MYC expression in the BM clonal PCs and with a subsequent shorter time to progression (TTP) to MM. Thus, measuring 2-HG levels in BM supernatant or peripheral blood plasma of SMM patients offers potential early identification of those patients at high risk of progression to MM, who could benefit from early therapeutic intervention.