Macronutrient-differential dietary pattern impacts on body weight, hepatic inflammation, and metabolism.

Introduction: Obesity is a multi-factorial disease frequently associated with poor nutritional habits and linked to many detrimental health outcomes. Individuals with obesity are more likely to have increased levels of persistent inflammatory and metabolic dysregulation. The goal of this study was to compare four dietary patterns differentiated by macronutrient content in a postmenopausal model. Dietary patterns were high carbohydrate (HC), high fat (HF), high carbohydrate plus high fat (HCHF), and high protein (HP) with higher fiber. Methods: Changes in body weight and glucose levels were measured in female, ovariectomized C57BL/6 mice after 15 weeks of feeding. One group of five mice fed the HCHF diet was crossed over to the HP diet on day 84, modeling a 21-day intervention. In a follow-up study comparing the HCHF versus HP dietary patterns, systemic changes in inflammation, using an 80-cytokine array and metabolism, by untargeted liquid chromatography-mass spectrometry (LCMS)-based metabolomics were evaluated. Results: Only the HF and HCHF diets resulted in obesity, shown by significant differences in body weights compared to the HP diet. Body weight gains during the two-diet follow-up study were consistent with the four-diet study. On Day 105 of the 4-diet study, glucose levels were significantly lower for mice fed the HP diet than for those fed the HC and HF diets. Mice switched from the HCHF to the HP diet lost an average of 3.7 grams by the end of the 21-day intervention, but this corresponded with decreased food consumption. The HCHF pattern resulted in dramatic inflammatory dysregulation, as all 80 cytokines were elevated significantly in the livers of these mice after 15 weeks of HCHF diet exposure. Comparatively, only 32 markers changed significantly on the HP diet (24 up, 8 down). Metabolic perturbations in several endogenous biological pathways were also observed based on macronutrient differences and revealed dysfunction in several nutritionally relevant biosynthetic pathways. Conclusion: Overall, the HCHF diet promoted detrimental impacts and changes linked to several diseases, including arthritis or breast neoplasms. Identification of dietary pattern-specific impacts in this model provides a means to monitor the effects of disease risk and test interventions to prevent poor health outcomes through nutritional modification.

Perspective: use and reuse of NMR-based metabolomics data: what works and what remains challenging.

BACKGROUND: The National Cancer Institute issued a Request for Information (RFI; NOT-CA-23-007) in October 2022, soliciting input on using and reusing metabolomics data. This RFI aimed to gather input on best practices for metabolomics data storage, management, and use/reuse. AIM OF REVIEW: The nuclear magnetic resonance (NMR) Interest Group within the Metabolomics Association of North America (MANA) prepared a set of recommendations regarding the deposition, archiving, use, and reuse of NMR-based and, to a lesser extent, mass spectrometry (MS)-based metabolomics datasets. These recommendations were built on the collective experiences of metabolomics researchers within MANA who are generating, handling, and analyzing diverse metabolomics datasets spanning experimental (sample handling and preparation, NMR/MS metabolomics data acquisition, processing, and spectral analyses) to computational (automation of spectral processing, univariate and multivariate statistical analysis, metabolite prediction and identification, multi-omics data integration, etc.) studies. KEY SCIENTIFIC CONCEPTS OF REVIEW: We provide a synopsis of our collective view regarding the use and reuse of metabolomics data and articulate several recommendations regarding best practices, which are aimed at encouraging researchers to strengthen efforts toward maximizing the utility of metabolomics data, multi-omics data integration, and enhancing the overall scientific impact of metabolomics studies.

Fatty acid metabolism promotes TRPV4 activity in lung microvascular endothelial cells in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by significant lung endothelial cell (EC) dysfunction. Prior work has shown that microvascular endothelial cells (MVECs) isolated from animals with experimental PAH and patients with PAH exhibit significant abnormalities in metabolism and calcium signaling. With regards to metabolism, we and others have shown evidence of increased aerobic glycolysis and evidence of increased utilization of alternate fuel sources (such as fatty acids) in PAH EC. In the realm of calcium signaling, our prior work linked increased activity of the transient receptor potential vanilloid-4 (TRPV4) channel to increased proliferation of MVECs isolated from the Sugen/Hypoxia rat model of PAH (SuHx-MVECs). However, the relationship between metabolic shifts and calcium abnormalities was not clear. Specifically, whether shifts in metabolism were responsible for increasing TRPV4 channel activity in SuHx-MVECs was not known. In this study, using human data, serum samples from SuHx rats, and SuHx-MVECs, we describe the consequences of increased MVEC fatty acid oxidation in PAH. In human samples, we observed an increase in long-chain fatty acid levels that was associated with PAH severity. Next, using SuHx rats and SuHx-MVECs, we observed increased intracellular levels of lipids. We also show that increasing intracellular lipid content increases TRPV4 activity, whereas inhibiting fatty acid oxidation normalizes basal calcium levels in SuHx-MVECs. By exploring the fate of fatty acid-derived carbons, we observed that the metabolite linking increased intracellular lipids to TRPV4 activity was ß-hydroxybutyrate (BOHB), a product of fatty acid oxidation. Finally, we show that BOHB supplementation alone is sufficient to sensitize the TRPV4 channel in rat and mouse MVECs. Returning to humans, we observe a transpulmonary BOHB gradient in human patients with PAH. Thus, we establish a link between fatty acid oxidation, BOHB production, and TRPV4 activity in MVECs in PAH. These data provide new insight into metabolic regulation of calcium signaling in lung MVECs in PAH.NEW & NOTEWORTHY In this paper, we explore the link between metabolism and intracellular calcium levels in microvascular endothelial cells (MVECs) in pulmonary arterial hypertension (PAH). We show that fatty acid oxidation promotes sensitivity of the transient receptor potential vanilloid-4 (TRPV4) calcium channel in MVECs isolated from a rodent model of PAH.

Multi-omics analyses reveal ClpP activators disrupt essential mitochondrial pathways in triple-negative breast cancer.

ClpP activators ONC201 and related small molecules (TR compounds, Madera Therapeutics), have demonstrated significant anti-cancer potential in vitro and in vivo studies, including clinical trials for refractory solid tumors. Though progress has been made in identifying specific phenotypic outcomes following ClpP activation, the exact mechanism by which ClpP activation leads to broad anti-cancer activity has yet to be fully elucidated. In this study, we utilized a multi-omics approach to identify the ClpP-dependent proteomic, transcriptomic, and metabolomic changes resulting from ONC201 or the TR compound TR-57 in triple-negative breast cancer cells. Applying mass spectrometry-based methods of proteomics and metabolomics, we identified ∼8,000 proteins and 588 metabolites, respectively. From proteomics data, 113 (ONC201) and 191 (TR-57) proteins significantly increased and 572 (ONC201) and 686 (TR-57) proteins significantly decreased in this study. Gene ontological (GO) analysis revealed strong similarities between proteins up- or downregulated by ONC201 or TR-57 treatment. Notably, this included the downregulation of many mitochondrial processes and proteins, including mitochondrial translation and mitochondrial matrix proteins. We performed a large-scale transcriptomic analysis of WT SUM159 cells, identifying ∼7,700 transcripts (746 and 1,100 significantly increasing, 795 and 1,013 significantly decreasing in ONC201 and TR-57 treated cells, respectively). Less than 21% of these genes were affected by these compounds in ClpP null cells. GO analysis of these data demonstrated additional similarity of response to ONC201 and TR-57, including a decrease in transcripts related to the mitochondrial inner membrane and matrix, cell cycle, and nucleus, and increases in other nuclear transcripts and transcripts related to metal-ion binding. Comparison of response between both compounds demonstrated a highly similar response in all -omics datasets. Analysis of metabolites also revealed significant similarities between ONC201 and TR-57 with increases in α-ketoglutarate and 2-hydroxyglutaric acid and decreased ureidosuccinic acid, L-ascorbic acid, L-serine, and cytidine observed following ClpP activation in TNBC cells. Further analysis identified multiple pathways that were specifically impacted by ClpP activation, including ATF4 activation, heme biosynthesis, and the citrulline/urea cycle. In summary the results of our studies demonstrate that ONC201 and TR-57 induce highly similar and broad effects against multiple mitochondrial processes required for cell proliferation.

Untargeted Metabolomics: Biochemical Perturbations in Golestan Cohort Study Opium Users Inform Intervention Strategies.

Objective: Over 50 million people worldwide are estimated to use opioids, of which ~30 million use opiates (opium and its derivatives). Use of opiates has been associated with a variety of adverse complications such as neurological and behavioral outcomes, addiction, cancers, diabetes, and cardiovascular disease. While it is well known that opiates exert their neurobiological effects through binding with mu, kappa, and delta receptors to exert analgesic and sedative effects, mechanistic links to other health effects are not well understood. Our study focuses on the identification of biochemical perturbations in Golestan Cohort Study (GCS) opium users. Methods: We used untargeted metabolomics to evaluate the metabolic profiles of 218 opium users and 80 non-users participating in the GCS. Urine samples were obtained from adult (age 40-75) opium users living in the Golestan Province of Iran. Untargeted analysis of urine was conducted using a UPLC-Q-Exactive HFx Mass Spectrometry and a 700 MHz NMR Spectrometry. Results: These GCS opium users had a significantly higher intake of tobacco and alcohol and a significantly decreased BMI compared with non-users. Metabolites derived from opium (codeine, morphine, and related glucuronides), nicotine, and curing or combustion of plant material were increased in opium users compared with non-users. Endogenous compounds which differentiated the opium users and non-users largely included vitamins and co-factors, metabolites involved in neurotransmission, Kreb's cycle, purine metabolism, central carbon metabolism, histone modification, and acetylation. Conclusions: Our study reveals biochemical perturbations in GCS opium users that are important to the development of intervention strategies to mitigate against the development of adverse effects of substance abuse.

Stable Isotope-Resolved Metabolomic Differences between Hormone-Responsive and Triple-Negative Breast Cancer Cell Lines.

PURPOSE: To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on differences in energy utilization. METHODS: Cells were cultured in media containing either [U-13C]-glucose or [U-13C]-glutamine for 48 hours. Conditioned media and cellular extracts were analyzed by 1H and 13C NMR spectroscopy. RESULTS: MCF-7 cells consumed the most glucose, producing the most lactate, demonstrating the greatest Warburg effect-associated energy utilization. BT474 cells had the highest tricarboxylic acid cycle (TCA) activity. The majority of energy utilization patterns in MCF-7 cells were more similar to MDA-MB-468 cells, while the patterns for BT474 cells were more similar to MDA-MB-231 cells. Compared to the Luminal A cell lines, TNBC cell lines consumed more glutamine and less glucose. BT474 and MDA-MB-468 cells produced high amounts of 13C-glycine from media [U-13C]-glucose which was integrated into glutathione, indicating de novo synthesis. CONCLUSIONS: Stable isotopic resolved metabolomics using 13C substrates provided mechanistic information about energy utilization that was difficult to interpret using 1H data alone. Overall, cell lines that have different hormone receptor status have different energy utilization requirements, even if they are classified by the same clinical BCa subtype; and these differences offer clues about optimizing treatment strategies.

Correlated metabolomic, genomic, and histologic phenotypes in histologically normal breast tissue.

Breast carcinogenesis is a multistep process accompanied by widespread molecular and genomic alterations, both in tumor and in surrounding microenvironment. It is known that tumors have altered metabolism, but the metabolic changes in normal or cancer-adjacent, nonmalignant normal tissues and how these changes relate to alterations in gene expression and histological composition are not well understood. Normal or cancer-adjacent normal breast tissues from 99 women of the Normal Breast Study (NBS) were evaluated. Data of metabolomics, gene expression and histological composition was collected by mass spectrometry, whole genome microarray, and digital image, respectively. Unsupervised clustering analysis determined metabolomics-derived subtypes. Their association with genomic and histological features, as well as other breast cancer risk factors, genomic and histological features were evaluated using logistic regression. Unsupervised clustering of metabolites resulted in two main clusters. The metabolite differences between the two clusters suggested enrichment of pathways involved in lipid metabolism, cell growth and proliferation, and migration. Compared with Cluster 1, subjects in Cluster 2 were more likely to be obese (body mass index ≥30 kg/m2, p<0.05), have increased adipose proportion (p<0.01) and associated with a previously defined Active genomic subtype (p<0.01). By the integrated analyses of histological, metabolomics and transcriptional data, we characterized two distinct subtypes of non-malignant breast tissue. Further research is needed to validate our findings, and understand the potential role of these alternations in breast cancer initiation, progression and recurrence.

A Microbiomic Analysis in African Americans with Colonic Lesions Reveals Streptococcus sp.VT162 as a Marker of Neoplastic Transformation.

Increasing evidence suggests a role of the gut microbiota in colorectal carcinogenesis (CRC). To detect bacterial markers of colorectal cancer in African Americans a metabolomic analysis was performed on fecal water extracts. DNA from stool samples of adenoma and healthy subjects and from colon cancer and matched normal tissues was analyzed to determine the microbiota composition (using 16S rDNA) and genomic content (metagenomics). Metagenomic functions with discriminative power between healthy and neoplastic specimens were established. Quantitative Polymerase Chain Reaction (q-PCR) using primers and probes specific to Streptococcus sp. VT_162 were used to validate this bacterium association with neoplastic transformation in stool samples from two independent cohorts of African Americans and Chinese patients with colorectal lesions. The metabolomic analysis of adenomas revealed low amino acids content. The microbiota in both cancer vs. normal tissues and adenoma vs. normal stool samples were different at the 16S rRNA gene level. Cross-mapping of metagenomic data led to 9 markers with significant discriminative power between normal and diseased specimens. These markers identified with Streptococcus sp. VT_162. Q-PCR data showed a statistically significant presence of this bacterium in advanced adenoma and cancer samples in an independent cohort of CRC patients. We defined metagenomic functions from Streptococcus sp. VT_162 with discriminative power among cancers vs. matched normal and adenomas vs. healthy subjects' stools. Streptococcus sp. VT_162 specific 16S rDNA was validated in an independent cohort. These findings might facilitate non-invasive screening for colorectal cancer.

Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences.

To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.

Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG) cross-links.

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.

Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.

The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn-protected diastereomerically pure aza-ENA-Ts (12a and 12b) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and equatorial N-H in 12a. The latter is thermodynamically preferred, while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is thus far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly modified 15-mer antisense oligonucleotides (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3' degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.