Tricarboxylic Acid Cycle Intermediates Research Articles

Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1β protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

A cell-free nutrient-supplemented perfusate allows four-day ex vivo metabolic preservation of human kidneys

The growing disparity between the demand for transplants and the available donor supply, coupled with an aging donor population and increasing prevalence of chronic diseases, highlights the urgent need for the development of platforms enabling reconditioning, repair, and regeneration of deceased donor organs. This necessitates the ability to preserve metabolically active kidneys ex vivo for days. However, current kidney normothermic machine perfusion (NMP) approaches allow metabolic preservation only for hours. Here we show that human kidneys discarded for transplantation can be preserved in a metabolically active state up to 4 days when perfused with a cell-free perfusate supplemented with TCA cycle intermediates at subnormothermia (25 °C). Using spatially resolved isotope tracing we demonstrate preserved metabolic fluxes in the kidney microenvironment up to Day 4 of perfusion. Beyond Day 4, significant changes were observed in renal cell populations through spatial lipidomics, and increases in injury markers such as LDH, NGAL and oxidized lipids. Finally, we demonstrate that perfused kidneys maintain functional parameters up to Day 4. Collectively, these findings provide evidence that this approach enables metabolic and functional preservation of human kidneys over multiple days, establishing a solid foundation for future clinical investigations.

Proline metabolic reprogramming modulates cardiac remodeling induced by pressure overload in the heart.

Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed after TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. In addition, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid cycle intermediates, enhance energy production, and restore glutathione redox balance. Our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis during cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.

Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Unveiling the altered metabolic pathways induced by nivolumab in non-small cell lung cancer via GC–MS metabolomics approach coupled with multivariate analysis

This research investigates the effects of the immunotherapeutic agent nivolumab on the metabolism of lung cancer cells (NCI-H1975) using GC–MS metabolomic profiling. Multivariate analysis such as unsupervised PCA and supervised OPLS-DA along with univariate analysis and pathway analysis were employed to explore the metabolomic data and identify altered metabolic pathways induced by nivolumab treatment. The study revealed distinct metabolic alterations in cancer cells, linked to proliferative and survival advantages, such as enhanced glycolysis, increased glutaminolysis, and modified amino acid metabolism. Key findings indicate elevated levels of glycolysis-related metabolites (glycine, alanine, pyruvate, and lactate) and TCA cycle intermediates (succinate, fumarate, malate) in cancer cells, with a significant decrease following nivolumab treatment. Additionally, lower levels of aspartic acid and citrate in cancer cells imply altered nucleotide synthesis and fatty acid production essential for tumor growth. Treatment with nivolumab also reduced oleic acid levels, indicative of its effect on disrupted lipid metabolism. Our research shows nivolumab's potential to modify metabolic pathways involved in lung cancer progression, suggesting its dual role in cancer therapy: as an immune response modulator and a metabolic pathway disruptor.

Direct Infusion Mass Spectrometry to Rapidly Map Metabolic Flux of Substrates Labeled with Stable Isotopes

Direct infusion–high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics.

Tricarboxylic Acid Cycle Regulation of Metabolic Program, Redox System, and Epigenetic Remodeling for Bone Health and Disease.

Imbalanced osteogenic cell-mediated bone gain and osteoclastic remodeling accelerates the development of osteoporosis, which is the leading risk factor of disability in the elderly. Harmonizing the metabolic actions of bone-making cells and bone resorbing cells to the mineralized matrix network is required to maintain bone mass homeostasis. The tricarboxylic acid (TCA) cycle in mitochondria is a crucial process for cellular energy production and redox homeostasis. The canonical actions of TCA cycle enzymes and intermediates are indispensable in oxidative phosphorylation and adenosine triphosphate (ATP) biosynthesis for osteogenic differentiation and osteoclast formation. Knockout mouse models identify these enzymes' roles in bone mass and microarchitecture. In the noncanonical processes, the metabolites as a co-factor or a substrate involve epigenetic modification, including histone acetyltransferases, DNA demethylases, RNA m6A demethylases, and histone demethylases, which affect genomic stability or chromatin accessibility for cell metabolism and bone formation and resorption. The genetic manipulation of these epigenetic regulators or TCA cycle intermediate supplementation compromises age, estrogen deficiency, or inflammation-induced bone mass loss and microstructure deterioration. This review sheds light on the metabolic functions of the TCA cycle in terms of bone integrity and highlights the crosstalk of the TCA cycle and redox and epigenetic pathways in skeletal tissue metabolism and the intermediates as treatment options for delaying osteoporosis.

Metabolic Profiling in Plasma and Brain Induced by 17β-Estradiol Supplementation in Ovariectomized Mice.

17β-Estradiol is an ovarian hormone that regulates energy circulation and storage by acting on the central nervous system. However, the metabolic differences between the blood and brain when stimulated by 17β-estradiol are poorly understood. Moreover, research using menopause-induced models to investigate primary metabolites in the blood and brain is limited. Thus, this study aimed to identify metabolic changes in the plasma and brain resulting from 17β-estradiol supplementation in an estrogen-deficient mouse model. Three groups of mice were utilized: sham-operated mice (Sham), ovariectomized mice (OVX), and ovariectomized mice that received a weekly supplementation of 17β-estradiol (E2). Plasma and brain samples from these mice were subjected to metabolic analysis using gas chromatography-time-of-flight-mass spectrometry. Compared with the plasma samples from the Sham and OVX groups, the plasma samples from the E2 group contained higher contents of branched-chain amino acids (BCAAs), such as valine, isoleucine, and leucine. Meanwhile, the brain samples from the E2 group contained higher contents of most metabolites, including BCAAs, neurotransmitters, tricarboxylic acid cycle intermediates, and fatty acids, than those from the two other groups. This study is the first to reveal differences in energy metabolism induced by 17β-estradiol supplementation through brain metabolic profiling of ovariectomized mice, emphasizing the importance of brain metabolic profiling in menopausal hormone research.

Inhibition of pyruvate dehydrogenase accelerates anaerobic glycolysis under postmortem simulating conditions

This research aimed to explore the potential influence of mitochondria on the rate of anaerobic glycolysis. We hypothesized that mitochondria could reduce the rate of anaerobic glycolysis and pH decline by metabolizing a portion of glycolytic pyruvate. We utilized an in vitro model and incorporated CPI-613 and Avidin to inhibit pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC), respectively. Four treatments were tested: 400 μM CPI-613, 1.5 U/ml Avidin, 400 μM CPI-613 + 1.5 U/ml Avidin, or control. Glycolytic metabolites and pH of the in vitro model were evaluated throughout a 1440-min incubation period. CPI-613-containing treatments, with or without Avidin, decreased pH levels and increased glycogen degradation and lactate accumulation compared to the control and Avidin treatments (P < 0.05), indicating increased glycolytic flux. In a different experiment, two treatments, 400 μM CPI-613 or control, were employed to track the fates of pyruvate using [13C6]glucose. CPI-613 reduced the contribution of glucose carbon to tricarboxylic acid cycle intermediates compared to control (P < 0.05). To test whether the acceleration of acidification in reactions containing CPI-613 was due to an increase in the activity of key enzymes of glycogenolysis and glycolysis, we evaluated the activities of glycogen phosphorylase, phosphofructokinase, and pyruvate kinase in the presence or absence of 400 μM CPI-613. The CPI-613 treatment did not elicit an alteration in the activity of these three enzymes. These findings indicate that inhibiting PDH increases the rate of anaerobic glycolysis and pH decline, suggesting that mitochondria are potential regulators of postmortem metabolism.

Abstract LB294: Targeting fatty acid oxidation to boost the anti-tumor effects of nutrient starvation in solid malignancies

Abstract Introduction Reducing glucose and growth factor provision to cancer cells through in vitro starvation (STV) or in vivo fasting/fasting-mimicking diets (FMDs) delays tumor progression and prolongs survival in tumor-bearing mice. More recently, cyclic FMD has been shown to be safe and associated with promising metabolic and immunomodulatory effects in cancer patients. However, cyclic fasting and FMD only retard tumor progression, but they do not result in tumor cure when used alone. Here, we hypothesized that tumor cells exposed to STV activate Fatty Acid Oxidation (FAO) as an alternative metabolic pathway that could be responsible for primary or acquired tumor cell resistance to STV. Methods We investigated the effects of FAO inhibition (FAOi) in combination with STV on the proliferation and survival of human or murine cell lines of Triple Negative Breast Cancer (TNBC) (n=16), HR+HER2- breast cancer (n=3), colorectal (n=1), pancreatic (n=1) and lung cancer (n=1), as well as of non-transformed human epithelial cells (n=3). STV consisted of a 50% reduction of extracellular glucose and serum in cell growth media. To inhibit FAO, we used the Carnitine Palmitoyl transferase 1 inhibitor Etomoxir or ST1326. Results STV+FAOi resulted in cooperative or highly synergistic antiproliferative effects in all cancer models tested, but not in normal cells. At longer time points, STV+FAOi enhanced tumor cell death through the activation of apoptosis. Depending on the specific tumor type, the cooperative effects of STV and FAOi were mostly mediated by glucose or fatty acid restriction in the STV medium. On the other hand, the reduction of extracellular growth factors did not affect cell proliferation upon STV+FAOi combination. Intracellular ATP levels were reduced upon cell exposure to the combined treatment at later time points. However, the supplementation of several sources of ATP, such as glutamine or TCA cycle intermediates, to tumor cells did not rescue the antitumor effects of STV+FAOi, thus excluding the strong impact of this combination on tumor cell bioenergetics. On the other hand, STV+FAOi caused an excessive intracellular antioxidant response, which in part mediated the synergistic effects of STV+FAOi. Indeed, Etomoxir alone mildly increased the NADPH/NADP+ ratio, whereas its combination with STV resulted in a remarkable increase of this ratio. In parallel, combining the antioxidant molecules n-acetylcysteine (NAC) and reduced glutathione (GSH) with FAOi caused antitumor effects that recapitulate those produced by STV+FAOi treatment. Conclusions FAO is a potential resistance mechanism to nutrient starvation, with broad antitumor activity in different models of murine and human malignancies, but not in normal cells. In cancer cells, FAOi synergizes with STV by strongly affecting intracellular redox balance. FAOi is a new strategy that should be explored in future preclinical in vivo experiments and in clinical studies to potentiate the antitumor effects of nutrient starvation in solid malignancies. Citation Format: Marzia Santamaria, Beatrice Cantarelli, Arta Ajazi, Chiara Milanese, Giulia Salvadori, Pier Giorgio Mastroberardino, Paola Antonia Corsetto, Giancarlo Pruneri, Filippo De Braud, Claudio Vernieri. Targeting fatty acid oxidation to boost the anti-tumor effects of nutrient starvation in solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB294.

Microbial Indoles: Key Regulators of Organ Growth and Metabolic Function.

Gut microbes supporting body growth are known but the mechanisms are less well documented. Using the microbial tryptophan metabolite indole, known to regulate prokaryotic cell division and metabolic stress conditions, we mono-colonized germ-free (GF) mice with indole-producing wild-type Escherichia coli (E. coli) or tryptophanase-encoding tnaA knockout mutant indole-non-producing E. coli. Indole mutant E. coli mice showed multiorgan growth retardation and lower levels of glycogen, cholesterol, triglycerides, and glucose, resulting in an energy deficiency despite increased food intake. Detailed analysis revealed a malfunctioning intestine, enlarged cecum, and reduced numbers of enterochromaffin cells, correlating with a metabolic phenotype consisting of impaired gut motility, diminished digestion, and lower energy harvest. Furthermore, indole mutant mice displayed reduction in serum levels of tricarboxylic acid (TCA) cycle intermediates and lipids. In stark contrast, a massive increase in serum melatonin was observed-frequently associated with accelerated oxidative stress and mitochondrial dysfunction. This observational report discloses functional roles of microbe-derived indoles regulating multiple organ functions and extends our previous report of indole-linked regulation of adult neurogenesis. Since indoles decline by age, these results imply a correlation with age-linked organ decline and levels of indoles. Interestingly, increased levels of indole-3-acetic acid, a known indole metabolite, have been shown to correlate with younger biological age, further supporting a link between biological age and levels of microbe-derived indole metabolites. The results presented in this resource paper will be useful for the future design of food intervention studies to reduce accelerated age-linked organ decline.

Systemic acylcarnitine levels are affected in response to multiple injuries and hemorrhagic shock - an analysis of lipidomic changes in a standardized porcine model.

Along with recent advances in analytical technologies, TCA-cycle intermediates are increasingly identified as promising makers for cellular ischemia and mitochondrial dysfunction during hemorrhagic shock (HS). For traumatized patients, the knowledge of the role of lipid oxidation substrates is sparse. In this study, we aimed to analyze the dynamics of systemic acylcarnitine (AcCa) release in a standardized polytrauma model with HS. 52 male pigs (50 ± 5 kg) were randomized into two groups: Group IF (isolated fracture) was subject to a standardized femur shaft fracture. Group PT (polytrauma) was subject to a femur fracture, followed by blunt chest trauma, liver laceration and a pressure controlled hemorrhagic shock for 60 min. Resuscitation was performed with crystalloids. Fractures were stabilized by intramedullary nailing. Venous samples were collected at 6 timepoints (baseline, trauma, resuscitation, 2 h, 4 h and 6 h). Lipidomic analysis was performed via liquid chromatography coupled mass spectrometry. Measurements were collated with clinical markers and near-infrared spectrometry measurements (NIRS) of tissue perfusion. Longitudinal analyses were performed with linear mixed models and spearman's correlations were calculated. A p-value of 0.05 was defined as threshold for statistical significance. From a total of 303 distinct lipids, we identified two species of long-chain AcCas. Both showed a highly significant (p < 0.001) two-fold increase after HS in Group PT that promptly normalized after resuscitation. This increase was associated with a significant decrease of the base excess (p = 0.005) but recovery after resuscitation was faster. For both AcCas, there were significant correlations with decreased muscle tissue oxygen delivery (p = 0.008, p = 0.003) and significant time-lagged correlations with the increase of creatine kinase (p < 0.001, p < 0.001). Our results point to plasma AcCas as a possible indicator for mitochondrial dysfunction and cellular ischemia in HS. The more rapid normalization after resuscitation in comparison to acid base changes may warrant further investigation. Experimental Animal Model. N/A.

Untargeted metabolomic reveals the changes in muscle metabolites of mice during exercise recovery and the mechanisms of whey protein and whey protein hydrolysate in promoting muscle repair

Our previous study indicated that whey protein hydrolysate (WPH) showed effective anti-fatigue properties, but its regulatory mechanism on recovery from exercise in mice is unclear. In the present study, we divided the mice into control, WP, and WPH groups and allowed them to rest for 1 h and 24 h after exercise, respectively. The changes in muscle metabolites of mice in the recovery period were investigated using metabolomics techniques. The results showed that the WPH group significantly up-regulated 94 muscle metabolites within 1 h of rest, which was 1.96 and 2.61 times more than the control and WP groups, respectively. In detail, significant decreases in TCA cycle intermediates, lipid metabolites, and carbohydrate metabolites were observed in the control group during exercise recovery. In contrast, administration with WP and WPH enriched more amino acid metabolites within 1 h of rest, which might provide a more comprehensive metabolic environment for muscle repair. Moreover, the WPH group remarkably stimulated the enhancement of lipid, carbohydrate, and vitamin metabolites in the recovery period which might provide raw materials and energy for anabolic reactions. The result of the western blot further demonstrated that WPH could promote muscle repair via activating the Sestrin2/Akt/mTOR/S6K signaling pathway within 1 h of rest. These findings deepen our understanding of the regulatory mechanisms by WPH to promote muscle recovery and may serve as a reference for comprehensive assessments of protein supplements on exercise.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Abstract 4440: Topographical investigation of metabolites in excised squares (TIMES2): Comprehensive cross-sectional metabolite quantification of pancreatic cancer in vivo

Abstract Purpose: Pancreatic cancer is a lethal malignancy characterized by complex intratumoral metabolic reprogramming and intercellular nutrient sharing between cells in the tumor microenvironment (TME) that promote pancreatic cancer progression. However, this crosstalk, as well as regional variation in perfusion and oxygenation, can lead to metabolic heterogeneity that has not been appreciated by metabolomics of whole tumors. Here we quantify amino acids and tricarboxylic acid cycle (TCA) intermediates using a novel methodology that allows us to portray global tumor metabolite heterogeneity in a tumor. Methods: Human PaTu-8902 or murine HY19636 (from female KPC mice LSL-KrasG12D; p53 L/+, Ptf1a-Cre+) pancreatic cancer cell lines were orthotopically injected into pancreata of NCr nude mice (n=3) or C57BL/6 mice (n=2). Mice were euthanized after 3-5 weeks and tumors were harvested. Tumor slices were further sectioned into 1mm x 1mm x 1mm cubes using a custom-made multisectional slicing device and each cube location was recorded. Each cube was extracted using methanol, water, and chloroform with labelled amino acid standards, derivitized, and resolved using gas chromatography-mass spectrometry (DB-35MS column with Agilent 7890B gas chromatograph coupled to a single quadrupole 5977B mass spectrometer). 22 metabolites (15 amino acids, 5 TCA intermediates, lactate, and pyruvate) were identified by unique fragments and retention time compared to known standards. Peaks were picked using OpenChrom and analyzed using MATLAB. Data was analyzed using Graphpad Prism. Principal Component Analysis (PCA) was visualized using Python on a Jupyter notebook. Results: Both orthotopic human and murine pancreatic tumors demonstrated striking levels of intratumoral metabolite heterogeneity. Glycine, glutamine, and proline were the amino acids with the highest coefficient of variance, while leucine, isoleucine, and serine had the lowest coefficient of variance. α-ketoglutarate and succinate were the TCA intermediates with highest coefficient of variance. Lactate had the lowest coefficient of variance among all examined metabolites. Spatial mapping of each metabolite demonstrated distinct regions with varying abundance levels of metabolites. PCA demonstrated 75% of variance was carried by PC1 and 10% carried by PC2. Conclusions: This study reveals insights into the degree of intratumoral heterogeneity present in pancreatic tumors that illustrate the difficulty of in vivo metabolomics analysis and suggest that high-resolution (single cell) metabolomics techniques will be critical to study metabolism in the complex TME. Citation Format: Peter Yu, Robert Banh, Albert Sohn, Stephen Martis, Douglas Biancur, Keisuke Yamamoto, Elaine Lin, Alec Kimmelman. Topographical investigation of metabolites in excised squares (TIMES2): Comprehensive cross-sectional metabolite quantification of pancreatic cancer in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4440.

Abstract 3069: Targeting lactic acid sensing GPR132 in ccRCC

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, accounting for 70-80% of the cases. Despite the improvements in tumor therapeutics, patients with metastatic clear cell tumors still have a five-year survival rate lower than 10%. Most of the ccRCC tumors harbor loss of VHL tumor suppressor gene on chromosome 3, leading to constitutively stabilized HIF, activation of downstream pathways involved in glycolysis, angiogenesis and metastasis and a highly acidic, vascularized, and immunosuppressive tumor microenvironment (TME). Such molecular alterations lead to the buildup of lactic acid within the tumor microenvironment. Long has been considered as a byproduct of hypoxic tumor metabolism, but recent studies have demonstrated a pro-tumorigenic role of lactic acid. Therefore, elucidating the molecular mechanisms on how tumor cells take advantage of lactic acid can identify potential novel therapeutic options. To understand how ccRCC cells sense extracellular lactic acid, I explored the concept of a lactate sensor. The currently known lactate sensors are GPR81 and GPR132. By querying TCGA data, GPR81 is reduced in ccRCC tumors and does not predict patient outcome while GPR132 is elevated in ccRCC tumors compared to normal kidney and demonstrates a strong negative effect on survival. Therefore, GPR132 stands out as a potential lactate sensor in ccRCC with meaningful impact on patient survival. Previously, GPR132 has been shown to be involved in tumor associated macrophage M1 to M2 transition upon lactic acid exposure. However, GPR132 exerts a striking impact on ccRCC tumors. Depleting GPR132 significantly decreased the HIF2α level in multiple ccRCC cell lines, establishing a molecular connection between lactic acid, GPR132 and HIF2α. Furthermore, due to suppressed HIF2α signaling, GPR132 knockout led to decreased lactic acid uptake through MCT1. Such decreased lactic transporter MCT1 abrogated the elevated aggressive phenotype in ccRCC cells when exposed to lactic acid. Metabolically, GPR132 knockout led to decreased lipid deposition. In addition, such deprivation of lipid droplet is not replenished when cells are exposed to lactic acid. Furthermore, by conducting an isotype-tracing experiment, GPR132 knockout actively decreased lactic acid uptake by the tumors and limit their resources to regenerate TCA cycle intermediates and build up lipid droplets. More importantly, GPR132 knockout led to defective mitochondria activities and activation of apoptosis, both of which led to significantly suppressed ccRCC growth in vitro and in vivo. This suggested that GPR132 likely affects ccRCC fitness through multiple layers of signaling mechanisms. Overall, our study has revealed that lactate sensing GPR132 is a critical signaling pathway for ccRCC tumor development in a glycolytic feed-forward context. As a membrane bound receptor, GPR132 holds immense potential as a novel therapeutic target in ccRCC. Citation Format: Dazhi Wang. Targeting lactic acid sensing GPR132 in ccRCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3069.

Abstract 6609: NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells

Abstract Esophageal diseases such as gastroesophageal reflux disease (GERD) and esophageal cancer have a strong association with alcohol drinking. However, the molecular mechanisms of alcohol-induced esophageal pathology remain unclear. In our previous study, we identified acyl-CoA synthetase short-chain family member 2 (ACSS2) as a transcriptional target of nuclear factor erythroid 2-related factor 2 (NRF2) in the esophageal epithelial cells in vitro and in vivo. Ethanol exposure enhanced de novo lipogenesis in NRF2high cells and generated a more energetic and invasive phenotype. In this study, we aimed to investigate the role of the NRF2/ACSS2 axis in regulating alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells. We first demonstrated that ethanol exposure activated NRF2 signaling and upregulated the expression of NRF2 and ACSS2 in human esophageal squamous epithelial cells. 13C-ethanol/13C-acetate metabolic flux assay showed a dramatic NRF2-dependent increase of labeling of the TCA cycle intermediates and de novo lipogenesis in NRF2high cells in comparison with NRF2null or NRF2low cells. Long-term ethanol exposure caused metabolic reprogramming, in particular induced de novo lipogenesis, in the mouse esophagus according to metabolomics profiling of 833 metabolites. PET/CT and autoradiography showed increased 11C-acetate uptake in the NRF2high esophagus of a conditional tissue-specific mouse (Sox2CreER;LSL-Nrf2E79Q) due to upregulation of transporters and metabolic enzymes. In summary, Our data demonstrated the functional role of the NRF2-ACSS2 axis in alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells both in vitro and in vivo, suggesting NRF2/ACSS2 inhibition as a novel mechanism-based prevention for alcohol-associated esophageal pathology. Citation Format: Zhaohui Xiong, Boopathi Subramaniyan, Chorlada Paiboonrungruang, Yahui Li, Wentao He, Hong Yuan, Guofang Zhang, Xiaoxin Chen. NRF2/ACSS2 axis regulates alcohol-induced metabolic reprogramming in esophageal squamous epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6609.

Abstract 1222: Mechanisms of succinate-mediated drug resistance in ER+ breast cancer

Abstract Breast cancer is the most prevalent cancer among women worldwide with estrogen receptor-positive (ER+) cancer constituting 70% of all breast cancers. Although ER+ breast cancers initially respond well to ER-targeted therapies and CDK4/6 inhibitors, approximately 30% patients acquire resistance to targeted therapies. In addition to intrinsic resistance that tumors develop, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment, also contribute to treatment-resistance and poor survival. The role of secreted metabolites by CAFs has been poorly studied in the context of targeted drug resistance. Therefore, to investigate the mechanism of stroma-mediated resistance to CDK4/6-inhibitors, we propagated patient-derived organoids (PDOs) and CAFs from treatment-naïve and treatment-resistant ER+ breast tumors. Our data show that the CAF conditioned media containing the polar metabolite fraction stimulates resistance to CDK4/6 inhibitors. Therefore, to identify metabolites that drive resistance, we performed an untargeted metabolomics on CAF conditioned media. We observed that the CAF conditioned media was enriched for TCA cycle intermediates, including succinate. Succinate is an oncometabolite that has an essential function in energy metabolism. Aside from causing metabolic reprogramming, succinate can be transported out of the mitochondria to perform other biological processes, such as epigenetic modifications and pseudohypoxia. Furthermore, succinate in the extracellular space binds to the succinate receptor SUCNR1 and induces G-protein coupled receptor-mediated signaling in cells expressing SUCNR1. To assess mechanism of resistance that these metabolites might induce, we treated breast cancer cells with the most enriched metabolites we had identified in our metabolomics assay. These experiments identified succinate as driver of CDK4/6 inhibitor resistance in ER+ breast cancer cells. Furthermore, treating tumor cells with SUCNR1 antagonist increased their sensitivity to CDK4/6-inhibitors, showing that succinate mediated resistance is conveyed via SUCNR1. This suggests that CAF secreted succinate plays a role in supporting a pro-tumor environment by promoting drug resistance towards CDK4/6 inhibitors. Defining this mechanism of resistance will elucidate potential therapeutic targets to augment patient response to therapy. Citation Format: Jenny Hogstrom, Boryana Petrova, Camila Perea, Madelyn Ward, Gerburg Wulf, Laura Collins, Naama Kanarek, Taru Muranen. Mechanisms of succinate-mediated drug resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1222.

Abstract 6627: Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge, mainly due to therapy resistance linked to the immunosuppressive tumor microenvironment (TME) within PDAC tumors. This TME is characterized by a fibrotic stroma and an abundance of immunosuppressive cells, notably tumor-associated macrophages (TAMs). TAMs crucially hinder effector T cell activity and contribute to T cell exhaustion. Redirecting TAMs toward an immunostimulatory state has emerged as a promising approach in PDAC treatment. Recent investigations, including our own studies, reveal a profound influence of metabolic pathways on the immune phenotype of TAMs. Specifically, dysregulation in branched-chain amino acid (BCAA) metabolism has been observed in PDAC patients, manifesting as elevated circulating BCAA levels correlating with a doubled risk of PDAC development. However, the extent to which BCAA metabolism influences TAM phenotype and anti-tumor immune responses remains a largely unexplored territory, presenting a challenge for PDAC treatment. Our focus revolves around understanding how heightened BCAA levels in PDAC affect TAM phenotype, the immunosuppressive tumor environment, and tumor progression. Employing an orthotopic PDAC model and genetically modified mice, specifically BCKDKfl/fl BCKDK LysMcre/+ (mimicking increased BCAA oxidation), alongside a BCKDK-targeting pharmacological inhibitor (BT-2), multicolor flow cytometry, and in vitro models, our research has revealed crucial insights. We found a diminished expression of BCAA oxidation genes in TAMs compared to normal pancreatic macrophages. Interestingly, M1 and M2 macrophages seemed to utilize BCAAs differently, reflected in varying levels of TCA cycle intermediates derived from BCAA oxidation. Notably, boosting BCAA oxidation specifically in TAMs through genetic modifications resulted in reduced PDAC growth associated with an enhanced immunostimulatory TAM phenotype and activation of CD8+ T cells in the TME. Conversely, administering BCAAs induced an immunosuppressive TAM phenotype, fostering PDAC growth, suggesting a direct influence of BCAAs on TAM phenotype. Pharmacologically enhancing BCAA oxidation via BT-2 bolstered immune activation within the tumor environment and reduced PDAC growth. Intriguingly, our analysis of publicly available data unveiled a significant correlation between high BCAA oxidation and favorable responses to anti-PD1 therapy in cancer patients. Our findings emphasize the pivotal role of BCAA metabolism in shaping the immune landscape of PDAC, offering potential avenues for therapeutic interventions targeting TAMs and mitigating TME immunosuppression. Citation Format: Gauri Mirji, Sajad Ahmad Bhat, Zachary Schug, Ben Z. Stanger, Zoltan Arany, Rahul S. Shinde. Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6627.

Rapid dissemination of host metabolism–manipulating genes via integrative and conjugative elements

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.