Metabolic Reprogramming Research Articles

Amidst Cytokine Storm in Metabolic Diseases: Can 2-deoxy-D-glucose (2-DG) Cure COVID-19?

Abstract: The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2)-related pandemic disease COVID-19, which began in China in 2019, became the leading cause of fatalities globally. The transmission rate of the virus is directly proportional to the so-called ‘dance dynamic’ energy of the spike protein as this phenomenon favours the ligand-receptor binding. The clinical spectrum of the COVID-19 disease, which includes (inflammation, cytokine storm, and multi-organ dysfunction), puts Corona Warriors active in various aspects of the disease, such as the efficient management of critically ill COVID-19 patients, in danger of losing their lives. Due to its effects on an ancient oxygen-independent glycolytic pathway, anti-inflammatory properties, and competitive interaction with viral proteins, 2-deoxy-D-glucose (2-DG), which is being developed in India at the Defence Research and Development Organization (DRDO), has emerged as a ground-breaking agent for COVID-19 treatment. After a virus gains entry into host cells, it is found that metabolic reprogramming takes place to meet the nutritional and energy requirements for virus reproduction. The recent approval of 2-DG for adjunctive emergency usage by the Drug Controller General of India (DCGI) may mark a turning point in the management of mild to moderate COVID-19 infection.

Bioinformatics and Experimental Study revealed LINC00982/ miR-183-5p/ABCA8 Axis Suppresses LUAD Progression.

Lung adenocarcinoma (LUAD) is a major health challenge worldwide with an undesirable prognosis. LINC00982 has been implicated as a tumor suppressor in diverse human cancers; however, its role in LUAD has not been fully characterized. Expression level and prognostic value of LINC00982 were investigated in pan-cancer and lung cancer from The Cancer Genome Atlas (TCGA) project. Differential expression analysis based on the LINC00982 expression level was performed in LUAD followed by gene set enrichment analysis (GSEA) and functional enrichment analyses. The association between LINC00982 expression and tumor immune microenvironment characteristics was evaluated. A potential ceRNA regulatory axis was identified and experimentally validated. We found that LINC00982 expression was downregulated and correlated with poor prognosis in LUAD. Enrichment analyses revealed that LINC00982 could inhibit DNA damage repair and cell proliferation, but enhance tumor metabolic reprogramming. We identified a competing endogenous RNA network involving LINC00982, miR-183-5p, and ATP-binding cassette subfamily A member 8 (ABCA8). Luciferase assays confirmed that miR-183-5p can interact with LINC00982 and ABCA8. Forced miR-183-5p expression reduced LINC00982 transcript levels and suppressed ABCA8 expression. Our findings revealed the LINC00982/miR-183-5p/ABCA8 axis as a potential therapeutic target in LUAD.

Recent Advances on PKM2 Inhibitors and Activators in Cancer Applications.

Metabolic reprogramming of cells, from the normal mode of glucose metabolism named glycolysis, is a pivotal characteristic of impending cancerous cells. Pyruvate kinase M2 (PKM2), an important enzyme that catalyzes the final rate-limiting stage during glycolysis, is highly expressed in numerous types of tumors and aids in development of favorable conditions for the survival of tumor cells. Increasing evidence has suggested that PKM2 is one of promising targets for innovative drug discovery, especially for the developments of antitumor therapeutics. Herein, we systematically summarize the recent advancement on PKM2 modulators including inhibitors and activators in cancer applications. We also discussed the classifications of pyruvate kinases in mammals and the biological functions of PKM2 in this review. We do hope that this review would provide a comprehensive understanding of the current research on PKM2 modulators, which may benefit the development of more potent PKM2-related drug candidates to treat PKM2-associated diseases including cancers in future.

Elucidating the Role of Lipid-Metabolism-Related Signal Transduction and Inhibitors in Skin Cancer

Lipids, as multifunctional molecules, play a crucial role in a variety of cellular processes. These include regulating membrane glycoprotein functions, controlling membrane trafficking, influencing apoptotic pathways, and affecting drug transport. In addition, lipid metabolites can alter the surrounding microenvironment in ways that might encourage tumor progression. The reprogramming of lipid metabolism is pivotal in promoting tumorigenesis and cancer progression, with tumors often displaying significant changes in lipid profiles. This review concentrates on the essential factors that drive lipid metabolic reprogramming, which contributes to the advancement and drug resistance in melanoma. Moreover, we discuss recent advances and current therapeutic strategies that employ small-molecule inhibitors to target lipid metabolism in skin cancers, particularly those associated with inflammation and melanoma.

Loss of Bcl-3 regulates macrophage polarization by promoting macrophage glycolysis.

M1/M2 macrophage polarization plays an important role in regulating the balance of the microenvironment within tissues. Moreover, macrophage polarization involves the reprogramming of metabolism, such as glucose and lipid metabolism. Transcriptional coactivator B-cell lymphoma-3 (Bcl-3) is an atypical member of the IκB family that controls inflammatory factor levels in macrophages by regulating nuclear factor kappa B pathway activation. However, the relationship between Bcl-3 and macrophage polarization and metabolism remains unclear. In this study, we show that the knockdown of Bcl-3 in macrophages can regulate glycolysis-related gene expression by promoting the activation of the nuclear factor kappa B pathway. Furthermore, the loss of Bcl-3 was able to promote the interferon gamma/lipopolysaccharide-induced M1 macrophage polarization by accelerating glycolysis. Taken together, these results suggest that Bcl-3 may be a candidate gene for regulating M1 polarization in macrophages.

Establishment of disulfidptosis-related LncRNA signature as biomarkers in colon adenocarcinoma.

Metabolic reprogramming is a hallmark of cancer and plays a key role in precision oncology treatment. Long non-coding RNAs (lncRNAs) regulate cancer cell behavior, including metabolism. Disulfidptosis, a newly identified form of regulated cell death triggered by glucose starvation, has yet to be fully understood in colon adenocarcinoma (COAD). This study aimed to confirm the existence and role of disulfidptosis in COAD and identify disulfidptosis-related lncRNAs that may be targeted to induce disulfidptosis in COAD. PI and F-actin staining were used to observe disulfidptosis in COAD cell lines. Disulfidptosis-related lncRNAs were identified based on the expression of disulfidptosis-associated genes in the TCGA-COAD database. A four-lncRNA signature for disulfidptosis was established. Subsequently, loss-of-function assays explored the roles of AC013652.1 and MCM3AP-AS1 in disulfidptosis. Disulfidptosis was observed in COAD cells under glucose starvation and could be reversed by agents that prevent disulfide stress, such as dithiothreitol (DTT) and tris-(2-carboxyethyl)-phosphine (TCEP). The prognostic value of disulfidptosis-associated genes in COAD patients was confirmed, with higher expression indicating longer survival. A disulfidptosis-related lncRNA signature comprising four lncRNAs was established based on the expression of these genes. Among these, AC013652.1 and MCM3AP-AS1 predicted worse prognoses. Furthermore, inhibiting AC013652.1 or MCM3AP-AS1 increased disulfidptosis-associated gene expression and cellular death, which could be reversed by DTT and TCEP. This study provides hitherto undocumented evidence of the existence of disulfidptosis and the prognostic value of disulfidptosis-associated genes in COAD. Importantly, we identified lncRNAs AC013652.1 and MCM3AP-AS1, which suppress disulfidptosis and may serve as potential therapeutic targets for COAD.

LncRNA TUG1 mediates microglial inflammatory activation by regulating glucose metabolic reprogramming

Microglia are natural immune cells in the central nervous system, and the activation of microglia is accompanied by a reprogramming of glucose metabolism. In our study, we investigated the role of long non-coding RNA taurine-upregulated gene 1 (TUG1) in regulating microglial glucose metabolism reprogramming and activation. BV2 cells were treated with Lipopolysaccharides (LPS)/Interferon-γ (IFN-γ) to establish a microglial activation model. The glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) was used as a control. The expression levels of TUG1 mRNA and proinflammatory cytokines such as Interleukin-1β (IL-1β), Interleukin -6, and Tumor Necrosis Factor-α mRNA and anti-inflammatory cytokines such as IL-4, Arginase 1(Arg1), CD206, and Ym1 were detected by RT-qPCR. TUG1 was silenced using TUG1 siRNA and knocked out using CRISPR/Cas9. The mRNA and protein expression levels of key enzymes involved in glucose metabolism, such as Hexokinase2, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Lactate dehydrogenase, Glucose 6 phosphate dehydrogenase, and Pyruvate dehydrogenase (PDH), were determined by RT-qPCR and Western blotting. The glycolytic rate of microglial cells was measured using Seahorse. Differential metabolites were determined by metabolomics, and pathway enrichment was performed using these differential metabolites. Our findings revealed that the expression of TUG1 was elevated in proinflammatory-activated microglia and positively correlated with the levels of inflammatory factors. The expression of anti-inflammatory cytokines such as IL-4, Arg1, CD206, and Ym1 were decreased when induced with LPS/IFN-γ. However, this decrease was reversed by the treatment with 2-DG. Silencing of GAPDH led to an increase in the expression of TUG1 and inflammatory factors. TUG1 knockout (TUG1KO) inhibited the expression of glycolytic key enzymes and promoted the expression of oxidative phosphorylation key enzymes, shifting the metabolic profile of activated microglia from glycolysis to oxidative phosphorylation. Additionally, TUG1KO reduced the accumulation of metabolites, facilitating the restoration of the tricarboxylic acid cycle and enhancing oxidative phosphorylation in microglia. Furthermore, the downregulation of TUG1 was found to reduce the expression of both proinflammatory and anti-inflammatory cytokines under normal conditions. Interestingly, when induced with LPS/IFN-γ, TUG1 downregulation showed a potentially beneficial effect on microglia in terms of inflammation. Downregulation of TUG1 expression inhibits glycolysis and facilitates the shift of microglial glucose metabolism from glycolysis to oxidative phosphorylation, promoting their transformation towards an anti-inflammatory phenotype and exerting anti-inflammatory effects in BV2.

Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.

Nano‐Fat Actuated Lipometabolic Reprogramming of Macrophages for Intracellular Infections in Biofilm Microenvironment

AbstractMetabolic competition is a zero‐sum game between bacterial biofilms and host immune responses on the surface of medical implants. In an in vitro biofilm‐macrophages co‐culture system, it is found that suppressed lipid metabolic processes in host macrophages in the biofilm microenvironment correlates with immune tolerance and intracellular persistence. Reactivation of immune cells against bacterial infection by reprogramming lipid metabolism through the supply of lipids such as oleic acid (OA) is a promising strategy, but this cannot completely destroy the bacterial biofilms. Nanomaterial‐based zinc ion interference therapy in antibacterial field emerges relying on the outstanding benefits of nanomaterials. Therefore, a targeted nano‐fat HSA‐IR820@OA@ZIF‐8 (HIROZ) with near infrared‐II (NIR‐II) photothermal capacities is developed for the destrcution of the biofilms via zinc ion combined photothermal therapy. The improved cellular compatibility and enhanced intracellular uptake make HIROZ induce intracellular lipid droplets (LDs) formation in macrophages. Photothermal combined zinc ion‐induced metabolic interference augments the antimicrobial effect of LDs and activates lipometabolic reprogramming‐mediated antibacterial immune responses via mitochondrial stress. In the mouse wound biofilm infections model and subcutaneous implant‐associated biofilm infections (IABIs) model, HIROZ demonstrates sustained and thorough biofilm scavenging based on reprogramming of lipid metabolism, providing a new idea for metabolic interference‐centered therapeutic strategies for full‐scale IABIs eliminations.

AGC1-mediated Metabolic Reprogramming and Autophagy Sustain Survival of Hepatocellular Carcinoma Cells under Glutamine Deprivation.

The dependence of hepatocellular carcinoma (HCC) cells on glutamine suggests the feasibility of targeting glutamine metabolism for therapy. However, drugs inhibiting glutamine uptake and breakdown have not shown promising outcomes. Therefore, investigating the mechanism of glutamine metabolism reprogramming in HCC cells is crucial. We used bioinformatics approaches to investigate the metabolic flux of glutamine in HCC cells and validated it using qRT-PCR and western blotting. HCC cells were cultured in glutamine-deprived medium, and changes in glutamate and ATP levels were monitored. Western blotting was employed to assess the expression of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) and autophagy-related proteins. The impact of Solute carrier family 25 member 12 (AGC1) on HCC cell proliferation was studied using CCK-8 and colony formation assays. Furthermore, the effects of AGC1 knockdown via siRNA on metabolic reprogramming and energy supply during glutamine deprivation in HCC were explored. During glutamine deprivation, HCC cells sustain cytosolic asparagine synthesis and ATP production through AGC1. Low ATP levels activate AMPK and inhibit mTOR activation, inducing autophagy to rescue HCC cell survival. Knockdown of AGC1 inhibits mitochondrial aspartate output and continuously activates autophagy, rendering HCC cells more sensitive to glutamine deprivation. AGC1 serves as a critical node in the reprogramming of glutamine metabolism and energy supply in HCC cells. This study provides theoretical support for overcoming resistance to drugs targeting glutamine metabolism.

Single-Cell RNA Sequencing Analysis Reveals Metabolic Changes in Epithelial Glycosphingolipids and Establishes a Prognostic Risk Model for Pancreatic Cancer

Objective: Metabolic reprogramming serves as a distinctive feature of cancer, impacting proliferation and metastasis, with aberrant glycosphingolipid expression playing a crucial role in malignancy. Nevertheless, limited research has investigated the connection between glycosphingolipid metabolism and pancreatic cancer. Methods: This study utilized a single-cell sequencing dataset to analyze the cell composition in pancreatic cancer tissues and quantified single-cell metabolism using a newly developed computational pipeline called scMetabolism. A gene signature developed from the differential expressed genes (DEGs), related to epithelial cell glycosphingolipid metabolism, was established to forecast patient survival, immune response, mutation status, and reaction to chemotherapy with pancreatic adenocarcinoma (PAAD). Results: The single-cell sequencing analysis revealed a significant increase in epithelial cell proportions in PAAD, with high glycosphingolipid metabolism occurring in the cancerous tissue. A six-gene signature prognostic model based on abnormal epithelial glycosphingolipid metabolism was created and confirmed using publicly available databases. Patients with PAAD were divided into high- and low-risk categories according to the median risk score, with those in the high-risk group demonstrating a more unfavorable survival outcome in all three cohorts, with higher rates of gene mutations (e.g., KRAS, CDKN2A), increased levels of immunosuppressive cells (macrophages, Th2 cells, regulatory T cells), and heightened sensitivity to Acetalax and Selumetinlb. Conclusions: Abnormal metabolism of glycosphingolipids in epithelial cells may promote the development of PAAD. A model utilizing a gene signature associated with epithelial glycosphingolipids metabolism has been established, serving as a valuable indicator for the prognostic stratification of patients with PAAD.

Comprehensive transcriptomic and metabolomic analysis of porcine intestinal epithelial cells after PDCoV infection

IntroductionPorcine deltacoronavirus (PDCoV), an emerging swine enteropathogenic coronavirus with worldwide distribution, mainly infects newborn piglets with severe diarrhea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. However, the underlying pathogenic mechanisms of PDCoV infection and the effects of PDCoV infection on host transcripts and metabolites remain incompletely understood.MethodsThis study investigated a combined transcriptomic and metabolomic analysis of porcine intestinal epithelial cells (IPEC-J2) following PDCoV infection by LC/MS and RNA-seq techniques. A total of 1,401 differentially expressed genes and 254 differentially accumulated metabolites were detected in the comparison group of PDCoV-infected vs. mock-infected.Results and discussionWe found that PDCoV infection regulates gene sets associated with multiple signaling pathways, including the neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, MAPK signaling pathway, chemokine signaling pathway, ras signaling pathway and so on. Besides, the metabolomic results showed that biosynthesis of cofactors, nucleotide metabolism, protein digestion and absorption, and biosynthesis of amino acid were involved in PDCoV infection. Moreover, integrated transcriptomics and metabolomics analyses revealed the involvement of ferroptosis in PDCoV infection, and exogenous addition of the ferroptosis activator erastin significantly inhibited PDCoV replication. Overall, these unique transcriptional and metabolic reprogramming features may provide a better understanding of PDCoV-infected IPEC-J2 cells and potential targets for antiviral treatment.

The metabolic characteristics and changes of chondrocytes in vivo and in vitro in osteoarthritis

Osteoarthritis (OA) is an intricate pathological condition that primarily affects the entire synovial joint, especially the hip, hand, and knee joints. This results in inflammation in the synovium and osteochondral injuries, ultimately causing functional limitations and joint dysfunction. The key mechanism responsible for maintaining articular cartilage function is chondrocyte metabolism, which involves energy generation through glycolysis, oxidative phosphorylation, and other metabolic pathways. Some studies have shown that chondrocytes in OA exhibit increased glycolytic activity, leading to elevated lactate production and decreased cartilage matrix synthesis. In OA cartilage, chondrocytes display alterations in mitochondrial activity, such as decreased ATP generation and increased oxidative stress, which can contribute to cartilage deterioration. Chondrocyte metabolism also involves anabolic processes for extracellular matrix substrate production and energy generation. During OA, chondrocytes undergo considerable metabolic changes in different aspects, leading to articular cartilage homeostasis deterioration. Numerous studies have been carried out to provide tangible therapies for OA by using various models in vivo and in vitro targeting chondrocyte metabolism, although there are still certain limitations. With growing evidence indicating the essential role of chondrocyte metabolism in disease etiology, this literature review explores the metabolic characteristics and changes of chondrocytes in the presence of OA, both in vivo and in vitro. To provide insight into the complex metabolic reprogramming crucial in chondrocytes during OA progression, we investigate the dynamic interaction between metabolic pathways, such as glycolysis, lipid metabolism, and mitochondrial function. In addition, this review highlights prospective future research directions for novel approaches to diagnosis and treatment. Adopting a multifaceted strategy, our review aims to offer a comprehensive understanding of the metabolic intricacies within chondrocytes in OA, with the ultimate goal of identifying therapeutic targets capable of modulating chondrocyte metabolism for the treatment of OA.

Research progress in the metabolic reprogramming of hepatocellular carcinoma (Review)

Research progress in the metabolic reprogramming of hepatocellular carcinoma (Review)

#2834 Analysis of cyst hetero- and homogeneity to unravel common pathways in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, with a prevalence of 1 in ca. 1000 individuals and accounting for 10% of the European dialysis population. It is most frequently (75-80%) caused by mutations in the PKD1 gene encoding Polycystin-1. Besides systemic manifestations such as liver cysts, hypertension and cardiac abnormalities, the most prominent phenotype is the gradual development of kidney cysts, leading to kidney enlargement and a decline in kidney function leading to end-stage kidney disease at an average age of ca. 50 years. The cysts arise from tubular epithelial cells in the nephron that start to dedifferentiate, proliferate and eventually secrete fluid into the cyst lumen promoting its swelling. This process is molecularly regulated by changes in Ca2+, cAMP, PKA and PI3K-Akt-mTOR signaling, among others. The renal cysts are further characterized by ciliary dysfunction, altered signal transduction, loss of polarity, apoptosis, mitochondrial abnormalities, metabolic reprogramming, inflammation, oxidative stress and epigenetic changes. However, the exact sequence of events and their relative importance in the process of cyst formation remains unclear. Therefore, we analyzed the transcriptomic cyst hetero- and homogeneity to identify the pathways that were different, but also shared among individual cysts. Method We performed RNA-Seq analysis on 40 individual cystic membranes of nephrectomized kidneys of 4 PKD1 patients. We included 4 samples of macroscopically healthy tissue from 2 ADPKD kidneys (histologically confirmed to have microcysts) and 4 samples from 4 healthy kidneys. Principal component, cluster and trajectory analysis was performed to explore cyst heterogeneity. Adjacent pieces of cystic membrane were analyzed for histology (H&E, Sirius Red) or stained with FITC-tagged Lotus Tetragonolobus Lectin (LTL) or Dolichos Biflorus Agglutinin (DBA), a proximal and distal tubular marker, respectively. Results Although the cysts clearly separated from the healthy tissues in the analyses, there was also a distinct heterogeneity observed among the cysts that was independent of patient, gender, cyst size, location or fluid color. Pathway analysis revealed that these changes were characterized by a gradual increase of interstitial (remodeling) and inflammation markers, and a gradual decrease in differentiation markers (e.g. development, intercellular junctions, basement membrane) along the trajectory pseudotime. We could identify 6 clusters of cystic samples, with 2 clusters deviating from the main trajectory. With respect to tubular/epithelial dedifferentiation, the decrease in proximal tubular markers already occurred in microcystic tissue and was stronger compared to the other tubular segments. Consequently, none of the analyzed cystic membranes stained positive for LTL, while others showed clear staining for DBA. Microcysts on the other hand stained positive for LTL or DBA. Several cysts showed RNA marker expression of multiple tubular segments. Indeed, smaller macrocysts (diameter in the mm range) were lined by both LTL- and DBA-positive regions. Conclusion By analyzing transcriptomic hetero- and homogeneity among individual cysts, the molecular alterations in ADPKD progression can be tracked down and determined whether they occur similarly in all cysts or at different levels. Our data also suggest that loss of proximal tubular markers in macrocysts is more prominent compared to the decrease in distal markers. Finally, inner cyst walls can consist of cyst-lining cells of multiple origins.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p < 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

The integration of metabolic and proteomic data uncovers an augmentation of the sphingolipid biosynthesis pathway during T-cell differentiation

Recent studies have highlighted the significance of cellular metabolism in the initiation of clonal expansion and effector differentiation of T cells. Upon exposure to antigens, naïve CD4+ T cells undergo metabolic reprogramming to meet their metabolic requirements. However, only few studies have simultaneously evaluated the changes in protein and metabolite levels during T cell differentiation. Our research seeks to fill the gap by conducting a comprehensive analysis of changes in levels of metabolites, including sugars, amino acids, intermediates of the TCA cycle, fatty acids, and lipids. By integrating metabolomics and proteomics data, we discovered that the quantity and composition of cellular lipids underwent significant changes in different effector Th cell subsets. Especially, we found that the sphingolipid biosynthesis pathway was commonly activated in Th1, Th2, Th17, and iTreg cells and that inhibition of this pathway led to the suppression of Th17 and iTreg cells differentiation. Additionally, we discovered that Th17 and iTreg cells enhance glycosphingolipid metabolism, and inhibition of this pathway also results in the suppression of Th17 and iTreg cell generation. These findings demonstrate that the utility of our combined metabolomics and proteomics analysis in furthering the understanding of metabolic transition during Th cell differentiation.

Ovarian Cancer Cell-Conditioning Medium Induces Cancer-Associated Fibroblast Phenoconversion through Glucose-Dependent Inhibition of Autophagy

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor–stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer.

#1914 Comprehensive single-cell sequencing and lipidomics identify diverse injury states and cholesterol dynamics of the PTs in AKI-to-CKD transition

Abstract Background and Aims The underlying cellular events and metabolic dysfunction of acute kidney injury (AKI) transformed to chronic kidney disease (CKD) is still largely unclear. Previous studies have suggested lipids and metabolites have a profound impact on acute and chronic inflammation. One of the main sites of renal lipid accumulation is the proximal tubule cells (PTs). Here we demonstrated the injury states and cholesterol dynamics of PTs and its potential function in AKI to CKD in unilateral ischemia-reperfusion injury (UIR) mice model. Method We employed single-cell combinatorial indexing lipidomics to analyze 15 mouse kidneys from a mouse model of failed repaired UIR at five time points. We identified cell types through unsupervised clustering analysis. Differential gene expression, enrichment analysis and main function scoring were applied to define functional heterogeneity and injury states of PTs. Next, pseudotime analysis of differentiation transitions was performed to create the diverse PT lineages. Finally, using SCENIC analysis, we identified the key transcription factors that drive the differentiation of PTs. Results We identified 13 main cell clusters and the dynamic changes in the process of AKI to CKD transition were analysed. A remarkable decrease in PTs after injury at day 1 followed with a recovery at day 3 post AKI were observed. Spatiotemporal profiling of the main cell types showed that the most injured areas in the acute stages of kidney injury are primarily concentrated in the outer stripe, specifically the region where the proximal straight tubules (PSTs) are located. In order to gain a higher resolution of the dynamics of PST subtypes during AKI to CKD, we performed sub-clustering analysis specifically on PSTs, partitioning them into 10 subclusters. Single-cell analysis identified maladaptive PSTs (PST-5) with the highest apoptosis and ferroptosis scores. Notably, pseudotime analysis revealed that PST-5 differentiated from PST-4, which has the highest lipid metabolism score. Indeed, among all the cells in the kidney, the PTs exhibited the most active metabolic reprogramming due to their differentiation and redifferentiation early after AKI. Lipid omics profiling reveals an abnormal elevation of the cholesterol metabolites 27-hydroxycholesterol (27-OH) and 25-hydroxycholesterol (25-OH) following AKI. Interestingly, the PST-4-specific expression of Cyp7b1 served as a key enzyme regulating the concentration of 27-OH. This gene has been found to be significantly absent after AKI. Moreover, the binding of 27HC, a natural ligand, to endogenous estrogen receptor alpha (ERα) leads to the upregulation of heme oxygenase 1 (Hmox1), thereby promoting ferroptosis. Finally, SCENIC analysis revealed that Cyp7b1-mediated accumulation of 27-HC is dependent on the transcription factor Atf3. Conclusion Our results indicate that Cyp7b1 deficiency confers ferroptosis, which are primarily dependent on the upregulation of cellular cholesterol homeostasis and 27-HC secretion, leading to the progression of AKI. These findings suggest that the Cyp7b1/Hmox1/Atf3/27-HC axis may serve as a potential therapeutic target for preventing the development of CKD.

#2160 Ketosis moderates the effect on kidney volume in dietary interventions for ADPKD—more insights on the KETO ADPKD trial

Abstract Background and Aims Autosomal-dominant polycystic kidney disease (ADPKD) is the most common monogenic disease leading to kidney failure. Tolvaptan, the only approved targeted treatment strategy, comes with adverse events such as hepatotoxicity and massive polyuria, limiting its use. Novel treatment strategies are urgently needed. Cyst-lining epithelial cells are glucose-dependent and metabolically inflexible. Evidence from polycystic kidney disease (PKD) animal models show that ketogenic dietary interventions (KDI) can ameliorate cyst growth and loss of kidney function. To enable clinical translation of these findings, our group set up a series of trials—from small cohorts and proof of principle studies to our most recent trial KETO-ADPKD, showing that KDIs are feasible and can work as a treatment for ADPKD [1]. This has received a lot of attention. With this post-hoc analyses, we aim to share further in-depth analyses of the factors moderating the effects we see on ADPKD. Method KETO-ADPKD is an exploratory randomized and controlled clinical trial (NCT04680780). Sixty-six patients were randomized to 3 months dietary intervention (ketogenic diet [KD] or water fasting [WF]) or the control group. Here, we explore correlations between biochemical readout parameters of ketosis and markers of disease progression. Results The KD group shows a promising, yet statistically not significant decline in height-adjusted total kidney volume (htTKV). Patients reaching high biochemical thresholds of ketosis however significantly reduced their htTKV in comparison with the control group (KD −16.3 ml/m, CG + 14.8 ml/m, p-value 0.049). This becomes even clearer when higher thresholds for adherence are administered: In a smaller group requiring not only beta-hydroxybutyrate (BHB) levels ≥0.5 mmol/l but breath acetone ≥5 ppm on 75% of daily measurements, htTKV could be reduced by −17.6 ml/m (KD) vs +14.8 ml/m (CG), p-value 0.026. The significant reduction of liver volume upon KD is not influenced by the level of ketosis. Beneficial effects on estimated glomerular filtration rate (eGFR) can be equally observed in all subsets. Weight loss ≥5% was nor associated with a more significant loss of kidney nor liver volume. Conclusion Subgroup analyses of the KETO-ADPKD trial show stronger impact of the dietary intervention with higher ketone body levels. In particular, ketogenesis as a marker of metabolic reprogramming strongly moderates the effects we see on kidney volume. The assessment of renal cyst fractions could further enlighten the effects on cyst burden. This is in line with preclinical data showing that ketosis rather than caloric intake is responsible for the amelioration of disease progression [2].