Biology subjects

Ilkayeva, O.

Publications and source records attributed to Ilkayeva, O..

10 recordsLinked to original sources

bioRxiv2025

SIRT4 Controls Macrophage Function and Wound Healing through Control of Protein Itaconylation in Mice

Proper regulation of inflammatory responses is essential for organismal health. Dysregulation can lead to accelerated development of the diseases of aging and the aging process itself. Here, we identify a novel enzymatic activity of the mitochondrial sirtuin SIRT4 as a lysine deitaconylase that regulates macrophage inflammatory responses. Itaconate is a metabolite abundantly produced in activated macrophages. We find it forms a protein modification called lysine itaconylation. Using biochemical and proteomics approaches, we demonstrate that SIRT4 efficiently removes this modification from target proteins both in vitro and in vivo. In macrophages, elevated protein itaconylation increases upon LPS stimulation, coinciding with elevated SIRT4 expression. SIRT4-deficient macrophages exhibit significantly increased IL-1{beta} production in response to LPS stimulation. This phenotype is intrinsic to macrophages, as demonstrated by both lentiviral over-expression and acute SIRT4 knockdown models. Mechanistically, we identify key enzymes in branched-chain amino acid (BCAA) metabolism as targets of hyperitaconylation in SIRT4-deficient macrophages. The BCKDH complex component dihydrolipoamide branched chain transacylase E2 (DBT) is hyperitaconylated and has reduced BCKDH activity in SIRT4KO macrophages. Physiologically, SIRT4-deficient mice exhibit significantly delayed wound healing, demonstrating a consequence of dysregulated macrophage function. Our data reveal a novel protein modification pathway in immune cells and establish SIRT4 as a critical regulator at the intersection of metabolism and inflammation. These findings have implications for understanding immune dysregulation in aging and metabolic disease.

molecular biology↗

bioRxiv2024

Integration of metabolomic and transcriptomic analyses reveals novel regulatory functions of the ChREBP transcription factor in energy metabolism

Carbohydrate Response Element-Binding Protein (ChREBP) is a transcription factor that activates key genes involved in glucose, fructose, and lipid metabolism in response to carbohydrate feeding, but its other potential roles in metabolic homeostasis have not been as well studied. We used liver-selective GalNAc-siRNA technology to suppress expression of ChREBP in rats fed a high fat/high sucrose diet and characterized hepatic and systemic responses by integrating transcriptomic and metabolomic analyses. GalNAc-siChREBP-treated rats had lower levels of multiple short-chain acyl CoA metabolites compared to rats treated with GalNAc-siCtrl containing a non-targeting siRNA sequence. These changes were related to a sharp decrease in free CoA levels in GalNAc-siChREBP treated-rats, accompanied by lower expression of transcripts encoding enzymes and transporters involved in CoA biosynthesis. These activities of ChREBP likely contribute to its complex effects on hepatic lipid and energy metabolism. While core enzymes of fatty acid (FA) oxidation are induced by ChREBP knockdown, accumulation of liver acylcarnitines and circulating ketones indicate diversion of acetyl CoA to ketone production rather than complete oxidation in the TCA cycle. Despite strong suppression of pyruvate kinase and activation of pyruvate dehydrogenase, pyruvate levels were maintained, likely via increased expression of pyruvate transporters, and decreased expression of lactate dehydrogenase and alanine transaminase. GalNAc-siChREBP treatment increased hepatic citrate and isocitrate levels while decreasing levels of distal TCA cycle intermediates. The drop in free CoA levels, needed for the 2-ketoglutarate dehydrogenase reaction, as well as a decrease in transcripts encoding the anaplerotic enzymes pyruvate carboxylase, glutamate dehydrogenase, and aspartate transaminase likely contributed to these effects. GalNAc-siChREBP treatment caused striking increases in PRPP and ZMP/AICAR levels, and decreases in GMP, IMP, AMP, NaNM, NAD(P), and NAD(P)H levels, accompanied by reduced expression of enzymes that catalyze late steps in purine and NAD synthesis. ChREBP suppression also increased expression of a set of plasma membrane amino acid transporters, possibly as an attempt to replenish TCA cycle intermediates. In sum, combining transcriptomic and metabolomic analyses has revealed regulatory functions of ChREBP that go well beyond its canonical roles in control of carbohydrate and lipid metabolism to now include mitochondrial metabolism and cellular energy balance.

physiology↗

bioRxiv2023

Prevotella copri-linked effects of a therapeutic food for malnutrition

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the metagenome assembled genomes (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed reverse translation experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

microbiology↗

bioRxiv2022

Temporal dynamics of the multi-omic response to endurance exercise training across tissues

Regular exercise promotes whole-body health and prevents disease, yet the underlying molecular mechanisms throughout a whole organism are incompletely understood. Here, the Molecular Transducers of Physical Activity Consortium (MoTrPAC) profiled the temporal transcriptome, proteome, metabolome, lipidome, phosphoproteome, acetylproteome, ubiquitylproteome, epigenome, and immunome in whole blood, plasma, and 18 solid tissues in Rattus norvegicus over 8 weeks of endurance exercise training. The resulting data compendium encompasses 9466 assays across 19 tissues, 25 molecular platforms, and 4 training time points in young adult male and female rats. We identified thousands of shared and tissue- and sex-specific molecular alterations. Temporal multi-omic and multi-tissue analyses demonstrated distinct patterns of tissue remodeling, with widespread regulation of immune, metabolism, heat shock stress response, and mitochondrial pathways. These patterns provide biological insights into the adaptive responses to endurance training over time. For example, exercise training induced heart remodeling via altered activity of the Mef2 family of transcription factors and tyrosine kinases. Translational analyses revealed changes that are consistent with human endurance training data and negatively correlated with disease, including increased phospholipids and decreased triacylglycerols in the liver. Sex differences in training adaptation were widespread, including those in the brain, adrenal gland, lung, and adipose tissue. Integrative analyses generated novel hypotheses of disease relevance, including candidate mechanisms that link training adaptation to non-alcoholic fatty liver disease, inflammatory bowel disease, cardiovascular health, and tissue injury and recovery. The data and analysis results presented in this study will serve as valuable resources for the broader community and are provided in an easily accessible public repository (https://motrpac-data.org/). HighlightsO_LIMulti-tissue resource identifies 35,439 analytes regulated by endurance exercise training at 5% FDR across 211 combinations of tissues and molecular platforms. C_LIO_LIInterpretation of systemic and tissue-specific molecular adaptations produced hypotheses to help describe the health benefits induced by exercise. C_LIO_LIRobust sex-specific responses to endurance exercise training are observed across multiple organs at the molecular level. C_LIO_LIDeep multi-omic profiling of six tissues defines regulatory signals for tissue adaptation to endurance exercise training. C_LIO_LIAll data are available in a public repository, and processed data, analysis results, and code to reproduce major analyses are additionally available in convenient R packages. C_LI

molecular biology↗

bioRxiv2022

Fitm2 is required for ER homeostasis and normal function of murine liver

The ER-resident protein fat-inducing transcript 2 (FIT2) catalyzes acyl-CoA cleavage in vitro and is required for endoplasmic reticulum (ER) homeostasis and normal lipid storage in cells. The gene encoding FIT2 is essential for the viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in vivo and how this activity affects liver, where the protein was discovered, are unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice exhibited elevated acyl-CoA levels, ER stress, and signs of liver injury. FIT2-LKO mice fed a chow diet had more triglycerides in their livers than control littermates due, in part, to impaired secretion of triglyceride-rich lipoproteins and reduced capacity for fatty acid oxidation. Challenging FIT2-LKO mice with a high-fat diet to increase FIT2 acyl-CoA substrates worsened hepatic ER stress and liver injury, but unexpectedly reversed the steatosis phenotype, similar to what is observed in FIT2-deficient cells loaded with fatty acids. Our findings support the model that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal hepatocyte function and ER homeostasis in murine liver.

cell biology↗

bioRxiv2022

Paradoxical activation of SREBP1c and de novo lipogenesis by hepatocyte-selective ACLY depletion in obese mice.

Hepatic steatosis associated with high fat diets, obesity and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl-coenzyme A (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high fat feeding is unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, while in contrast ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element-binding protein 1c (SREBP1c) and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels due to its increased conversion to Malonyl CoA (MalCoA) and palmitate. Together, these data indicate that in HFD fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or MalCoA, but rather by activities of DNL enzymes.

molecular biology↗

bioRxiv2021

Liver enhancer signature and regulatory network of non-alcoholic steatohepatitis resistance in humans with obesity

The molecular underpinnings of non-alcoholic steatohepatitis (NASH) development in patients are poorly understood. Active enhancer landscapes are known to determine cell states and behaviors. Super-enhancers, in particular, have helped reveal key disease drivers in several cancer types; however, they remain unexplored in human NASH. To define the enhancer signature of NASH-prone (NP) and NASH-resistant (NR) phenotypes in humans with obesity, we performed chromatin run-on sequencing (ChRO-seq) analysis on liver biopsies of individuals with obesity who were stratified into either NP or NR. We first demonstrated that NP and NR groups exhibit distinct active enhancer signatures. The subsequent identification of NP- and NR-specific super-enhancers revealed the specific genes that are likely the most critical for each of the phenotypes, including HES1 for NP and GATM for NR. Integrative analysis with results from genome-wide association studies of NAFLD and related traits identified disease/trait-loci specific to NP or NR enhancers. Further analysis of the ChRO-seq data pointed to critical roles for serine/glycine metabolism in NASH resistance, which was corroborate by profiling of circulating amino acids in the same patients. Overall, the distinct enhancer signatures of human NP and NR phenotypes revealed key genes, pathways, and transcription factor networks that promote NASH development.

genomics↗

bioRxiv2020

A Precision Medicine Approach to Stress Testing Using Metabolomics and MicroRibonucleic Acids

BackgroundAcute coronary syndrome (ACS) is a growing global health problem, and precision medicine techniques hold promise for the development of diagnostic indicators of ACS. In this pilot, we sought to assess the utility of an integrated analysis of metabolomic and microRNA data in peripheral blood to distinguish patients with abnormal cardiac stress testing from matched controls. MethodsWe used prospectively collected samples from emergency department (ED) patients placed in an ED-based observation unit who underwent stress testing for ACS. We isolated microRNA and quantified metabolites from plasma collected before and after stress testing in patients with myocardial ischemia on stress testing versus those with normal stress tests. The combined metabolomic and microRNA data were analyzed jointly for case (ischemia) and 1:1 matched control patients in a supervised, dimension-reducing discriminant analysis. Two integrative models were implemented: a baseline model utilizing data collected prior to stress-testing (T0) and a stress-delta model, which included the difference between post-stress test (T1) and pre-stress test (T0). ResultsSeven case patients with myocardial ischemia on ED cardiac stress testing (6 females, 85% Caucasian, mean Thrombolysis In Myocardial Infarction Score=3, 4 patients ultimately received percutaneous coronary intervention) were 1:1 age and sex-matched to controls. Several metabolites and microRNAs were differentially expressed between cases and controls. Integrative analysis of the baseline levels of metabolites and microRNA expression showed modest performance for distinguishing cases from controls with an overall error rate of 0.143. The stress-delta model showed worse performance for distinguishing cases from controls, with an overall error rate of 0.500. ConclusionsGiven our small sample size, results are hypothesis-generating. However, this pilot study shows a potential method for a precision medicine approach to cardiac stress testing in patients undergoing workup for ACS.

physiology↗

bioRxiv2019

Type-2-diabetes alters CSF but not plasma metabolomic and AD risk profiles in vervet monkeys

Epidemiological studies suggest that individuals with type 2 diabetes (T2D) have a 2-4 fold increased risk for developing Alzheimers disease (AD), however the exact mechanisms linking the two disease is unknown. In both conditions, the majority of pathophysiological changes (including glucose and insulin dysregulation, insulin resistance, and AD-related changes in A{beta} and tau) occur decades before the onset of clinical symptoms and diagnosis. In this study, we investigated the relationship between metabolic biomarkers associated with T2D and AD-related pathology, including A{beta} levels, from cerebrospinal fluid (CSF) and fasting plasma of healthy, prediabetic (PreD), and T2D vervet monkeys (Chlorocebus aethiops sabeus). Consistent with the human disease, T2D monkeys have increased plasma and CSF glucose levels as they transition from normoglycemia to pre-diabetic and diabetic states. Although plasma levels of acylcarnitines and amino acids remained largely unchanged, peripheral hyperglycemia correlated with decreased CSF acylcarnitines and CSF amino acids, including branched chain amino acid (BCAA) concentrations, suggesting profound changes in cerebral metabolism coincident with systemic glucose dysregulation. Moreover, CSF A{beta}40 and CSF A{beta}42 levels decreased in T2D monkeys, a phenomenon observed in the human course of AD which coincides with increased amyloid deposition within the brain. In agreement with our previous studies in mice, CSF A{beta}40 and CSF A{beta}42 were highly correlated with CSF glucose levels, suggesting that glucose levels in the brain are associated with changes in A{beta} metabolism. Interestingly, CSF A{beta}40 and CSF A{beta}42 levels were also highly correlated with plasma but not CSF lactate levels, suggesting that plasma lactate might serve as a potential biomarker of disease progression in AD. Moreover, CSF glucose and plasma lactate levels were correlated with CSF amino acid and acylcarnitine levels, demonstrating alterations in cerebral metabolism occurring with the onset of T2D. Together, these data suggest that peripheral metabolic changes associated with the development of T2D produce alterations in brain metabolism that lead to early changes in the amyloid cascade, similar to those observed in pre-symptomatic AD.

neuroscience↗

bioRxiv2018

Multi-Omic Profiling Reveals the Opposing Forces of Excess Dietary Sugar and Fat on Liver Mitochondria Protein Acetylation and Succinylation

Dietary macronutrient composition alters metabolism through several mechanisms, including post-translational modification (PTM) of proteins. To connect diet and molecular changes, here we performed short- and long-term feeding of mice with standard chow diet (SCD) and high-fat diet (HFD), with or without glucose or fructose supplementation, and quantified liver metabolites, 861 proteins, and 1,815 protein level-corrected mitochondrial acetylation and succinylation sites. Nearly half the acylation sites were altered by at least one diet; nutrient-specific changes in protein acylation sometimes encompass entire pathways. Although acetyl-CoA is an intermediate in both sugar and fat metabolism, acetyl-CoA had a dichotomous fate depending on its source; chronic feeding of dietary sugars induced protein hyperacetylation, whereas the same duration of HFD did not. Instead, HFD resulted in citrate accumulation, anaplerotic metabolism of amino acids, and protein hypo-succinylation. Together, our results demonstrate novel connections between dietary macronutrients, protein post-translational modifications, and regulation of fuel selection in liver.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC=\"FIGDIR/small/263426_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (53K):\norg.highwire.dtl.DTLVardef@1419c64org.highwire.dtl.DTLVardef@82a874org.highwire.dtl.DTLVardef@16023org.highwire.dtl.DTLVardef@4ed44e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract\n\nC_FIG

systems biology↗