Pooja Jha

Systems proteomics of liver mitochondria function

Expanded proteomic analysis of metabolism Combined analysis of large data sets characterizing genes, transcripts, and proteins can elucidate biological functions and disease processes. Williams et al. report an exceptionally detailed characterization of mitochondrial function in a genetic reference panel of recombinant inbred mice. They measured the metabolic function of nearly 400 mice under various environmental conditions and collected detailed quantitative information from livers of the animals on over 25,000 transcripts. These data were integrated with quantitation of over 2500 proteins and nearly 1000 metabolites. Such analysis showed a frequent lack of correlation of transcript and protein abundance, enabled the identification of genomic variants of mitochondrial enzymes that caused inborn errors in metabolism, and revealed two genes that appear to function in cholesterol metabolism. Science, this issue p. 10.1126/science.aad0189 Advances in mass spectrometry yield insights into mitochondrial function. INTRODUCTION Over the past two decades, continuous improvements in “omics” technologies have driven an ever-greater capacity to define the relationships between genetics, molecular pathways, and overall phenotypes. Despite this progress, the majority of genetic factors influencing complex traits remain unknown. This is exemplified by mitochondrial supercomplex assembly, a critical component of the electron transport chain, which remains poorly characterized. Recent advances in mass spectrometry have expanded the scope and reliability of proteomics and metabolomics measurements. These tools are now capable of identifying thousands of factors driving diverse molecular pathways, their mechanisms, and consequent phenotypes and thus substantially contribute toward the understanding of complex systems. RATIONALE Genome-wide association studies (GWAS) have revealed many causal loci associated with specific phenotypes, yet the identification of such genetic variants has been generally insufficient to elucidate the molecular mechanisms linking these genetic variants with specific phenotypes. A multitude of control mechanisms differentially affect the cellular concentrations of different classes of biomolecules. Therefore, the identification of the causal mechanisms underlying complex trait variation requires quantitative and comprehensive measurements of multiple layers of data—principally of transcripts, proteins, and metabolites and the integration of the resulting data. Recent technological developments now support such multiple layers of measurements with a high degree of reproducibility across diverse sample or patient cohorts. In this study, we applied a multilayered approach to analyze metabolic phenotypes associated with mitochondrial metabolism. RESULTS We profiled metabolic fitness in 386 individuals from 80 cohorts of the BXD mouse genetic reference population across two environmental states. Specifically, this extensive phenotyping program included the analysis of metabolism, mitochondrial function, and cardiovascular function. To understand the variation in these phenotypes, we quantified multiple, detailed layers of systems-scale measurements in the livers of the entire population: the transcriptome (25,136 transcripts), proteome (2622 proteins), and metabolome (981 metabolites). Together with full genomic coverage of the BXDs, these layers provide a comprehensive view on overall variances induced by genetics and environment regarding metabolic activity and mitochondrial function in the BXDs. Among the 2600 transcript-protein pairs identified, 85% of observed quantitative trait loci uniquely influenced either the transcript or protein level. The transomic integration of molecular data established multiple causal links between genotype and phenotype that could not be characterized by any individual data set. Examples include the link between D2HGDH protein and the metabolite D-2-hydroxyglutarate, the BCKDHA protein mapping to the gene Bckdhb, the identification of two isoforms of ECI2, and mapping mitochondrial supercomplex assembly to the protein COX7A2L. These respective measured variants in these mitochondrial proteins were in turn associated with varied complex metabolic phenotypes, such as heart rate, cholesterol synthesis, and branched-chain amino acid metabolism. Of note, our transomics approach clarified the contested role of COX7A2L in mitochondrial supercomplex formation and identified and validated Echdc1 and Mmab as involved in the cholesterol pathway. CONCLUSION Overall, these findings indicate that data generated by next-generation proteomics and metabolomics techniques have reached a quality and scope to complement transcriptomics, genomics, and phenomics for transomic analyses of complex traits. Using mitochondria as a case in point, we show that the integrated analysis of these systems provides more insights into the emergence of the observed phenotypes than any layer can by itself, highlighting the complementarity of a multilayered approach. The increasing implementation of these omics technologies as complements, rather than as replacements, will together move us forward in the integrative analysis of complex traits. Model of the transomics analysis. A transomics approach was taken to analyze genetic and environmental variation in metabolic and mitochondrial phenotypes by measuring five distinct layers of biology in a diverse population of BXD mice. The combined analysis of all layers together provides additional information not yielded by any single omics approach. Recent improvements in quantitative proteomics approaches, including Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS), permit reproducible large-scale protein measurements across diverse cohorts. Together with genomics, transcriptomics, and other technologies, transomic data sets can be generated that permit detailed analyses across broad molecular interaction networks. Here, we examine mitochondrial links to liver metabolism through the genome, transcriptome, proteome, and metabolome of 386 individuals in the BXD mouse reference population. Several links were validated between genetic variants toward transcripts, proteins, metabolites, and phenotypes. Among these, sequence variants in Cox7a2l alter its protein’s activity, which in turn leads to downstream differences in mitochondrial supercomplex formation. This data set demonstrates that the proteome can now be quantified comprehensively, serving as a key complement to transcriptomics, genomics, and metabolomics—a combination moving us forward in complex trait analysis.

Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents

The biological effects of urolithins remain poorly characterized, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which are found in the pomegranate fruit, as well as in nuts and berries. We identified urolithin A (UA) as a first-in-class natural compound that induces mitophagy both in vitro and in vivo following oral consumption. In C. elegans, UA prevented the accumulation of dysfunctional mitochondria with age and extended lifespan. Likewise, UA prolonged normal activity during aging in C. elegans, including mobility and pharyngeal pumping, while maintaining mitochondrial respiratory capacity. These effects translated to rodents, where UA improved exercise capacity in two different mouse models of age-related decline of muscle function, as well as in young rats. Our findings highlight the health benefits of urolithin A and its potential application in strategies to improve mitochondrial and muscle function.

Inhibiting poly ADP-ribosylation increases fatty acid oxidation and protects against fatty liver disease

BACKGROUND & AIMS To date, no pharmacological therapy has been approved for non-alcoholic fatty liver disease (NAFLD). The aim of the present study was to evaluate the therapeutic potential of poly ADP-ribose polymerase (PARP) inhibitors in mouse models of NAFLD. METHODS As poly ADP-ribosylation (PARylation) of proteins by PARPs consumes nicotinamide adenine dinucleotide (NAD+), we hypothesized that overactivation of PARPs drives NAD+ depletion in NAFLD. Therefore, we assessed the effectiveness of PARP inhibition to replenish NAD+ and activate NAD+-dependent sirtuins, hence improving hepatic fatty acid oxidation. To do this, we examined the preventive and therapeutic benefits of the PARP inhibitor (PARPi), olaparib, in different models of NAFLD. RESULTS The induction of NAFLD in C57BL/6J mice using a high-fat high-sucrose (HFHS)-diet increased PARylation of proteins by PARPs. As such, increased PARylation was associated with reduced NAD+ levels and mitochondrial function and content, which was concurrent with elevated hepatic lipid content. HFHS diet supplemented with PARPi reversed NAFLD through repletion of NAD+, increasing mitochondrial biogenesis and β-oxidation in liver. Furthermore, PARPi reduced reactive oxygen species, endoplasmic reticulum stress and fibrosis. The benefits of PARPi treatment were confirmed in mice fed with a methionine- and choline-deficient diet and in mice with lipopolysaccharide-induced hepatitis; PARP activation was attenuated and the development of hepatic injury was delayed in both models. Using Sirt1hep-/- mice, the beneficial effects of a PARPi-supplemented HFHS diet were found to be Sirt1-dependent. CONCLUSIONS Our study provides a novel and practical pharmacological approach for treating NAFLD, fueling optimism for potential clinical studies. LAY SUMMARY Non-alcoholic fatty liver disease (NAFLD) is now considered to be the most common liver disease in the Western world and has no approved pharmacological therapy. PARP inhibitors given as a treatment in two different mouse models of NAFLD confer a protection against its development. PARP inhibitors may therefore represent a novel and practical pharmacological approach for treating NAFLD.

Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE).

Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in‐gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN‐PAGE).

Analysis of mtDNA/nDNA Ratio in Mice

Mitochondrial DNA (mtDNA) lacks the protection provided by the nucleosomes in the nuclear DNA and does not have a DNA repair mechanism, making it highly susceptible to damage, which can lead to mtDNA depletion. mtDNA depletion compromises the efficient function of cells and tissues and thus impacts negatively on health. Here, we describe a brief and easy protocol to quantify mtDNA copy number by determining the mtDNA/nDNA ratio. The procedure has been validated using a cohort of young and aged mice.

Increased Hepatic PDGF-AA Signaling Mediates Liver Insulin Resistance in Obesity Associated Type 2 Diabetes

In type 2 diabetes (T2D), hepatic insulin resistance is strongly associated with nonalcoholic fatty liver disease (NAFLD). In this study, we hypothesized that the DNA methylome of livers from patients with T2D compared with livers of individuals with normal plasma glucose levels can unveil some mechanism of hepatic insulin resistance that could link to NAFLD. Using DNA methylome and transcriptome analyses of livers from obese individuals, we found that hypomethylation at a CpG site in PDGFA (encoding platelet-derived growth factor α) and PDGFA overexpression are both associated with increased T2D risk, hyperinsulinemia, increased insulin resistance, and increased steatohepatitis risk. Genetic risk score studies and human cell modeling pointed to a causative effect of high insulin levels on PDGFA CpG site hypomethylation, PDGFA overexpression, and increased PDGF-AA secretion from the liver. We found that PDGF-AA secretion further stimulates its own expression through protein kinase C activity and contributes to insulin resistance through decreased expression of insulin receptor substrate 1 and of insulin receptor. Importantly, hepatocyte insulin sensitivity can be restored by PDGF-AA–blocking antibodies, PDGF receptor inhibitors, and by metformin, opening therapeutic avenues. Therefore, in the liver of obese patients with T2D, the increased PDGF-AA signaling contributes to insulin resistance, opening new therapeutic avenues against T2D and possibly NAFLD.

Systems Analyses Reveal Physiological Roles and Genetic Regulators of Liver Lipid Species

The genetics of individual lipid species and their relevance in disease is largely unresolved. We profiled a subset of storage, signaling, membrane, and mitochondrial liver lipids across 385 mice from 47 strains of the BXD mouse population fed chow or high-fat diet and integrated these data with complementary multi-omics datasets. We identified several lipid species and lipid clusters with specific phenotypic and molecular signatures and, in particular, cardiolipin species with signatures of healthy and fatty liver. Genetic analyses revealed quantitative trait loci for 68% of the lipids (lQTL). By multi-layered omics analyses, we show the reliability of lQTLs to uncover candidate genes that can regulate the levels of lipid species. Additionally, we identified lQTLs that mapped to genes associated with abnormal lipid metabolism in human GWASs. This work provides a foundation and resource for understanding the genetic regulation and physiological significance of lipid species.

Genetic Regulation of Plasma Lipid Species and Their Association with Metabolic Phenotypes

SUMMARY The genetic regulation and physiological impact of most lipid species are unexplored. Here, we profiled 129 plasma lipid species across 49 strains of the BXD mouse genetic reference population fed either chow or a high-fat diet. By integrating these data with genomics and phenomics datasets, we elucidated genes by environment (diet) interactions that regulate systemic metabolism. We found quantitative trait loci (QTLs) for ~94% of the lipids measured. Several QTLs harbored genes associated with blood lipid levels and abnormal lipid metabolism in human genome-wide association studies. Lipid species from different classes provided signatures of metabolic health, including seven plasma triglyceride species that associated with either healthy or fatty liver. This observation was further validated in an independent mouse model of non-alcoholic fatty liver disease (NAFLD) and in plasma from NAFLD patients. This work provides a resource to identify plausible genes regulating the measured lipid species and their association with metabolic traits.