ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD ratio, also referred to as reductive stress.

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS.

The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states - glucose, galactose, OXPHOS inhibition, and absence of pyruvate - designed to unmask the inter-dependence of these genes.

Hypoxia ameliorates brain hyperoxia and NAD deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4 mouse model of Leigh syndrome, continuously breathing 11% O (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4 mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood.

Evolutionary divergence reveals the molecular basis of EMRE dependence of the human MCU.

The mitochondrial calcium uniporter (MCU) is a calcium-activated calcium channel critical for signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan protein essential MCU regulator (EMRE) for conductance.

Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD ratio, also known as reductive stress.

Leigh Syndrome Mouse Model Can Be Rescued by Interventions that Normalize Brain Hyperoxia, but Not HIF Activation.

Leigh syndrome is a devastating mitochondrial disease for which there are no proven therapies. We previously showed that breathing chronic, continuous hypoxia can prevent and even reverse neurological disease in the Ndufs4 knockout (KO) mouse model of complex I (CI) deficiency and Leigh syndrome. Here, we show that genetic activation of the hypoxia-inducible factor transcriptional program via any of four different strategies is insufficient to rescue disease.

Exploring the In Vivo Role of the Mitochondrial Calcium Uniporter in Brown Fat Bioenergetics.

The mitochondrial calcium uniporter has been proposed to coordinate the organelle's energetics with calcium signaling. Uniporter current has previously been reported to be extremely high in brown adipose tissue (BAT), yet it remains unknown how the uniporter contributes to BAT physiology. Here, we report the generation and characterization of a mouse model lacking Mcu, the pore forming subunit of the uniporter, specifically in BAT (BAT-Mcu-KO).

Impaired hypoxic pulmonary vasoconstriction in a mouse model of Leigh syndrome.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children.

Cardiovascular homeostasis dependence on MICU2, a regulatory subunit of the mitochondrial calcium uniporter.

Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of , a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if expression was cardioprotective, we produced and characterized mice. Mutant mice had left atrial enlargement and cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction.

Hypoxia treatment reverses neurodegenerative disease in a mouse model of Leigh syndrome.

The most common pediatric mitochondrial disease is Leigh syndrome, an episodic, subacute neurodegeneration that can lead to death within the first few years of life, for which there are no proven general therapies. Mice lacking the complex I subunit, Ndufs4, develop a fatal progressive encephalopathy resembling Leigh syndrome and die at ≈60 d of age. We previously reported that continuously breathing normobaric 11% O from an early age prevents neurological disease and dramatically improves survival in these mice.

Mitochondrial dysfunction remodels one-carbon metabolism in human cells.

Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways.

Hypoxia as a therapy for mitochondrial disease.

Defects in the mitochondrial respiratory chain (RC) underlie a spectrum of human conditions, ranging from devastating inborn errors of metabolism to aging. We performed a genome-wide Cas9-mediated screen to identify factors that are protective during RC inhibition. Our results highlight the hypoxia response, an endogenous program evolved to adapt to limited oxygen availability.

EMRE is an essential component of the mitochondrial calcium uniporter complex.

The mitochondrial uniporter is a highly selective calcium channel in the organelle's inner membrane. Its molecular components include the EF-hand-containing calcium-binding proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore-forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex).

Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation.

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency.

Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter.

Mitochondria from diverse organisms are capable of transporting large amounts of Ca(2+) via a ruthenium-red-sensitive, membrane-potential-dependent mechanism called the uniporter. Although the uniporter's biophysical properties have been studied extensively, its molecular composition remains elusive. We recently used comparative proteomics to identify MICU1 (also known as CBARA1), an EF-hand-containing protein that serves as a putative regulator of the uniporter.

High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency.

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function.

A plasma signature of human mitochondrial disease revealed through metabolic profiling of spent media from cultured muscle cells.

Mutations in either the mitochondrial or nuclear genomes can give rise to respiratory chain disease (RCD), a large class of devastating metabolic disorders. Their clinical management is challenging, in part because we lack facile and accurate biomarkers to aid in diagnosis and in the monitoring of disease progression. Here we introduce a sequential strategy that combines biochemical analysis of spent media from cell culture with analysis of patient plasma to identify disease biomarkers.

Syndecan-1 expression in epithelial cells is induced by transforming growth factor beta through a PKA-dependent pathway.

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different.

Insulin promotes shedding of syndecan ectodomains from 3T3-L1 adipocytes: a proposed mechanism for stabilization of extracellular lipoprotein lipase.

Syndecans are a family of four transmembrane heparan sulfate proteoglycans that act as coreceptors for a variety of cell-surface ligands and receptors. Receptor activation in several cell types leads to shedding of syndecan-1 and syndecan-4 ectodomains into the extracellular space by metalloproteinase-mediated cleavage of the syndecan core protein. We have found that 3T3-L1 adipocytes express syndecan-1 and syndecan-4 and that their ectodomains are shed in response to insulin in a dose-, time-, and metalloproteinase-dependent manner.

Systematic identification of human mitochondrial disease genes through integrative genomics.

The majority of inherited mitochondrial disorders are due to mutations not in the mitochondrial genome (mtDNA) but rather in the nuclear genes encoding proteins targeted to this organelle. Elucidation of the molecular basis for these disorders is limited because only half of the estimated 1,500 mitochondrial proteins have been identified. To systematically expand this catalog, we experimentally and computationally generated eight genome-scale data sets, each designed to provide clues as to mitochondrial localization: targeting sequence prediction, protein domain enrichment, presence of cis-regulatory motifs, yeast homology, ancestry, tandem-mass spectrometry, coexpression and transcriptional induction during mitochondrial biogenesis.

Chondroitin sulfate chains on syndecan-1 and syndecan-4 from normal murine mammary gland epithelial cells are structurally and functionally distinct and cooperate with heparan sulfate chains to bind growth factors. A novel function to control binding of midkine, pleiotrophin, and basic fibroblast growth factor.

A comparative analysis was carried out of heparan sulfate (HS) and chondroitin sulfate (CS) chains of the ectodomains of hybrid type transmembrane proteoglycans, syndecan-1 and -4, synthesized simultaneously by normal murine mammary gland epithelial cells. Although the HS chains were structurally indistinguishable, intriguingly the CS chains were structurally and functionally distinct, probably reflecting the differential regulation of sulfotransferases involved in the synthesis of HS and CS. The CS chains of the two syndecans comprised nonsulfated, 4-O-, 6-O-, and 4,6-O-disulfated N-acetylgalactosamine-containing disaccharide units and were significantly different, with a higher degree of sulfation for syndecan-4.

Syndecan-1 and -4 synthesized simultaneously by mouse mammary gland epithelial cells bear heparan sulfate chains that are apparently structurally indistinguishable.

Many of the biological functions attributed to cell surface heparan sulfate (HS) proteoglycans, including the Syndecan family, are elicited through the interaction of their HS chains with soluble extracellular molecules. Tightly controlled, cell-specific sulfation and epimerization of HS precursors endows these chains with highly sulfated, iduronate-rich regions, which are major determinants of cytokine and matrix-protein binding and which are interspersed by N-acetylated, poorly sulfated regions. Until this study, there have been no comprehensive structural comparisons made on HS chains decorating simultaneously expressed, but different, syndecan core proteins.

Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse.

Mice lacking syndecan-1 are viable, fertile and have morphologically normal skin, hair and ocular surface epithelia. While studying the response of these mice to corneal epithelial and skin wounding, we identified defects in epithelial cell proliferation and regulation of integrin expression. mRNA profiling of corneal epithelial tissues obtained from wild-type and syndecan-1(-/-) mice suggest that these defects result from differences in overall gene transcription.