ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules.

Worldwide, over 800 million people are affected by kidney disease, yet its pathogenesis remains elusive, hindering the development of novel therapeutics. In this study, we used kidney-specific expression of quantitative traits and single-nucleus open chromatin analysis to show that genetic variants linked to kidney dysfunction on chromosome 20 target the acyl-CoA synthetase short-chain family 2 (ACSS2). By generating ACSS2-KO mice, we demonstrated their protection from kidney fibrosis in multiple disease models. Our analysis of primary tubular cells revealed that ACSS2 regulated de novo lipogenesis (DNL), causing NADPH depletion and increasing ROS levels, ultimately leading to NLRP3-dependent pyroptosis. Additionally, we discovered that pharmacological inhibition or genetic ablation of fatty acid synthase safeguarded kidney cells against profibrotic gene expression and prevented kidney disease in mice. Lipid accumulation and the expression of genes related to DNL were elevated in the kidneys of patients with fibrosis. Our findings pinpoint ACSS2 as a critical kidney disease gene and reveal the role of DNL in kidney disease.

NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury.

Our understanding of how global changes in cellular metabolism contribute to human kidney disease remains incompletely understood. Here we show that nicotinamide adenine dinucleotide (NAD+) deficiency drives mitochondrial dysfunction causing inflammation and kidney disease development. Using unbiased global metabolomics in healthy and diseased human kidneys, we identify NAD+ deficiency as a disease signature. Furthermore using models of cisplatin- or ischaemia-reperfusion induced kidney injury in male mice we observed NAD+ depletion Supplemental nicotinamide riboside or nicotinamide mononucleotide restores NAD+ levels and improved kidney function. We find that cisplatin exposure causes cytosolic leakage of mitochondrial RNA (mtRNA) and activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), both of which can be ameliorated by restoring NAD+. Male mice with RIG-I knock-out (KO) are protected from cisplatin-induced kidney disease. In summary, we demonstrate that the cytosolic release of mtRNA and RIG-I activation is an NAD+-sensitive mechanism contributing to kidney disease.

NAD precursors cycle between host tissues and the gut microbiome.

Nicotinamide adenine dinucleotide (NAD) is an essential redox cofactor in mammals and microbes. Here we use isotope tracing to investigate the precursors supporting NAD synthesis in the gut microbiome of mice. We find that dietary NAD precursors are absorbed in the proximal part of the gastrointestinal tract and not available to microbes in the distal gut. Instead, circulating host nicotinamide enters the gut lumen and supports microbial NAD synthesis. The microbiome converts host-derived nicotinamide into nicotinic acid, which is used for NAD synthesis in host tissues and maintains circulating nicotinic acid levels even in the absence of dietary consumption. Moreover, the main route from oral nicotinamide riboside, a widely used nutraceutical, to host NAD is via conversion into nicotinic acid by the gut microbiome. Thus, we establish the capacity for circulating host micronutrients to feed the gut microbiome, and in turn be transformed in a manner that enhances host metabolic flexibility.

NAD+ flux is maintained in aged mice despite lower tissue concentrations.

NAD+ is an essential coenzyme for all living cells. NAD+ concentrations decline with age, but whether this reflects impaired production or accelerated consumption remains unclear. We employed isotope tracing and mass spectrometry to probe age-related changes in NAD+ metabolism across tissues. In aged mice, we observed modest tissue NAD+ depletion (median decrease ∼30%). Circulating NAD+ precursors were not significantly changed, and isotope tracing showed the unimpaired synthesis of nicotinamide from tryptophan. In most tissues of aged mice, turnover of the smaller tissue NAD+ pool was modestly faster such that absolute NAD+ biosynthetic flux was maintained, consistent with more active NAD+-consuming enzymes. Calorie restriction partially mitigated age-associated NAD+ decline by decreasing consumption. Acute inflammatory stress induced by LPS decreased NAD+ by impairing synthesis in both young and aged mice. Thus, the decline in NAD+ with normal aging is relatively subtle and occurs despite maintained NAD+ production, likely due to increased consumption.

SIRT3 is required for liver regeneration but not for the beneficial effect of nicotinamide riboside.

Liver regeneration is critical to survival after traumatic injuries, exposure to hepatotoxins, or surgical interventions, yet the underlying signaling and metabolic pathways remain unclear. In this study, we show that hepatocyte-specific loss of the mitochondrial deacetylase SIRT3 drastically impairs regeneration and worsens mitochondrial function after partial hepatectomy. Sirtuins, including SIRT3, require NAD as a cosubstrate. We previously showed that the NAD precursor nicotinamide riboside (NR) promotes liver regeneration, but whether this involves sirtuins has not been tested. Here, we show that despite their NAD dependence and critical roles in regeneration, neither SIRT3 nor its nuclear counterpart SIRT1 is required for NR to enhance liver regeneration. NR improves mitochondrial respiration in regenerating WT or mutant livers and rapidly increases oxygen consumption and glucose output in cultured hepatocytes. Our data support a direct enhancement of mitochondrial redox metabolism as the mechanism mediating improved liver regeneration after NAD supplementation and exclude signaling via SIRT1 and SIRT3. Therefore, we provide the first evidence to our knowledge for an essential role for a mitochondrial sirtuin during liver regeneration and insight into the beneficial effects of NR.

FoxA-dependent demethylation of DNA initiates epigenetic memory of cellular identity.

Tissue-specific DNA methylation patterns are created by transcription factors that recruit methylation and demethylation enzymes to cis-regulatory elements. To date, it is not known whether transcription factors are needed to continuously maintain methylation profiles in development and mature tissues or whether they only establish these marks during organ development. We queried the role of the pioneer factor FoxA in generating hypomethylated DNA at liver enhancers. We discovered a set of FoxA-binding sites that undergo regional, FoxA-dependent demethylation during organ development. Conditional ablation of FoxA genes in the adult liver demonstrated that continued FoxA presence was not required to maintain the hypomethylated state, even when massive cell proliferation was induced. This study provides strong evidence for the stable, epigenetic nature of tissue-specific DNA methylation patterns directed by lineage-determining transcription factors during organ development.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

mTORC1 restrains adipocyte lipolysis to prevent systemic hyperlipidemia.

OBJECTIVE: Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo. METHODS: We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes. RESULTS: Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test. CONCLUSIONS: Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.

Hypothalamic mTORC2 is essential for metabolic health and longevity.

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that regulates growth and metabolism. mTOR is found in two protein complexes, mTORC1 and mTORC2, that have distinct components and substrates and are both inhibited by rapamycin, a macrolide drug that robustly extends lifespan in multiple species including worms and mice. Although the beneficial effect of rapamycin on longevity is generally attributed to reduced mTORC1 signaling, disruption of mTORC2 signaling can also influence the longevity of worms, either positively or negatively depending on the temperature and food source. Here, we show that loss of hypothalamic mTORC2 signaling in mice decreases activity level, increases the set point for adiposity, and renders the animals susceptible to diet-induced obesity. Hypothalamic mTORC2 signaling normally increases with age, and mice lacking this pathway display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice. Our results have implications for the use of mTORC2-inhibiting pharmaceuticals in the treatment of brain cancer and diseases of aging.

Nicotinamide mononucleotide preserves mitochondrial function and increases survival in hemorrhagic shock.

Hemorrhagic shock depletes nicotinamide adenine dinucleotide (NAD) and causes metabolic derangements that, in severe cases, cannot be overcome, even after restoration of blood volume and pressure. However, current strategies to treat acute blood loss do not target cellular metabolism. We hypothesized that supplemental nicotinamide mononucleotide (NMN), the immediate biosynthetic precursor to NAD, would support cellular energetics and enhance physiologic resilience to hemorrhagic shock. In a rodent model of decompensated hemorrhagic shock, rats receiving NMN displayed significantly reduced lactic acidosis and serum IL-6 levels, two strong predictors of mortality in human patients. In both livers and kidneys, NMN increased NAD levels and prevented mitochondrial dysfunction. Moreover, NMN preserved mitochondrial function in isolated hepatocytes cocultured with proinflammatory cytokines, indicating a cell-autonomous protective effect that is independent from the reduction in circulating IL-6. In kidneys, but not in livers, NMN was sufficient to prevent ATP loss following shock and resuscitation. Overall, NMN increased the time animals could sustain severe shock before requiring resuscitation by nearly 25% and significantly improved survival after resuscitation (P = 0.018), whether NMN was given as a pretreatment or only as an adjunct during resuscitation. Thus, we demonstrate that NMN substantially mitigates inflammation, improves cellular metabolism, and promotes survival following hemorrhagic shock.

Clock Regulation of Metabolites Reveals Coupling between Transcription and Metabolism.

The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.

Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR. CONCLUSION: NAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630).

Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy.

Liver sinusoidal endothelial cells (LSECs) are the main endothelial cells in the liver and are important for maintaining liver homeostasis as well as responding to injury. LSECs express cellular fibronectin containing the alternatively spliced extra domain A (EIIIA-cFN) and increase expression of this isoform after liver injury, although its function is not well understood. Here, we examined the role of EIIIA-cFN in liver regeneration following partial hepatectomy. We carried out two-thirds partial hepatectomies in mice lacking EIIIA-cFN and in their wild type littermates, studied liver endothelial cell adhesion on decellularized, EIIIA-cFN-containing matrices and investigated the role of cellular fibronectins in liver endothelial cell tubulogenesis. We found that liver weight recovery following hepatectomy was significantly delayed and that sinusoidal repair was impaired in EIIIA-cFN null mice, especially females, as was the lipid accumulation typical of the post-hepatectomy liver. In vitro, we found that liver endothelial cells were more adhesive to cell-deposited matrices containing the EIIIA domain and that cellular fibronectin enhanced tubulogenesis and vascular cord formation. The integrin α9ß1, which specifically binds EIIIA-cFN, promoted tubulogenesis and adhesion of liver endothelial cells to EIIIA-cFN. Our findings identify a role for EIIIA-cFN in liver regeneration and tubulogenesis. We suggest that sinusoidal repair is enhanced by increased LSEC adhesion, which is mediated by EIIIA-cFN.

Rapamycin Blocks Induction of the Thermogenic Program in White Adipose Tissue.

Rapamycin extends life span in mice, yet paradoxically causes lipid dysregulation and glucose intolerance through mechanisms that remain incompletely understood. Whole-body energy balance can be influenced by beige/brite adipocytes, which are inducible by cold and other stimuli via ß-adrenergic signaling in white adipose depots. Induction of beige adipocytes is considered a promising strategy to combat obesity because of their ability to metabolize glucose and lipids, dissipating the resulting energy as heat through uncoupling protein 1. Here, we report that rapamycin blocks the ability of ß-adrenergic signaling to induce beige adipocytes and expression of thermogenic genes in white adipose depots. Rapamycin enhanced transcriptional negative feedback on the ß3-adrenergic receptor. However, thermogenic gene expression remained impaired even when the receptor was bypassed with a cell-permeable cAMP analog, revealing the existence of a second inhibitory mechanism. Accordingly, rapamycin-treated mice are cold intolerant, failing to maintain body temperature and weight when shifted to 4°C. Adipocyte-specific deletion of the mTORC1 subunit Raptor recapitulated the block in ß-adrenergic signaling. Our findings demonstrate a positive role for mTORC1 in the recruitment of beige adipocytes and suggest that inhibition of ß-adrenergic signaling by rapamycin may contribute to its physiological effects.

Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor-mediated disease.

Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA.

Genomic and genetic variation in E2F transcription factor-1 in men with nonobstructive azoospermia.

OBJECTIVE: To identify gene dosage changes associated with nonobstructive azoospermia (NOA) using array comparative genomic hybridization (aCGH). DESIGN: Prospective study. SETTING: Medical school. PATIENT(S): One hundred ten men with NOA and 78 fertile controls. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The study has four distinct analytic components: aCGH, a molecular karyotype that detects copy number variations (CNVs); Taqman CNV assays to validate CNVs; mutation identification by Sanger sequencing; and histological analyses of testicular tissues. RESULT(S): A microduplication at 20q11.22 encompassing E2F transcription factor-1 (E2F1) was identified in one of eight men with NOA analyzed using aCGH. CNVs were confirmed and in an additional 102 men with NOA screened using Taqman CNV assays, for a total of 110 NOA men analyzed for CNVs in E2F1. Eight of 110 (7.3%) NOA men had microduplications or microdeletions of E2F1 that were absent in fertile controls. CONCLUSION(S): E2F1 microduplications or microdeletions are present in men with NOA (7.3%). Duplications or deletions of E2F1 occur very rarely in the general population (0.011%), but E2F1 gene dosage changes, previously reported only in cancers, are present in a subset of NOA men. These results recapitulate the infertility phenotype seen in mice lacking or overexpressing E2f1.

Dynamic recruitment of microRNAs to their mRNA targets in the regenerating liver.

BACKGROUND: Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs. RESULTS: miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate 'competing endogenous RNA' (ceRNA) networks. CONCLUSION: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver.

The in vivo role of androgen receptor SUMOylation as revealed by androgen insensitivity syndrome and prostate cancer mutations targeting the proline/glycine residues of synergy control motifs.

The androgen receptor (AR) mediates the effects of male sexual hormones on development and physiology. Alterations in AR function are central to reproductive disorders, prostate cancer, and Kennedy disease. AR activity is influenced by post-translational modifications, but their role in AR-based diseases is poorly understood. Conjugation by small ubiquitin-like modifier (SUMO) proteins at two synergy control (SC) motifs in AR exerts a promoter context-dependent inhibitory role. SC motifs are composed of a four-amino acid core that is often preceded and/or followed by nearby proline or glycine residues. The function of these flanking residues, however, has not been examined directly. Remarkably, several AR mutations associated with oligospermia and androgen insensitivity syndrome map to Pro-390, the conserved proline downstream of the first SC motif in AR. Similarly, mutations at Gly-524, downstream of the second SC motif, were recovered in recurrent prostate cancer samples. We now provide evidence that these clinically isolated substitutions lead to a partial loss of SC motif function and AR SUMOylation that affects multiple endogenous genes. Consistent with a structural role as terminators of secondary structure elements, substitution of Pro-390 by Gly fully supports both SC motif function and SUMOylation. As predicted from the functional properties of SC motifs, the clinically isolated mutations preferentially enhance transcription driven by genomic regions harboring multiple AR binding sites. The data support the view that alterations in AR SUMOylation play significant roles in AR-based diseases and offer novel SUMO-based therapeutic opportunities.

DNA mismatch repair and infertility.

PURPOSE OF REVIEW: To highlight the significance of the abnormal DNA repair mechanism in male infertility. RECENT FINDINGS: DNA repair defects cause a variety of spermatogenic defects in mouse models. Evidence is accumulating to demonstrate the importance of DNA repair defects in human nonobstructive azoospermia. Epigenetic changes may also play a crucial role in infertility. SUMMARY: The DNA in the cell needs to be constantly repaired to ensure fidelity of DNA replication, to maintain genome stability and to ensure propagation of species. The DNA repair and recombination machineries are highly conserved across the species and inactivation of these pathways may lead to replication and recombination errors. This review summarizes the different types of DNA lesions and DNA repair pathways, particularly focusing on highly conserved meiotic regulators, the DNA mismatch repair proteins. Targeted deletions of some of these proteins result in infertility and predisposes to tumor in mutant mouse models. There is evidence for loss of some of these proteins in human male infertility. Because defective DNA repair is associated with a mutator phenotype, the risk of transmission to the offspring of these otherwise infertile men conceived using an assisted reproductive technology needs further evaluation.