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Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

ObjectiveMitophagy removes damaged mitochondria to maintain cellular homeostasis. Aryl hydrocarbon receptor (AhR) expression in the liver plays a crucial role in supporting normal liver functions, but its impact on mitochondrial function is unclear. Here, we identified a new role of AhR in the regulation of mitophagy to control hepatic energy homeostasis.MethodsIn this study, we utilized primary hepatocytes from AhR knockout (KO) mice and AhR knockdown AML12 hepatocytes. An endogenous AhR ligand, kynurenine (Kyn), was used to activate AhR in AML12 hepatocytes. Mitochondrial function and mitophagy process were comprehensively assessed by MitoSOX and mt-Keima fluorescence imaging, Seahorse XF-based oxygen consumption rate measurement, and Mitoplate S-1 mitochondrial substrate utilization analysis.ResultsTranscriptomic analysis indicated that mitochondria-related gene sets were dysregulated in AhR KO liver. In both primary mouse hepatocytes and AML12 hepatocyte cell lines, AhR inhibition strongly suppressed mitochondrial respiration rate and substrate utilization. AhR inhibition also blunted the fasting response of several essential autophagy genes and the mitophagy process. We further identified BCL2 interacting protein 3 (BNIP3), a mitophagy receptor that senses nutrient stress, as an AhR target gene. AhR is directly recruited to the Bnip3 genomic locus, and Bnip3 transcription was enhanced by AhR endogenous ligand treatment in wild-type liver and abolished entirely in AhR KO liver. Mechanistically, overexpression of Bnip3 in AhR knockdown cells mitigated the production of mitochondrial reactive oxygen species (ROS) and restored functional mitophagy.ConclusionsAhR regulation of the mitophagy receptor BNIP3 coordinates hepatic mitochondrial function. Loss of AhR induces mitochondrial ROS production and impairs mitochondrial respiration. These findings provide new insight into how endogenous AhR governs hepatic mitochondrial homeostasis.

Acute hepatic injury is observed in response to various stressors, including trauma, ingestion of hepatic toxins, and hepatitis. Investigations to date have focused on extrinsic and intrinsic signals required for hepatocytes to proliferate and regenerate the liver in response to injury, though there is a more limited understanding of induced stress responses promoting hepatocyte survival upon acute injury. In this issue of the JCI, Sun and colleagues detail a mechanism by which local activation of the nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) directly induces de novo asparagine synthesis and expression of asparagine synthetase (ASNS) in response to injury and show that this response restrains hepatic damage. This work opens up several avenues for inquiry, including the potential for asparagine supplementation to ameliorate acute hepatic injury.

Androgen receptor (AR) signaling is crucial for driving prostate cancer (PCa), the most diagnosed and the second leading cause of death in male patients with cancer in the United States. Androgen deprivation therapy is initially effective in most instances of AR-positive advanced or metastatic PCa. However, patients inevitably develop lethal castration-resistant PCa (CRPC), which is also resistant to the next-generation AR signaling inhibitors. Most CRPCs maintain AR expression, and blocking AR signaling remains a main therapeutic approach. GATA2 is a pioneer transcription factor emerging as a key therapeutic target for PCa because it promotes AR expression and activation. While directly inhibiting GATA2 transcriptional activity remains challenging, enhancing GATA2 degradation is a plausible therapeutic strategy. How GATA2 protein stability is regulated in PCa remains unknown. Here, we show that constitutive photomorphogenesis protein 1 (COP1), an E3 ubiquitin ligase, drives GATA2 ubiquitination at K419/K424 for degradation. GATA2 lacks a conserved [D/E](x)xxVP[D/E] degron but uses alternate BR1/BR2 motifs to bind COP1. By promoting GATA2 degradation, COP1 inhibits AR expression and activation and represses PCa cell and xenograft growth and castration resistance. Accordingly, GATA2 overexpression or COP1 mutations that disrupt COP1-GATA2 binding block COP1 tumor-suppressing activities. We conclude that GATA2 is a major COP1 substrate in PCa and that COP1 promotion of GATA2 degradation is a direct mechanism for regulating AR expression and activation, PCa growth, and castration resistance.

Neural stem cells, the source of newborn neurons in the adult hippocampus, are intimately involved in learning and memory, mood, and stress response. Despite considerable progress in understanding the biology of neural stem cells and neurogenesis, regulating the neural stem cell population precisely has remained elusive because we have lacked the specific targets to stimulate their proliferation and neurogenesis. The orphan nuclear receptor TLX/NR2E1 governs neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is not well understood because its endogenous ligand is not known. Here, we identify oleic acid (18:1ω9 monounsaturated fatty acid) as such a ligand. We first show that oleic acid is critical for neural stem cell survival. Next, we demonstrate that it binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell-cycle and neurogenesis genes, which in turn increases neural stem cell mitotic activity and drives hippocampal neurogenesis in mice. Interestingly, oleic acid-activated TLX strongly up-regulates cell cycle genes while only modestly up-regulating neurogenic genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation and drive the progeny toward neuronal lineage. Oleic acid thus serves as a metabolic regulator of TLX activity that can be used to selectively target neural stem cells, paving the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.

The nutrient sensing nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) regulates the host response to short-term fasting by inducing hepatic transcriptional programming of ketogenesis, fatty acid oxidation and transport, and autophagy. This adaptation is ineffective in chronically undernourished individuals, among whom dyslipidemia and hepatic steatosis are common. We recently reported that hepatic PPARα protein is profoundly depleted in male mice undernourished by a low-protein, low-fat diet. Here, we identify PPARα as a deacetylation target of the NAD-dependent deacetylase sirtuin-1 (SIRT1) and link this to the decrease in PPARα protein levels in undernourished liver. Livers from undernourished male mice expressed high levels of SIRT1, with decreased PPARα acetylation and strongly decreased hepatic PPARα protein. In cultured hepatocytes, PPARα protein levels were decreased by transiently transfecting constitutively active SIRT1 or by treating cells with the potent SIRT1 activator resveratrol, while silencing SIRT1 increased PPARα protein levels. SIRT1 expression is correlated with increased PPARα ubiquitination, suggesting that protein loss is due to proteasomal degradation. In accord with these findings, the dramatic loss of hepatic PPARα in undernourished male mice was completely restored by treating mice with the proteasome inhibitor bortezomib. Similarly, treating undernourished mice with the SIRT1 inhibitor selisistat/EX-527 completely restored hepatic PPARα protein. These data suggest that induction of SIRT1 in undernutrition results in hepatic PPARα deacetylation, ubiquitination, and degradation, highlighting a new mechanism that mediates the liver's failed adaptive metabolic responses in chronic undernutrition.

About 15-20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade.

Mitogen-activated protein kinase 6 (MAPK6) is an atypical MAPK. Its function in regulating cancer growth remains elusive. Here, we reported that MAPK6 directly activated AKT and induced oncogenic outcomes. MAPK6 interacted with AKT through its C34 region and the C-terminal tail and phosphorylated AKT at S473 independent of mTORC2, the major S473 kinase. mTOR kinase inhibitors have not made notable progress in the clinic. Our identified MAPK6-AKT axis may provide a major resistance pathway. Besides repressing growth, inhibiting MAPK6 sensitized cancer cells to mTOR kinase inhibitors. MAPK6 overexpression is associated with decreased overall survival and the survival of patients with lung adenocarcinoma, mesothelioma, uveal melanoma, and breast cancer. MAPK6 expression also correlated with AKT phosphorylation at S473 in human cancer tissues. We conclude that MAPK6 can promote cancer by activating AKT independent of mTORC2 and targeting MAPK6, either alone or in combination with mTOR blockade, may be effective in cancers.

ObjectiveWhite adipose tissue (WAT) expansion regulates energy balance and overall metabolic homeostasis. The absence or loss of WAT occurring through lipodystrophy and lipoatrophy contributes to the development of hepatic steatosis and insulin resistance. We previously demonstrated that sole small ubiquitin-like modifier (SUMO) E2-conjugating enzyme Ube2i represses human adipocyte differentiation. The role of Ube2i during WAT development remains unknown.MethodsTo determine how Ube2i impacts body composition and energy balance, we generated adipocyte-specific Ube2i knockout mice (Ube2ia-KO). CRISPR/Cas9 gene editing inserted loxP sites flanking exons 3 and 4 at the Ube2i locus. Subsequent genetic crosses to Adipoq-Cre transgenic mice allowed deletion of Ube2i in white and brown adipocytes. We measured multiple metabolic endpoints that describe energy balance and carbohydrate metabolism in Ube2ia-KO and littermate controls during postnatal growth.ResultsSurprisingly, Ube2ia-KO mice developed hyperinsulinemia and hepatic steatosis. Global energy balance defects emerged from dysfunctional WAT marked by pronounced local inflammation, loss of serum adipokines, hepatomegaly, and near absence of major adipose tissue depots. We observed progressive lipoatrophy that commences in the early adolescent period.ConclusionsOur results demonstrate that Ube2i expression in mature adipocytes allows WAT expansion during postnatal growth. Deletion of Ube2i in fat cells compromises and diminishes adipocyte function that induces WAT inflammation and ectopic lipid accumulation in the liver. Our findings reveal an indispensable role for Ube2i during white adipocyte expansion and endocrine control of energy balance.

Background & aimsThe accumulation of neutral lipids within hepatocytes underlies non-alcoholic fatty liver disease (NAFLD), which affects a quarter of the world's population and is associated with hepatitis, cirrhosis, and hepatocellular carcinoma. Despite insights gained from both human and animal studies, our understanding of NAFLD pathogenesis remains limited. To better study the molecular changes driving the condition we aimed to generate a humanised NAFLD mouse model.MethodsWe generated TIRF (transgene-free Il2rg -/-/Rag2 -/-/Fah -/-) mice, populated their livers with human hepatocytes, and fed them a Western-type diet for 12 weeks.ResultsWithin the same chimeric liver, human hepatocytes developed pronounced steatosis whereas murine hepatocytes remained normal. Unbiased metabolomics and lipidomics revealed signatures of clinical NAFLD. Transcriptomic analyses showed that molecular responses diverged sharply between murine and human hepatocytes, demonstrating stark species differences in liver function. Regulatory network analysis indicated close agreement between our model and clinical NAFLD with respect to transcriptional control of cholesterol biosynthesis.ConclusionsThese NAFLD xenograft mice reveal an unexpected degree of evolutionary divergence in food metabolism and offer a physiologically relevant, experimentally tractable model for studying the pathogenic changes invoked by steatosis.Lay summaryFatty liver disease is an emerging health problem, and as there are no good experimental animal models, our understanding of the condition is poor. We here describe a novel humanised mouse system and compare it with clinical data. The results reveal that the human cells in the mouse liver develop fatty liver disease upon a Western-style fatty diet, whereas the mouse cells appear normal. The molecular signature (expression profiles) of the human cells are distinct from the mouse cells and metabolic analysis of the humanised livers mimic the ones observed in humans with fatty liver. This novel humanised mouse system can be used to study human fatty liver disease.

Bile acids are lipid-emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine1. As detergents, bile acids can cause toxicity and inflammation in enterohepatic tissues2. Nuclear receptors maintain bile acid homeostasis in hepatocytes and enterocytes3, but it is unclear how mucosal immune cells tolerate high concentrations of bile acids in the small intestine lamina propria (siLP). CD4+ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1 (encoded by Abcb1a) in the siLP to prevent bile acid toxicity and suppress Crohn's disease-like small bowel inflammation4. Here we identify the nuclear xenobiotic receptor CAR (encoded by Nr1i3) as a regulator of MDR1 expression in T cells that can safeguard against bile acid toxicity and inflammation in the mouse small intestine. Activation of CAR induced large-scale transcriptional reprogramming in Teff cells that infiltrated the siLP, but not the colon. CAR induced the expression of not only detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine IL-10. Accordingly, CAR deficiency in T cells exacerbated bile acid-driven ileitis in T cell-reconstituted Rag1-/- or Rag2-/- mice, whereas pharmacological activation of CAR suppressed it. These data suggest that CAR acts locally in T cells that infiltrate the small intestine to detoxify bile acids and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn's disease and defines lymphocyte sub-specialization in the small intestine.

Although macroautophagy/autophagy deficiency causes degenerative diseases, the deletion of essential autophagy genes in adipocytes paradoxically reduces body weight. Brown adipose tissue (BAT) plays an important role in body weight regulation and metabolic control. However, the key cellular mechanisms that maintain BAT function remain poorly understood. in this study, we showed that global or brown adipocyte-specific deletion of pink1, a Parkinson disease-related gene involved in selective mitochondrial autophagy (mitophagy), induced BAT dysfunction, and obesity-prone type in mice. Defective mitochondrial function is among the upstream signals that activate the NLRP3 inflammasome. NLRP3 was induced in brown adipocyte precursors (BAPs) from pink1 knockout (KO) mice. Unexpectedly, NLRP3 induction did not induce canonical inflammasome activity. Instead, NLRP3 induction led to the differentiation of pink1 KO BAPs into white-like adipocytes by increasing the expression of white adipocyte-specific genes and repressing the expression of brown adipocyte-specific genes. nlrp3 deletion in pink1 knockout mice reversed BAT dysfunction. Conversely, adipose tissue-specific atg7 KO mice showed significantly lower expression of Nlrp3 in their BAT. Overall, our data suggest that the role of mitophagy is different from general autophagy in regulating adipose tissue and whole-body energy metabolism. Our results uncovered a new mitochondria-NLRP3 pathway that induces BAT dysfunction. The ability of the nlrp3 knockouts to rescue BAT dysfunction suggests the transcriptional function of NLRP3 as an unexpected, but a quite specific therapeutic target for obesity-related metabolic diseases.Abbreviations: ACTB: actin, beta; BAPs: brown adipocyte precursors; BAT: brown adipose tissue; BMDMs: bone marrow-derived macrophages; CASP1: caspase 1; CEBPA: CCAAT/enhancer binding protein (C/EBP), alpha; ChIP: chromatin immunoprecipitation; EE: energy expenditure; HFD: high-fat diet; IL1B: interleukin 1 beta; ITT: insulin tolerance test; KO: knockout; LPS: lipopolysaccharide; NLRP3: NLR family, pyrin domain containing 3; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RD: regular diet; ROS: reactive oxygen species; RT: room temperature; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); WT: wild-type.

Prostate cancer (PCa) is the second leading cause of cancer death in American men. Androgen receptor (AR) signaling is essential for PCa cell growth/survival and remains a key therapeutic target for lethal castration-resistant PCa (CRPC). GATA2 is a pioneer transcription factor crucial for inducing AR expression/activation. We recently reported that MAPK4, an atypical MAPK, promotes tumor progression via noncanonical activation of AKT. Here, we demonstrated that MAPK4 activated AR by enhancing GATA2 transcriptional expression and stabilizing GATA2 protein through repression of GATA2 ubiquitination/degradation. MAPK4 expression correlated with AR activation in human CRPC. Concerted activation of both GATA2/AR and AKT by MAPK4 promoted PCa cell proliferation, anchorage-independent growth, xenograft growth, and castration resistance. Conversely, knockdown of MAPK4 decreased activation of both AR and AKT and inhibited PCa cell and xenograft growth, including castration-resistant growth. Both GATA2/AR and AKT activation were necessary for MAPK4 tumor-promoting activity. Interestingly, combined overexpression of GATA2 plus a constitutively activated AKT was sufficient to drive PCa growth and castration resistance, shedding light on an alternative, MAPK4-independent tumor-promoting pathway in human PCa. We concluded that MAPK4 promotes PCa growth and castration resistance by cooperating parallel pathways of activating GATA2/AR and AKT and that MAPK4 is a novel therapeutic target in PCa, especially CRPC.

Vertical sleeve gastrectomy (VSG) is one of the most effective and durable therapies for morbid obesity and its related complications. Although bile acids (BAs) have been implicated as downstream mediators of VSG, the specific mechanisms through which BA changes contribute to the metabolic effects of VSG remain poorly understood. Here, we confirm that high fat diet-fed global farnesoid X receptor (Fxr) knockout mice are resistant to the beneficial metabolic effects of VSG. However, the beneficial effects of VSG were retained in high fat diet-fed intestine- or liver-specific Fxr knockouts, and VSG did not result in Fxr activation in the liver or intestine of control mice. Instead, VSG decreased expression of positive hepatic Fxr target genes, including the bile salt export pump (Bsep) that delivers BAs to the biliary pathway. This reduced small intestine BA levels in mice, leading to lower intestinal fat absorption. These findings were verified in sterol 27-hydroxylase (Cyp27a1) knockout mice, which exhibited low intestinal BAs and fat absorption and did not show metabolic improvements following VSG. In addition, restoring small intestinal BA levels by dietary supplementation with taurocholic acid (TCA) partially blocked the beneficial effects of VSG. Altogether, these findings suggest that reductions in intestinal BAs and lipid absorption contribute to the metabolic benefits of VSG.

Bile acids have recently emerged as key metabolic hormones with beneficial impacts in multiple metabolic diseases. We previously discovered that hepatic bile acid overload distally modulates glucose and fatty acid metabolism in adipose tissues to exert anti-obesity effects. However, the detailed mechanisms that explain the salutary effects of serum bile acid elevation remain unclear. Here, proteomic profiling identified a new hepatokine, Orosomucoid (ORM) that governs liver-adipose tissue crosstalk. Hepatic ORMs were highly induced by both genetic and dietary bile acid overload. To address the direct metabolic effects of ORM, purified ORM proteins were administered during adipogenic differentiation of 3T3-L1 cells and mouse stromal vascular fibroblasts. ORM suppressed adipocyte differentiation and strongly inhibited gene expression of adipogenic transcription factors such as C/EBPβ, KLF5, C/EBPα, and PPARγ. Taken together, our data clearly suggest that bile acid-induced ORM secretion from the liver blocks adipocyte differentiation, potentially linked to anti-obesity effect of bile acids.

Liver dysfunction, including coagulopathy, is a prominent feature of protein-energy malnutrition. To identify mechanisms underlying malnutrition-associated coagulopathy, we administered a low-protein low-fat diet to lactating dams and examined hepatic transcription and plasma coagulation parameters in young adult weanlings. Malnutrition impacted body composition to a greater extent in male versus female mice. Transcriptional profiles suggested opposing effects of nutrient-sensing nuclear receptors, namely induction of peroxisome proliferator-activated receptor α (PPARα) targets and repression of farnesoid-X-receptor (FXR) targets. Coagulopathy with decreased synthesis of fibrinogen-α (FGA) and factor 11 (F11) was observed in malnourished male animals but not female animals. In primary mouse hepatocytes, FXR agonist increased and PPARα agonist decreased Fga and F11 messenger RNA expression. Nuclear receptor DNA response elements were identified in the Fga and F11 gene regulatory regions, and opposing effects of FXR and PPARα were confirmed with luciferase assays. Unexpectedly, hepatic PPARα protein was markedly depleted in malnourished male liver and was not enriched on Fga or F11 response elements. Rather, there was loss of FXR binding at these response elements. Reduced PPARα protein was associated with loss of hepatocyte peroxisomes, which are necessary for bile acid biosynthesis, and with decreased concentrations of bile acids that function as FXR ligands, most notably the FXR agonist chenodeoxycholic acid. Conclusion: Malnutrition impairs growth and liver synthetic function more severely in male mice than in female mice. Malnourished male mice are coagulopathic and exhibit decreased hepatocyte peroxisomes, FXR agonist bile acids, FXR binding on Fga and F11 gene regulatory elements, and coagulation factor synthesis. These effects are absent in female mice, which have low baseline levels of PPARα, suggesting that nutrient-sensing nuclear receptors regulate coagulation factor synthesis in response to host nutritional status in a sex-specific manner.

Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.

Dynamin-Related-Protein 1 (DRP1) critically regulates mitochondrial and peroxisomal fission in multicellular organisms. However, the impact of DRP1 on other organelles, especially its direct influence on ER functions remains largely unclear. Here, we report that DRP1 translocates to endoplasmic reticulum (ER) in response to β-adrenergic stimulation. To further investigate the function of DRP1 on ER-lipid droplet (LD) dynamics and the metabolic subsequences, we generated an adipose tissue-specific DRP1 knockout model (Adipo-Drp1flx/flx ). We found that the LDs in adipose tissues of Adipo-Drp1flx/flx mice exhibited more unilocular morphology with larger sizes, and formed less multilocular structures upon cold exposure. Mechanistically, we discovered that abnormal LD morphology occurs because newly generated micro-LDs fail to dissociate from the ER due to DRP1 ablation. Conversely, the ER retention of LDs can be rescued by the overexpressed DRP1 in the adipocytes. The alteration of LD dynamics, combined with abnormal mitochondrial and autophagy functions in adipose tissue, ultimately lead to abnormalities in lipid metabolism in Adipo-Drp1flx/flx mice.

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.

Background and aimsLiver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates metabolic homeostasis in the liver. Previous studies identified phosphatidylcholines as potential endogenous agonist ligands for LRH-1. In the liver, distinct subsets of phosphatidylcholine species are generated by two different pathways: choline addition to phosphatidic acid through the Kennedy pathway and trimethylation of phosphatidylethanolamine through phosphatidylethanolamine N-methyl transferase (PEMT).Approach and resultsHere, we report that a PEMT-LRH-1 pathway specifically couples methyl metabolism and mitochondrial activities in hepatocytes. We show that the loss of Lrh-1 reduces mitochondrial number, basal respiration, beta-oxidation, and adenosine triphosphate production in hepatocytes and decreases expression of mitochondrial biogenesis and beta-oxidation genes. In contrast, activation of LRH-1 by its phosphatidylcholine agonists exerts opposite effects. While disruption of the Kennedy pathway does not affect the LRH-1-mediated regulation of mitochondrial activities, genetic or pharmaceutical inhibition of the PEMT pathway recapitulates the effects of Lrh-1 knockdown on mitochondria. Furthermore, we show that S-adenosyl methionine, a cofactor required for PEMT, is sufficient to induce Lrh-1 transactivation and consequently mitochondrial biogenesis.ConclusionsA PEMT-LRH-1 axis regulates mitochondrial biogenesis and beta-oxidation in hepatocytes.

Inactivating mutations in the copper transporter Atp7b result in Wilson's disease. The Atp7b -/- mouse develops hallmarks of Wilson's disease. The activity of several nuclear receptors decreased in Atp7b -/- mice, and nuclear receptors are critical for maintaining metabolic homeostasis. Therefore, we anticipated that Atp7b -/- mice would exhibit altered progression of diet-induced obesity, fatty liver, and insulin resistance. Following 10 wk on a chow or Western-type diet (40% kcal fat), parameters of glucose and lipid homeostasis were measured. Hepatic metabolites were measured by liquid chromatography-mass spectrometry and correlated with transcriptomic data. Atp7b -/- mice fed a chow diet presented with blunted body-weight gain over time, had lower fat mass, and were more glucose tolerant than wild type (WT) littermate controls. On the Western diet, Atp7b -/- mice exhibited reduced body weight, adiposity, and hepatic steatosis compared with WT controls. Atp7b -/- mice fed either diet were more insulin sensitive than WT controls; however, fasted Atp7b -/- mice exhibited hypoglycemia after administration of insulin due to an impaired glucose counterregulatory response, as evidenced by reduced hepatic glucose production. Coupling gene expression with metabolomic analyses, we observed striking changes in hepatic metabolic profiles in Atp7b -/- mice, including increases in glycolytic intermediates and components of the tricarboxylic acid cycle. In addition, the active phosphorylated form of AMP kinase was significantly increased in Atp7b -/- mice relative to WT controls. Alterations in hepatic metabolic profiles and nuclear receptor signaling were associated with improved glucose tolerance and insulin sensitivity as well as with impaired fasting glucose production in Atp7b -/- mice.

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, liver receptor homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases (IBD). Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of IBD, the new agonist increases expression of LRH-1-controlled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility.

MAPK4 is an atypical MAPK. Currently, little is known about its physiological function and involvement in diseases, including cancer. A comprehensive analysis of 8887 gene expression profiles in The Cancer Genome Atlas (TCGA) revealed that MAPK4 overexpression correlates with decreased overall survival, with particularly marked survival effects in patients with lung adenocarcinoma, bladder cancer, low-grade glioma, and thyroid carcinoma. Interestingly, human tumor MAPK4 overexpression also correlated with phosphorylation of AKT, 4E-BP1, and p70S6K, independent of the loss of PTEN or mutation of PIK3CA. This led us to examine whether MAPK4 activates the key metabolic, prosurvival, and proliferative kinase AKT and mTORC1 signaling, independent of the canonical PI3K pathway. We found that MAPK4 activated AKT via a novel, concerted mechanism independent of PI3K. Mechanistically, MAPK4 directly bound and activated AKT by phosphorylation of the activation loop at threonine 308. It also activated mTORC2 to phosphorylate AKT at serine 473 for full activation. MAPK4 overexpression induced oncogenic outcomes, including transforming prostate epithelial cells into anchorage-independent growth, and MAPK4 knockdown inhibited cancer cell proliferation, anchorage-independent growth, and xenograft growth. We concluded that MAPK4 can promote cancer by activating the AKT/mTOR signaling pathway and that targeting MAPK4 may provide a novel therapeutic approach for cancer.

Bile acid (BA) homeostasis is tightly regulated by multiple transcription factors, including farnesoid X receptor (FXR) and small heterodimer partner (SHP). We previously reported that loss of the FXR/SHP axis causes severe intrahepatic cholestasis, similar to human progressive familial intrahepatic cholestasis type 5 (PFIC5). In this study, we found that constitutive androstane receptor (CAR) is endogenously activated in Fxr:Shp double knockout (DKO) mice. To test the hypothesis that CAR activation protects DKO mice from further liver damage, we generated Fxr;Shp;Car triple knockout (TKO) mice. In TKO mice, residual adenosine triphosphate (ATP) binding cassette, subfamily B member 11 (ABCB11; alias bile salt export pump [BSEP]) function and fecal BA excretion are completely impaired, resulting in severe hepatic and biliary damage due to excess BA overload. In addition, we discovered that pharmacologic CAR activation has different effects on intrahepatic cholestasis of different etiologies. In DKO mice, CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP; here on TC) treatment attenuated cholestatic liver injury, as expected. However, in the PFIC2 model Bsep knockout (BKO) mice, TC treatment exhibited opposite effects that reflect increased BA accumulation and liver injury. These contrasting results may be linked to differential regulation of systemic cholesterol homeostasis in DKO and BKO livers. TC treatment selectively up-regulated hepatic cholesterol levels in BKO mice, supporting de novo BA synthesis. Conclusion: CAR activation in DKO mice is generally protective against cholestatic liver injury in these mice, which model PFIC5, but not in the PFIC2 model BKO mice. Our results emphasize the importance of the genetic and physiologic background when implementing targeted therapies to treat intrahepatic cholestasis.

Chronic inflammation accompanies obesity and limits subcutaneous white adipose tissue (WAT) expandability, accelerating the development of insulin resistance and type 2 diabetes mellitus. MicroRNAs (miRNAs) influence expression of many metabolic genes in fat cells, but physiological roles in WAT remain poorly characterized. Here, we report that expression of the miRNA miR-30a in subcutaneous WAT corresponds with insulin sensitivity in obese mice and humans. To examine the hypothesis that restoration of miR-30a expression in WAT improves insulin sensitivity, we injected adenovirus (Adv) expressing miR-30a into the subcutaneous fat pad of diabetic mice. Exogenous miR-30a expression in the subcutaneous WAT depot of obese mice coupled improved insulin sensitivity and increased energy expenditure with decreased ectopic fat deposition in the liver and reduced WAT inflammation. High-throughput proteomic profiling and RNA-Seq suggested that miR-30a targets the transcription factor STAT1 to limit the actions of the proinflammatory cytokine interferon-γ (IFN-γ) that would otherwise restrict WAT expansion and decrease insulin sensitivity. We further demonstrated that miR-30a opposes the actions of IFN-γ, suggesting an important role for miR-30a in defending adipocytes against proinflammatory cytokines that reduce peripheral insulin sensitivity. Together, our data identify a critical molecular signaling axis, elements of which are involved in uncoupling obesity from metabolic dysfunction.

Background & aimsDespite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts.MethodsIn this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product.ResultsWe used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse.ConclusionsThis approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

Hepatocellular carcinoma (HCC) is a heterogeneous disease. Mouse models are commonly used as preclinical models to study hepatocarcinogenesis, but how well these models recapitulate molecular subtypes of human HCC is unclear. Here, integration of genomic signatures from molecularly and clinically defined human HCC (n = 11) and mouse models of HCC (n = 9) identified the mouse models that best resembled subtypes of human HCC and determined the clinical relevance of each model. Mst1/2 knockout (KO), Sav1 KO, and SV40 T antigen mouse models effectively recapitulated subtypes of human HCC with a poor prognosis, whereas the Myc transgenic model best resembled human HCCs with a more favorable prognosis. The Myc model was also associated with activation of β-catenin. E2f1, E2f1/Myc, E2f1/Tgfa, and diethylnitrosamine (DEN)-induced models were heterogeneous and were unequally split into poor and favorable prognoses. Mst1/2 KO and Sav1 KO models best resemble human HCC with hepatic stem cell characteristics. Applying a genomic predictor for immunotherapy, the six-gene IFNγ score, the Mst1/2 KO, Sav1 KO, SV40, and DEN models were predicted to be the least responsive to immunotherapy. Further analysis showed that elevated expression of immune-inhibitory genes (Cd276 and Nectin2/Pvrl2) in Mst1/2 KO, Sav1 KO, and SV40 models and decreased expression of immune stimulatory gene (Cd86) in the DEN model might be accountable for the lack of predictive response to immunotherapy.Implication: The current genomic approach identified the most relevant mouse models to human liver cancer and suggests immunotherapeutic potential for the treatment of specific subtypes. Mol Cancer Res; 16(11); 1713-23. ©2018 AACR.

Epithelial dysfunction and crypt destruction are defining features of inflammatory bowel disease (IBD). However, current IBD therapies targeting epithelial dysfunction are lacking. The nuclear receptor LRH-1 (NR5A2) is expressed in intestinal epithelium and thought to contribute to epithelial renewal. Here we show that LRH-1 maintains intestinal epithelial health and protects against inflammatory damage. Knocking out LRH-1 in murine intestinal organoids reduces Notch signaling, increases crypt cell death, distorts the cellular composition of the epithelium, and weakens the epithelial barrier. Human LRH-1 (hLRH-1) rescues epithelial integrity and when overexpressed, mitigates inflammatory damage in murine and human intestinal organoids, including those derived from IBD patients. Finally, hLRH-1 greatly reduces disease severity in T-cell-mediated murine colitis. Together with the failure of a ligand-incompetent hLRH-1 mutant to protect against TNFα-damage, these findings provide compelling evidence that hLRH-1 mediates epithelial homeostasis and is an attractive target for intestinal disease.

IntroductionAdministration of cholic acid, or its synthetic derivative, 6-alpha-ethyl-23(S)-methylcholic acid (INT-777), activates the membrane GPCR, TGR5, influences whole body metabolism, reduces atherosclerosis, and benefits the cardiovascular physiology in mice. Direct effects of TGR5 agonists, and the role for TGR5, on myocardial cell biology and stress response are unknown.MethodsMice were fed chow supplemented with 0.5% cholic acid (CA) or 0.025% INT-777, a specific TGR5 agonist, or regular chow for 3 weeks. Anthropometric, biochemical, physiologic (electrocardiography and echocardiography), and molecular analysis was performed at baseline. CA and INT-777 fed mice were challenged with acute exercise-induced stress, acute catecholamine-induced stress, and hemodynamic stress induced by transverse aortic constriction (TAC) for a period of 8 weeks. In separate experiments, mice born with constitutive deletion of TGR5 in cardiomyocytes (CM-TGR5del ) were exposed to exercise, inotropic, and TAC-induced stress.ResultsAdministration of CA and INT-777 supplemented diets upregulated TGR5 expression and activated Akt, PKA, and ERK1/2 in the heart. CA and INT-777 fed mice showed improved exercise tolerance, improved sensitivity to catecholamine and attenuation in pathologic remodeling of the heart under hemodynamic stress. In contrast, CM-TGR5del showed poor response to exercise and catecholamine challenge as well as higher mortality and signs of accelerated cardiomyopathy under hemodynamic stress.ConclusionsBile acids, specifically TGR5 agonists, induce cytoprotective changes in the heart and improve myocardial response to physiologic, inotropic, and hemodynamic stress in mice. TGR5 plays a critical role in myocardial adaptability, and TGR5 activation may represent a potentially attractive treatment option in heart failure.

Asprosin is a recently discovered fasting-induced hormone that promotes hepatic glucose production. Here we demonstrate that asprosin in the circulation crosses the blood-brain barrier and directly activates orexigenic AgRP+ neurons via a cAMP-dependent pathway. This signaling results in inhibition of downstream anorexigenic proopiomelanocortin (POMC)-positive neurons in a GABA-dependent manner, which then leads to appetite stimulation and a drive to accumulate adiposity and body weight. In humans, a genetic deficiency in asprosin causes a syndrome characterized by low appetite and extreme leanness; this is phenocopied by mice carrying similar mutations and can be fully rescued by asprosin. Furthermore, we found that obese humans and mice had pathologically elevated concentrations of circulating asprosin, and neutralization of asprosin in the blood with a monoclonal antibody reduced appetite and body weight in obese mice, in addition to improving their glycemic profile. Thus, in addition to performing a glucogenic function, asprosin is a centrally acting orexigenic hormone that is a potential therapeutic target in the treatment of both obesity and diabetes.

The nuclear receptors farnesoid X receptor (FXR; NR1H4) and small heterodimer partner (SHP; NR0B2) play crucial roles in bile acid homeostasis. Global double knockout of FXR and SHP signaling (DKO) causes severe cholestasis and liver injury at early ages. Here, we report an unexpected beneficial impact on glucose and fatty acid metabolism in aged DKO mice, which show suppressed body weight gain and adiposity when maintained on normal chow. This phenotype was not observed in single Fxr or Shp knockouts. Liver-specific Fxr/Shp double knockout mice fully phenocopied the DKO mice, with lower hepatic triglyceride accumulation, improved glucose/insulin tolerance, and accelerated fatty acid use. In both DKO and liver-specific Fxr/Shp double knockout livers, these metabolic phenotypes were associated with altered expression of fatty acid metabolism and autophagy-machinery genes. Loss of the hepatic FXR/SHP axis reprogrammed white and brown adipose tissue gene expression to boost fatty acid usage.ConclusionCombined deletion of the hepatic FXR/SHP axis improves glucose/fatty acid homeostasis in aged mice, reversing the aging phenotype of body weight gain, increased adiposity, and glucose/insulin tolerance, suggesting a central role of this axis in whole-body energy homeostasis. (Hepatology 2017;66:498-509).

RCAS avian viruses have been used to deliver oncogene expression and induce tumors in transgenic mice expressing the virus receptor TVA. Here we report the generation and characterization of a novel RCAS-Cre-IRES-PyMT (RCI-PyMT) virus designed to specifically knockout genes of interest in tumors generated in appropriate mutant mouse hosts. FGF receptor 1 (FGFR1) is a gene that is amplified in human breast cancer, but there have been no definitive studies on its function in mammary tumorigenesis, progression, and metastasis in vivo in spontaneous tumors in mice. We used the retroviral tumor knockout, or TuKO, strategy to delete fgfr1 in PyMT-induced mammary tumors in K19-tva/fgfr1loxP/loxP mice. The similarly injected control K19-tva mice developed mammary tumors exhibiting high metastasis to lung, making this an ideal model for breast cancer metastasis. The fgfr1 TuKO tumors showed significantly decreased primary tumor growth and, most importantly, greatly reduced metastasis to lung. In contrast to previous reports, FGFR1 action in this spontaneous mammary tumor model does not significantly induce epithelial-to-mesenchymal transition. Loss of FGFR1 does generate a gene signature that is reverse correlated with FGFR1 gene amplification and/or upregulation in human breast cancer. Our results suggest that FGFR1 signaling is a key pathway driving breast cancer lung metastasis and that targeting FGFR1 in breast cancer is an exciting approach to inhibit metastasis.

The nuclear receptors PPARα (encoded by NR1C1) and farnesoid X receptor (FXR, encoded by NR1H4) are activated in the liver in the fasted and fed state, respectively. PPARα activation induces fatty acid oxidation, while FXR controls bile acid homeostasis, but both nuclear receptors also regulate numerous other metabolic pathways relevant to liver energy balance. Here we review evidence that they function coordinately to control key nutrient pathways, including fatty acid oxidation and gluconeogenesis in the fasted state and lipogenesis and glycolysis in the fed state. We have also recently reported that these receptors have mutually antagonistic impacts on autophagy, which is induced by PPARα but suppressed by FXR. Secretion of multiple blood proteins is a major drain on liver energy and nutrient resources, and we present preliminary evidence that the liver secretome may be directly suppressed by PPARα, but induced by FXR. Finally, previous studies demonstrated a striking deficiency in bile acid levels in malnourished mice that is consistent with results in malnourished children. We present evidence that hepatic targets of PPARα and FXR are dysregulated in chronic undernutrition. We conclude that PPARα and FXR function coordinately to integrate liver energy balance.

Cardiac dysfunction in patients with liver cirrhosis is strongly associated with increased serum bile acid concentrations. Here we show that excess bile acids decrease fatty acid oxidation in cardiomyocytes and can cause heart dysfunction, a cardiac syndrome that we term cholecardia. Farnesoid X receptor; Small Heterodimer Partner double knockout mice, a model for bile acid overload, display cardiac hypertrophy, bradycardia, and exercise intolerance. In addition, double knockout mice exhibit an impaired cardiac response to catecholamine challenge. Consistent with this decreased cardiac function, we show that elevated serum bile acids reduce cardiac fatty acid oxidation both in vivo and ex vivo. We find that increased bile acid levels suppress expression of proliferator-activated receptor-γ coactivator 1α, a key regulator of fatty acid metabolism, and that proliferator-activated receptor-γ coactivator 1α overexpression in cardiac cells was able to rescue the bile acid-mediated reduction in fatty acid oxidation genes. Importantly, intestinal bile acid sequestration with cholestyramine was sufficient to reverse the observed heart dysfunction in the double knockout mice.ConclusionsDecreased proliferator-activated receptor-γ coactivator 1α expression contributes to the metabolic dysfunction in cholecardia so that reducing serum bile acid concentrations may be beneficial against the metabolic and pathological changes in the heart. (Hepatology 2017;65:189-201).

Targeted mutant models are common in mechanistic toxicology experiments investigating the absorption, metabolism, distribution, or elimination (ADME) of chemicals from individuals. Key models include those for xenosensing transcription factors and cytochrome P450s (CYP). Here we investigated changes in transcript levels, protein expression, and steroid hydroxylation of several xenobiotic detoxifying CYPs in constitutive androstane receptor (CAR)-null and two CYP-null mouse models that have subfamily members regulated by CAR; the Cyp3a-null and a newly described Cyp2b9/10/13-null mouse model. Compensatory changes in CYP expression that occur in these models may also occur in polymorphic humans, or may complicate interpretation of ADME studies performed using these models. The loss of CAR causes significant changes in several CYPs probably due to loss of CAR-mediated constitutive regulation of these CYPs. Expression and activity changes include significant repression of Cyp2a and Cyp2b members with corresponding drops in 6α- and 16β-testosterone hydroxylase activity. Further, the ratio of 6α-/15α-hydroxylase activity, a biomarker of sexual dimorphism in the liver, indicates masculinization of female CAR-null mice, suggesting a role for CAR in the regulation of sexually dimorphic liver CYP profiles. The loss of Cyp3a causes fewer changes than CAR. Nevertheless, there are compensatory changes including gender-specific increases in Cyp2a and Cyp2b. Cyp2a and Cyp2b were down-regulated in CAR-null mice, suggesting activation of CAR and potentially PXR following loss of the Cyp3a members. However, the loss of Cyp2b causes few changes in hepatic CYP transcript levels and almost no significant compensatory changes in protein expression or activity with the possible exception of 6α-hydroxylase activity. This lack of a compensatory response in the Cyp2b9/10/13-null mice is probably due to low CYP2B hepatic expression, especially in male mice. Overall, compensatory and regulatory CYP changes followed the order CAR-null > Cyp3a-null > Cyp2b-null mice.

Chronic jet lag induces spontaneous hepatocellular carcinoma (HCC) in wild-type mice following a mechanism very similar to that observed in obese humans. The process initiates with non-alcoholic fatty liver disease (NAFLD) that progresses to steatohepatitis and fibrosis before HCC detection. This pathophysiological pathway is driven by jet-lag-induced genome-wide gene deregulation and global liver metabolic dysfunction, with nuclear receptor-controlled cholesterol/bile acid and xenobiotic metabolism among the top deregulated pathways. Ablation of farnesoid X receptor dramatically increases enterohepatic bile acid levels and jet-lag-induced HCC, while loss of constitutive androstane receptor (CAR), a well-known liver tumor promoter that mediates toxic bile acid signaling, inhibits NAFLD-induced hepatocarcinogenesis. Circadian disruption activates CAR by promoting cholestasis, peripheral clock disruption, and sympathetic dysfunction.

Despite clear associations between vitamin D deficiency and obesity and/or type 2 diabetes, a causal relationship is not established. Vitamin D receptors (VDRs) are found within multiple tissues, including the brain. Given the importance of the brain in controlling both glucose levels and body weight, we hypothesized that activation of central VDR links vitamin D to the regulation of glucose and energy homeostasis. Indeed, we found that small doses of active vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3) (calcitriol), into the third ventricle of the brain improved glucose tolerance and markedly increased hepatic insulin sensitivity, an effect that is dependent upon VDR within the paraventricular nucleus of the hypothalamus. In addition, chronic central administration of 1,25D3 dramatically decreased body weight by lowering food intake in obese rodents. Our data indicate that 1,25D3-mediated changes in food intake occur through action within the arcuate nucleus. We found that VDR colocalized with and activated key appetite-regulating neurons in the arcuate, namely proopiomelanocortin neurons. Together, these findings define a novel pathway for vitamin D regulation of metabolism with unique and divergent roles for central nervous system VDR signaling. Specifically, our data suggest that vitamin D regulates glucose homeostasis via the paraventricular nuclei and energy homeostasis via the arcuate nuclei.

Hepatic glucose release into the circulation is vital for brain function and survival during periods of fasting and is modulated by an array of hormones that precisely regulate plasma glucose levels. We have identified a fasting-induced protein hormone that modulates hepatic glucose release. It is the C-terminal cleavage product of profibrillin, and we name it Asprosin. Asprosin is secreted by white adipose, circulates at nanomolar levels, and is recruited to the liver, where it activates the G protein-cAMP-PKA pathway, resulting in rapid glucose release into the circulation. Humans and mice with insulin resistance show pathologically elevated plasma asprosin, and its loss of function via immunologic or genetic means has a profound glucose- and insulin-lowering effect secondary to reduced hepatic glucose release. Asprosin represents a glucogenic protein hormone, and therapeutically targeting it may be beneficial in type II diabetes and metabolic syndrome.

Neonatal cholestasis is a potentially life-threatening condition requiring prompt diagnosis. Mutations in several different genes can cause progressive familial intrahepatic cholestasis, but known genes cannot account for all familial cases. Here we report four individuals from two unrelated families with neonatal cholestasis and mutations in NR1H4, which encodes the farnesoid X receptor (FXR), a bile acid-activated nuclear hormone receptor that regulates bile acid metabolism. Clinical features of severe, persistent NR1H4-related cholestasis include neonatal onset with rapid progression to end-stage liver disease, vitamin K-independent coagulopathy, low-to-normal serum gamma-glutamyl transferase activity, elevated serum alpha-fetoprotein and undetectable liver bile salt export pump (ABCB11) expression. Our findings demonstrate a pivotal function for FXR in bile acid homeostasis and liver protection.

UnlabelledBalance of labile methyl groups (choline, methionine, betaine, and folate) is important for normal liver function. Quantitatively, a significant use of labile methyl groups is in the production of phosphatidylcholines (PCs), which are ligands for the nuclear liver receptor homolog-1 (LRH-1). We studied the role of LRH-1 in methyl-pool homeostasis and determined its metabolic effects using the methionine and choline-deficient (MCD) diet, which depletes methyl groups and results in a deleterious decrease in the PC-to-phosphatidylethanolamine ratio. We found that MCD diet-fed, liver-specific LRH-1 knockout mice (Lrh-1(-/-) ) do not show the expected decreased methyl-pool and PC/phosphatidylethanolamine ratio and are resistant to the hepatitis and fibrosis normally induced by the diet. Adaptive responses observed in wild-type mice on the MCD diet were also observed in Lrh-1(-/-) mice on a normal diet. This includes reduced expression of the highly active glycine-n-methyltransferase and the biliary phospholipid floppase multidrug-resistance protein 2 (Mdr2/Abcb4), resulting in reduced consumption of methyl groups and biliary PC secretion. In vitro studies confirm that Gnmt and Mdr2 are primary LRH-1 target genes. Additional similarities between hepatic gene expression profiles in MCD diet-fed wild-type and untreated Lrh-1(-/-) mice suggest that methyl-pool deficiency decreases LRH-1 activity, and this was confirmed by in vitro functional results in cells maintained in MCD medium.ConclusionLRH-1 is a novel transcriptional regulator of methyl-pool balance; when the methyl-pool is depleted, decreased LRH-1 transactivation suppresses expression of key genes to minimize loss of labile methyl groups. (Hepatology 2016;63:95-106).

BackgroundExpression of hepatic cholesterol 7α-hydroxylase (CYP7A1) is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP). In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1).MethodsWe injected thyroid hormone (triiodothyronine, T3) to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR). Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA) were also performed using human hepatoma cell line.ResultsInitial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor β/retinoid X receptor α/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding.ConclusionWe found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1.

BackgroundSmall heterodimer partner (SHP, NR0B2) is involved in diverse metabolic pathways, including hepatic bile acid, lipid and glucose homeostasis, and has been implicated in effects on the peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis and the receptor for antidiabetic drugs thiazolidinediones (TZDs). In this study, we aim to investigate the role of SHP in TZD response by comparing TZD-treated leptin-deficient (ob/ob) and leptin-, SHP-deficient (ob/ob;Shp(-/-)) double mutant mice.ResultsBoth ob/ob and double mutant ob/ob;Shp(-/-) mice developed hyperglycemia, insulin resistance, and hyperlipidemia, but hepatic fat accumulation was decreased in the double mutant ob/ob;Shp(-/-) mice. PPARγ2 mRNA levels were markedly lower in ob/ob;Shp(-/-) liver and decreased to a lesser extent in adipose tissue. The TZD troglitazone did not reduce glucose or circulating triglyceride levels in ob/ob;Shp(-/-) mice. Expression of the adipocytokines, such as adiponectin and resistin, was not stimulated by troglitazone treatment. Expression of hepatic lipogenic genes was also reduced in ob/ob;Shp(-/-) mice. Moreover, overexpression of SHP by adenovirus infection increased PPARγ2 mRNA levels in mouse primary hepatocytes.ConclusionsOur results suggest that SHP is required for both antidiabetic and hypolipidemic effects of TZDs in ob/ob mice through regulation of PPARγ expression.

Circadian disruption is associated with obesity, implicating the central clock in body weight control. Our comprehensive screen of wild-type and three circadian mutant mouse models, with or without chronic jet lag, shows that distinct genetic and physiologic interventions differentially disrupt overall energy homeostasis and Leptin signaling. We found that BMAL1/CLOCK generates circadian rhythm of C/EBPα-mediated leptin transcription in adipose. Per and Cry mutant mice show similar disruption of peripheral clock and deregulation of leptin in fat, but opposite body weight and composition phenotypes that correlate with their distinct patterns of POMC neuron deregulation in the arcuate nucleus. Chronic jet lag is sufficient to disrupt the endogenous adipose clock and also induce central Leptin resistance in wild-type mice. Thus, coupling of the central and peripheral clocks controls Leptin endocrine feedback homeostasis. We propose that Leptin resistance, a hallmark of obesity in humans, plays a key role in circadian dysfunction-induced obesity and metabolic syndromes.

Wilson's disease (WD) is an autosomal recessive disorder that results in accumulation of copper in the liver as a consequence of mutations in the gene encoding the copper-transporting P-type ATPase (ATP7B). WD is a chronic liver disorder, and individuals with the disease present with a variety of complications, including steatosis, cholestasis, cirrhosis, and liver failure. Similar to patients with WD, Atp7b⁻/⁻ mice have markedly elevated levels of hepatic copper and liver pathology. Previous studies have demonstrated that replacement of zinc in the DNA-binding domain of the estrogen receptor (ER) with copper disrupts specific binding to DNA response elements. Here, we found decreased binding of the nuclear receptors FXR, RXR, HNF4α, and LRH-1 to promoter response elements and decreased mRNA expression of nuclear receptor target genes in Atp7b⁻/⁻ mice, as well as in adult and pediatric WD patients. Excessive hepatic copper has been described in progressive familial cholestasis (PFIC), and we found that similar to individuals with WD, patients with PFIC2 or PFIC3 who have clinically elevated hepatic copper levels exhibit impaired nuclear receptor activity. Together, these data demonstrate that copper-mediated nuclear receptor dysfunction disrupts liver function in WD and potentially in other disorders associated with increased hepatic copper levels.