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Background & Aims: The fatty acid translocase, also known as CD36, is a well-established scavenger receptor for fatty acid (FA) uptake and is abundantly expressed in many metabolically active tissues. In the liver, CD36 is known to contribute to the progression of non-alcoholic fatty liver disease and to the more severe non-alcoholic steatohepatitis, by promoting triglyceride accumulation and subsequent lipid-induced endoplasmic reticulum (ER) stress. Given the recent discovery that the hepatocyte-secreted proprotein convertase subtilisin/kexin type 9 (PCSK9) blocks CD36 expression, we sought to investigate the role of PCSK9 in liver fat accumulation and injury in response to saturated FAs and in a mouse model of diet-induced hepatic steatosis. Methods: In this study, we investigated the role of PCSK9 on the uptake and accumulation of FAs, as well as FA-induced toxicity, in a variety of cultured hepatocytes. Diet-induced hepatic steatosis and liver injury were also assessed in Pcsk9-/- mice. Results: Our results indicate that PCSK9 deficiency in cultured hepatocytes increased the uptake and accumulation of saturated and unsaturated FAs. In the presence of saturated FAs, PCSK9 also protected cultured hepatocytes from ER stress and cytotoxicity. In line with these findings, a metabolic challenge using a high-fat diet caused severe hepatic steatosis, ER stress inflammation and fibrosis in the livers of Pcsk9-/- mice compared to controls. Given that inhibition of CD36 ablated the observed accumulation of lipid in vitro and in vivo, our findings also highlight CD36 as a strong contributor to steatosis and liver injury in the context of PCSK9 deficiency. Conclusions: Collectively, our findings demonstrate that PCSK9 regulates hepatic triglyceride content in a manner dependent on CD36. In the presence of excess dietary fats, PCSK9 can also protect against hepatic steatosis and liver injury. Lay summary: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a circulating protein known to reduce the abundance of receptors on the surface of liver cells charged with the task of lipid uptake from the circulation. Although PCSK9 deficiency is known to cause lipid accumulation in mice and in cultured cells, the toxicological implications of this observation have not yet been reported. In this study, we demonstrate that PCSK9 can protect against cytotoxicity in cultured liver cells treated with a saturated fatty acid and we also show that Pcsk9 knockout mice develop increased liver injury in response to a high-fat diet. Key words: ER stress, CD36, HFD, NAFLD, Liver disease, Cardiometabolic disease, CVD

Objective: Growth differentiation factors (GDFs) and bone-morphogenic proteins (BMPs) are members of the transforming growth factor β (TGFβ) superfamily and are known to play a central role in the growth and differentiation of developing tissues. Accumulating evidence, however, demonstrates that many of these factors, such as BMP-2 and -4, as well as GDF15, also regulate lipid metabolism. GDF10 is a divergent member of the TGFβ superfamily with a unique structure and is abundantly expressed in brain and adipose tissue; it is also secreted by the latter into the circulation. Although previous studies have demonstrated that overexpression of GDF10 reduces adiposity in mice, the role of circulating GDF10 on other tissues known to regulate lipid, like the liver, has not yet been examined. Methods: Accordingly, GDF10−/− mice and age-matched GDF10+/+ control mice were fed either normal control diet (NCD) or high-fat diet (HFD) for 12 weeks and examined for changes in liver lipid homeostasis. Additional studies were also carried out in primary and immortalized human hepatocytes treated with recombinant human (rh)GDF10. Results: Here, we show that circulating GDF10 levels are increased in conditions of diet-induced hepatic steatosis and, in turn, that secreted GDF10 can prevent excessive lipid accumulation in hepatocytes. We also report that GDF10−/− mice develop an obese phenotype as well as increased liver triglyceride accumulation when fed a NCD. Furthermore, HFD-fed GDF10−/− mice develop increased steatosis, endoplasmic reticulum (ER) stress, fibrosis, and injury of the liver compared to HFD-fed GDF10+/+ mice. To explain these observations, studies in cultured hepatocytes led to the observation that GDF10 attenuates nuclear peroxisome proliferator-activated receptor γ (PPARγ) activity; a transcription factor known to induce de novo lipogenesis. Conclusion: Our work delineates a hepatoprotective role of GDF10 as an adipokine capable of regulating hepatic lipid levels by blocking de novo lipogenesis to protect against ER stress and liver injury. Keywords: BMP-3b, ER stress, Hepatic steatosis, HFD, NAFLD, NASH, TGFβ

Objective: Non-alcoholic fatty liver disease (NAFLD) risk begins in utero in offspring of obese mothers. A critical unmet need in this field is to understand the pathways and biomarkers underlying fetal hepatic lipotoxicity and whether maternal dietary intervention during pregnancy is an effective countermeasure. Methods: We utilized a well-established non-human primate model of chronic, maternal, Western-style diet induced obesity (OB-WSD) compared with mothers on a healthy control diet (CON) or a subset of OB-WSD mothers switched to the CON diet (diet reversal; OB-DR) prior to and for the duration of the next pregnancy. Fetuses were studied in the early 3rd trimester. Results: Fetuses from OB-WSD mothers had higher circulating triglycerides (TGs) and lower arterial oxygenation suggesting hypoxemia, compared with fetuses from CON and OB-DR mothers. Hepatic TG content, oxidative stress (TBARs), and de novo lipogenic genes were increased in fetuses from OB-WSD compared with CON mothers. Fetuses from OB-DR mothers had lower lipogenic gene expression and TBARs yet persistently higher TGs. Metabolomic profiling of fetal liver and serum (umbilical artery) revealed distinct separation of CON and OB-WSD groups, and an intermediate phenotype in fetuses from OB-DR mothers. Pathway analysis identified decreased tricarboxylic acid cycle intermediates, increased amino acid (AA) metabolism and byproducts, and increased gluconeogenesis, suggesting an increased reliance on AA metabolism to meet energy needs in the liver of fetuses from OB-WSD mothers. Components in collagen synthesis, including serum protein 5-hydroxylysine and hepatic lysine and proline, were positively correlated with hepatic TGs and TBARs, suggesting early signs of fibrosis in livers from the OB-WSD group. Importantly, hepatic gluconeogenic and arginine related intermediates and serum levels of lactate, pyruvate, several AAs, and nucleotide intermediates were normalized in the OB-DR group. However, hepatic levels of CDP-choline and total ceramide levels remained high in fetuses from OB-DR mothers. Conclusions: Our data provide new metabolic evidence that, in addition to fetal hepatic steatosis, maternal WSD creates fetal hypoxemia and increases utilization of AAs for energy production and early activation of gluconeogenic pathways in the fetal liver. When combined with hyperlipidemia and limited antioxidant activity, the fetus suffers from hepatic oxidative stress and altered intracellular metabolism which can be improved with maternal diet intervention. Our data reinforce the concept that multiple “first hits” occur in the fetus prior to development of obesity and demonstrate new biomarkers with potential clinical implications for monitoring NAFLD risk in offspring. Keywords: Liver, Fetus, Steatosis, Hypoxemia, Fetal programming, Metabolomic

During pregnancy, estrogen (E2) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H2S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E2 stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E2 stimulation. We quantified serum and uterine artery NO metabolites (NOx), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E2 stimulation of UBF. Renal blood flow was E2-insensitive for both genotypes. While serum and uterine artery NOx were similar between genotypes at baseline, E2 decreased NOx in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E2 and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E2 or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E2 stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds.

In addition to its role in the endogenous synthesis of cysteine, cystathionine gamma-lyase (CGL) is a major physiological source of the vasorelaxant hydrogen sulfide. Cgl null mice are potentially useful for studying the influence of this compound upon vascular tone and endothelial function. Here, we confirm a previous report that female Cgl null mice exhibit an approximate 45-fold increase in plasma total homocysteine compared to wild type controls. This level of homocysteine is approximately 3.5-fold higher than that observed in male Cgl null mice and is essentially equivalent to that observed in mouse models of cystathionine beta synthase deficient homocystinuria. Cgl null mice of both sexes exhibited decreased expression of methylenetetrahydrofolate reductase and cysteinesulfinate decarboxylase compared to WT controls. Female Cgl null mice exhibited a sex-specific induction of betaine homocysteine S-methyltransferase and methionine adenosyltransferase 1, alpha and a 70% decrease in methionine synthase expression accompanied by significantly decreased plasma methionine. Decreased plasma cysteine levels in female Cgl null mice were associated with sex-specific dysregulation of cysteine dioxygenase expression. Comparative histological assessment between cystathionine beta-synthase and Cgl null mice indicated that the therapeutic potential of cystathionine against liver injury merits possible further investigation. Collectively, our data demonstrates the importance of considering sex when investigating mouse models of inborn errors of metabolism and indicate that while female Cgl null mice are of questionable utility for studying the physiological role of hydrogen sulfide, they could serve as a useful model for studying the consequences of methionine synthase deficiency and the methylfolate trap.

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

Alcoholic liver disease is a significant contributor to global liver failure. In murine models, chronic ethanol consumption dysregulates PTEN/Akt signaling. Hepatospecific deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTENLKO) mice possess constitutive activation of Akt(s) and increased de novo lipogenesis resulting in increased hepatocellular steatosis. This makes PTENLKO a viable model to examine the effects of ethanol in an environment of preexisting steatosis. The aim of this study was to determine the impact of chronic ethanol consumption and the absence of PTEN (PTENLKO) compared to Alb-Cre control mice (PTENf/f) on hepatocellular damage as evidenced by changes in lipid accumulation, protein carbonylation and alanine amino transferase (ALT). In the control PTENf/f animals, ethanol significantly increased ALT, liver triglycerides and steatosis. In contrast, chronic ethanol consumption in PTENLKO mice decreased hepatocellular damage when compared to PTENLKO pair-fed controls. Consumption of ethanol elevated protein carbonylation in PTENf/f animals but had no effect in PTENLKO animals. In PTENLKO mice, overall hepatic mRNA expression of genes that contribute to GSH homeostasis as well as reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were significantly elevated compared to respective PTENf/f counterparts. These data indicate that during conditions of constitutive Akt activation and steatosis, increased GSH homeostasis assists in mitigation of ethanol-dependent induction of oxidative stress and hepatocellular damage. Furthermore, data herein suggest a divergence in EtOH-induced hepatocellular damage and increases in steatosis due to polyunsaturated fatty acids downstream of PTEN.

Down syndrome (DS) is a chromosomal disorder caused by trisomy of chromosome 21 (Ts21). Unbalanced karyotypes can lead to dysfunction of the proteostasis network (PN) and disrupted proteostasis is mechanistically associated with multiple DS comorbidities. Autophagy is a critical component of the PN that has not previously been investigated in DS. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts and other DS cell models. Following induction of autophagy by serum starvation, DS fibroblasts displayed impaired autophagic flux indicated by autophagolysosome accumulation and elevated p62, NBR1, and LC3-II abundance, compared to age- and sex-matched, euploid (CTL) fibroblasts. While lysosomal physiology was unaffected in both groups after serum starvation, we observed decreased basal abundance of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) family members syntaxin 17 (STX17) and Vesicle Associated Membrane Protein 8 (VAMP8) indicating that decreased autophagic flux in DS is due at least in part to a possible impairment of autophagosome-lysosome fusion. This conclusion was further supported by the observation that over-expression of either STX17 or VAMP8 in DS fibroblasts restored autophagic degradation and reversed p62 accumulation. Collectively, our results indicate that impaired autophagic clearance is a characteristic of DS cells that can be reversed by enhancement of SNARE protein expression and provides further evidence that PN disruption represents a candidate mechanism for multiple aspects of pathogenesis in DS and a possible future target for therapeutic intervention.

Environmental exposures strongly influence the development and progression of asthma. We have previously demonstrated that mice exposed to a diet enriched with methyl donors during vulnerable periods of fetal development can enhance the heritable risk of allergic airway disease through epigenetic changes. There is conflicting evidence on the role of folate (one of the primary methyl donors) in modifying allergic airway disease.We hypothesized that blocking folate metabolism through the loss of methylene-tetrahydrofolate reductase (Mthfr) activity would reduce the allergic airway disease phenotype through epigenetic mechanisms.Allergic airway disease was induced in C57BL/6 and C57BL/6Mthfr-/- mice through house dust mite (HDM) exposure. Airway inflammation and airway hyperresponsiveness (AHR) were measured between the two groups. Gene expression and methylation profiles were generated for whole lung tissue. Disease and molecular outcomes were evaluated in C57BL/6 and C57BL/6Mthfr-/- mice supplemented with betaine.Loss of Mthfr alters single carbon metabolite levels in the lung and serum including elevated homocysteine and cystathionine and reduced methionine. HDM-treated C57BL/6Mthfr-/- mice demonstrated significantly less airway hyperreactivity (AHR) compared to HDM-treated C57BL/6 mice. Furthermore, HDM-treated C57BL/6Mthfr-/- mice compared to HDM-treated C57BL/6 mice have reduced whole lung lavage (WLL) cellularity, eosinophilia, and Il-4/Il-5 cytokine concentrations. Betaine supplementation reversed parts of the HDM-induced allergic airway disease that are modified by Mthfr loss. 737 genes are differentially expressed and 146 regions are differentially methylated in lung tissue from HDM-treated C57BL/6Mthfr-/- mice and HDM-treated C57BL/6 mice. Additionally, analysis of methylation/expression relationships identified 503 significant correlations.Collectively, these findings indicate that the loss of folate as a methyl donor is a modifier of allergic airway disease, and that epigenetic and expression changes correlate with this modification. Further investigation into the mechanisms that drive this observation is warranted.

Down syndrome (DS) is a genetic disorder caused by trisomy of chromosome 21. Abnormalities in chromosome number have the potential to lead to disruption of the proteostasis network (PN) and accumulation of misfolded proteins. DS individuals suffer from several comorbidities, and we hypothesized that disruption of proteostasis could contribute to the observed pathology and decreased cell viability in DS. Our results confirm the presence of a disrupted PN in DS, as several of its elements, including the unfolded protein response, chaperone system, and proteasomal degradation exhibited significant alterations compared to euploid controls in both cell and mouse models. Additionally, when cell models were treated with compounds that promote disrupted proteostasis, we observed diminished levels of cell viability in DS compared to controls. Collectively our findings provide a cellular-level characterization of PN dysfunction in DS and an improved understanding of the potential pathogenic mechanisms contributing to disrupted cellular physiology in DS. Lastly, this study highlights the future potential of designing therapeutic strategies that mitigate protein quality control dysfunction.

As much of the aberrant neural development in Down syndrome (DS) occurs postnatally, an early opportunity exists to intervene and influence life-long cognitive development. Recent success using neural progenitor cells (NPC) in models of adult neurodegeneration indicate such therapy may be a viable option in diseases such as DS. Murine NPC (mNPC, C17.2 cell line) or saline were implanted bilaterally into the dorsal hippocampus of postnatal day 2 (PND 2) Ts65Dn pups to explore the feasibility of early postnatal treatment in this mouse model of DS. Disomic littermates provided karyotype controls for trisomic pups. Pups were monitored for developmental milestone achievement, and then underwent adult behavior testing at 14 weeks of age. We found that implanted mNPC survived into adulthood and migrated beyond the implant site in both karyotypes. The implantation of mNPC resulted in a significant increase in the density of dentate granule cells. However, mNPC implantation did not elicit cognitive changes in trisomic mice either neonatally or in adulthood. To the best of our knowledge, these results constitute the first assessment of mNPC as an early intervention on cognitive ability in a DS model.