Hepatic DDAH1 mitigates hepatic steatosis and insulin resistance in obese mice: Involvement of reduced S100A11 expression.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an important regulator of plasma asymmetric dimethylarginine (ADMA) levels, which are associated with insulin resistance in patients with nonalcoholic fatty liver disease (NAFLD). To elucidate the role of hepatic DDAH1 in the pathogenesis of NAFLD, we used hepatocyte-specific Ddah1-knockout mice (Ddah1HKO) to examine the progress of high-fat diet (HFD)-induced NAFLD. Compared to diet-matched flox/flox littermates (Ddah1f/f), Ddah1HKO mice exhibited higher serum ADMA levels. After HFD feeding for 16 weeks, Ddah1HKO mice developed more severe liver steatosis and worse insulin resistance than Ddah1f/f mice. On the contrary, overexpression of DDAH1 attenuated the NAFLD-like phenotype in HFD-fed mice and ob/ob mice. RNA-seq analysis showed that DDAH1 affects NF-κB signaling, lipid metabolic processes, and immune system processes in fatty livers. Furthermore, DDAH1 reduces S100 calcium-binding protein A11 (S100A11) possibly via NF-κB, JNK and oxidative stress-dependent manner in fatty livers. Knockdown of hepatic S100a11 by an AAV8-shS100a11 vector alleviated hepatic steatosis and insulin resistance in HFD-fed Ddah1HKO mice. In summary, our results suggested that the liver DDAH1/S100A11 axis has a marked effect on liver lipid metabolism in obese mice. Strategies to increase liver DDAH1 activity or decrease S100A11 expression could be a valuable approach for NAFLD therapy.

Genetic and Pharmacological Inhibition of GCN2 Ameliorates Hyperglycemia and Insulin Resistance in Type 2 Diabetic Mice.

It is well recognized that there is a strong and complex association between nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D). We previously demonstrated that genetic knockout or pharmacological inhibition of general control nondepressible kinase 2 (GCN2), a well-known amino acid sensor, alleviated hepatic steatosis and insulin resistance in obese mice. However, whether GCN2 affects the development of T2D remains unclear. After a high-fat diet (HFD) plus low-dose streptozotocin (STZ) treatments, Gcn2-/- mice developed less hyperglycemia, insulin resistance, hepatic steatosis, and oxidative stress than wild-type (WT) mice. Inhibition of GCN2 by intraperitoneal injection of 3 mg/kg GCN2iB (a specific inhibitor of GCN2) every other day for 6 weeks also ameliorated hyperglycemia, insulin resistance, hepatic steatosis, and oxidative stress in HFD/STZ- and leptin receptor deletion (db/db)-induced T2D mice. Moreover, depletion of hepatic GCN2 in db/db mice by tail vein injection of an AAV8-shGcn2 vector resulted in similar improvement in those metabolic disorders. The protective mechanism of GCN2 inhibition in T2D mice was associated with regulation of the glucose metabolic pathway, repression of lipogenesis genes, and activation of the Nrf2 pathway. Together, our data provide evidence that strategies to inhibit hepatic GCN2 activity may be novel approaches for T2D therapy.

DDAH1 Protects against Acetaminophen-Induced Liver Hepatoxicity in Mice.

In many developed countries, acetaminophen (APAP) overdose-induced acute liver injury is a significant therapeutic problem. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is a critical enzyme for asymmetric dimethylarginine (ADMA) metabolism. Growing evidence suggests that liver dysfunction is associated with increased plasma ADMA levels and reduced hepatic DDAH1 activity/expression. The purpose of this study was to investigate the involvement of DDAH1 in APAP-mediated hepatotoxicity using Ddah1-/- and DDAH1 transgenic mice. After APAP challenge, Ddah1-/- mice developed more severe liver injury than wild type (WT) mice, which was associated with a greater induction of fibrosis, oxidative stress, inflammation, cell apoptosis and phosphorylation of JNK. In contrast, overexpression of DDAH1 attenuated APAP-induced liver injury. RNA-seq analysis showed that DDAH1 affects xenobiotic metabolism and glutathione metabolism pathways in APAP-treated livers. Furthermore, we found that DDAH1 knockdown aggravated APAP-induced cell death, oxidative stress, phosphorylation of JNK and p65, upregulation of CYP2E1 and downregulation of GSTA1 in HepG2 cells. Collectively, our data suggested that DDAH1 has a marked protective effect against APAP-induced liver oxidative stress, inflammation and injury. Strategies to increase hepatic DDAH1 expression/activity may be novel approaches for drug-induced acute liver injury therapy.

Inhibition of GCN2 alleviates hepatic steatosis and oxidative stress in obese mice: Involvement of NRF2 regulation.

The development of nonalcoholic fatty liver disease (NAFLD) is associated with increased reactive oxygen species (ROS) production. Previous observations on the contradictory roles of general control nonderepressible 2 (GCN2) in regulating the hepatic redox state under different nutritional conditions prompted an investigation of the underlying mechanism by which GCN2 regulates ROS homeostasis. In the present study, GCN2 was found to interact with NRF2 and decrease NRF2 expression in a KEAP1-dependent manner. Activation of GCN2 by halofuginone treatment or leucine deprivation decreased NRF2 expression in hepatocytes by increasing GSK-3ß activity. In response to oxidative stress, GCN2 repressed NRF2 transcriptional activity. Knockdown of hepatic GCN2 by tail vein injection of an AAV8-shGcn2 vector attenuated hepatic steatosis and oxidative stress in leptin-deficient (ob/ob) mice in an NRF2-dependent manner. Inhibition of GCN2 by GCN2iB also ameliorated hepatic steatosis and oxidative stress in both ob/ob mice and high fat diet-fed mice, which was associated with significant changes in lipid and amino acid metabolic pathways. Untargeted metabolomics analysis revealed that GCN2iB decreased fatty acid and sphingomyelin levels but increased aliphatic amino acid and phosphatidylcholine levels in fatty livers. Collectively, our results provided the first direct evidence that GCN2 is a novel regulator of NRF2 and that specific GCN2 inhibitors might be potential drugs for NAFLD therapy.

Metformin protects against PM2.5-induced lung injury and cardiac dysfunction independent of AMP-activated protein kinase α2.

Fine particulate matter (PM2.5) airborne pollution increases the risk of respiratory and cardiovascular diseases. Although metformin is a well-known antidiabetic drug, it also confers protection against a series of diseases through the activation of AMP-activated protein kinase (AMPK). However, whether metformin affects PM2.5-induced adverse health effects has not been investigated. In this study, we exposed wild-type (WT) and AMPKα2-/- mice to PM2.5 every other day via intratracheal instillation for 4 weeks. After PM2.5 exposure, the AMPKα2-/- mice developed more severe lung injury and cardiac dysfunction than were developed in the WT mice; however the administration of metformin was effective in attenuating PM2.5-induced lung injury and cardiac dysfunction in both the WT and AMPKα2-/- mice. In the PM2.5-exposed mice, metformin treatment resulted in reduced systemic and pulmonary inflammation, preserved left ventricular ejection fraction, suppressed induction of pulmonary and myocardial fibrosis and oxidative stress, and increased levels of mitochondrial antioxidant enzymes. Moreover, pretreatment with metformin significantly attenuated PM2.5-induced cell death and oxidative stress in control and AMPKα2-depleted BEAS-2B and H9C2 cells, and was associated with preserved expression of mitochondrial antioxidant enzymes. These data support the notion that metformin protects against PM2.5-induced adverse health effects through a pathway that appears independent of AMPKα2. Our findings suggest that metformin may also be a novel drug for therapies that treat air pollution associated disease.

The effect of exposure time and concentration of airborne PM2.5 on lung injury in mice: A transcriptome analysis.

The association between airborne fine particulate matter (PM2.5) concentration and the risk of respiratory diseases has been well documented by epidemiological studies. However, the mechanism underlying the harmful effect of PM2.5 has not been fully understood. In this study, we exposed the C57BL/6J mice to airborne PM2.5 for 3 months (mean daily concentration ~50 or ~110 µg/m3, defined as PM2.5-3L or PM2.5-3H) or 6 months (mean daily concentration ~50 µg/m3, defined as PM2.5-6L) through a whole-body exposure system. Histological and biochemical analysis revealed that PM2.5-3H exposure caused more severe lung injury than did PM2.5-3L, and the difference was greater than that of PM2.5-6L vs PM2.5-3L exposure. With RNA-sequencing technique, we found that the lungs exposed with different concentration of PM2.5 have distinct transcriptional profiles. PM2.5-3H exposure caused more differentially expressed genes (DEGs) in lungs than did PM2.5-3L or PM2.5-6L. The DEGs induced by PM2.5-3L or PM2.5-6L exposure were mainly enriched in immune pathways, including Hematopoietic cell lineage and Cytokine-cytokine receptor interaction, while the DEGs induced by PM2.5-3H exposure were mainly enriched in cardiovascular disease pathways, including Hypertrophic cardiomyopathy and Dilated cardiomyopathy. In addition, we found that upregulation of Cd5l and reduction of Hspa1 and peroxiredoxin-4 was associated with PM2.5-induced pulmonary inflammation and oxidative stress. These results may provide new insight into the cytotoxicity mechanism of PM2.5 and help to development of new strategies to attenuate air pollution associated respiratory disease.

GCN2 deficiency ameliorates cardiac dysfunction in diabetic mice by reducing lipotoxicity and oxidative stress.

Excessive myocardial lipid accumulation is a major feature of diabetic cardiomyopathy (DCM). Although general control nonderepressible 2 (GCN2) has been identified as a sensor of amino acid availability, it also functions as an important regulator of hepatic lipid metabolism. Our previous studies have reported that GCN2 promotes pressure overload or doxorubicin-induced cardiac dysfunction by increasing cardiomyocyte apoptosis and myocardial oxidative stress. However, the impact of GCN2 on the development of DCM remains unclear. In this study, we investigated the effect of GCN2 on DCM in type 1 and type 2 diabetes animal models. After streptozotocin (STZ) or high-fat diet (HFD) plus low-dose STZ treatments, GCN2-/- mice developed less cardiac dysfunction, hyperlipidemia, myocardial hypertrophy, fibrosis, lipid accumulation, oxidative stress, inflammation and apoptosis compared with wild-type (WT) mice. In diabetic hearts, GCN2 deficiency attenuated the upregulation of peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ), the phosphorylation of eIF2α and the induction of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), as well as the reduction of Bcl-2. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, high glucose or palmitic acid-induced cell death, oxidative and endoplasmic reticulum stress and lipid accumulation in H9C2 cells. Collectively, our data provide evidence that GCN2 deficiency protects cardiac function by reducing lipid accumulation, oxidative stress and cell death. Our findings suggest that strategies to inhibit GCN2 activity in the heart may be novel approaches for DCM therapy.

GCN2 deficiency protects against high fat diet induced hepatic steatosis and insulin resistance in mice.

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic lipid deposition and oxidative stress. It has been demonstrated that general control nonderepressible 2 (GCN2) is required to maintain hepatic fatty acid homeostasis under conditions of amino acid deprivation. However, the impact of GCN2 on the development of NAFLD has not been investigated. In this study, we used Gcn2-/- mice to investigate the effect of GCN2 on high fat diet (HFD)-induced hepatic steatosis. After HFD feeding for 12 weeks, Gcn2-/- mice were less obese than wild-type (WT) mice, and Gcn2-/- significantly attenuated HFD-induced liver dysfunction, hepatic steatosis and insulin resistance. In the livers of the HFD-fed mice, GCN2 deficiency resulted in higher levels of lipolysis genes, lower expression of genes related to FA synthesis, transport and lipogenesis, and less induction of oxidative stress. Furthermore, we found that knockdown of GCN2 attenuated, whereas overexpression of GCN2 exacerbated, palmitic acid-induced steatosis, oxidative & ER stress, and changes of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS) and metallothionein (MT) expression in HepG2 cells. Collectively, our data provide evidences that GCN2 deficiency protects against HFD-induced hepatic steatosis by inhibiting lipogenesis and reducing oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in the liver may provide a novel approach to attenuate NAFLD development.

AMPKα2 deficiency exacerbates long-term PM2.5 exposure-induced lung injury and cardiac dysfunction.

Previous studies have demonstrated that long-term exposure to fine particulate matter (PM2.5) increases the risk of respiratory and cardiovascular diseases. As a metabolic sensor, AMP-activated protein kinase (AMPK) is a promising target for cardiovascular disease. However, the impact of AMPK on the adverse health effects of PM2.5 has not been investigated. In this study, we exposed wild-type (WT) and AMPKα2-/- mice to either airborne PM2.5 (mean daily concentration ~64 µg/m3) or filtered air for 6 months through a whole-body exposure system. After exposure, AMPKα2-/- mice developed severe lung injury and left ventricular dysfunction. In the PM2.5-exposed lungs and hearts, loss of AMPKα2 resulted in higher levels of fibrotic genes, more collagen deposition, lower levels of peroxiredoxin 5 (Prdx5), and greater induction of oxidative stress and inflammation than observed in the lungs and hearts of WT mice. In PM2.5-exposed BEAS-2B and H9C2 cells, inhibition of AMPK activity significantly decreased cell viability and Prdx5 expression, and increased the intracellular ROS and p-NF-κB levels. Collectively, our results provide the first direct evidence that AMPK has a marked protective effect on the adverse health effects induced by long-term PM2.5 exposure. Our findings suggest that strategies to increase AMPK activity may provide a novel approach to attenuate air pollution associated disease.

GCN2 deficiency ameliorates doxorubicin-induced cardiotoxicity by decreasing cardiomyocyte apoptosis and myocardial oxidative stress.

The clinical use of doxorubicin for cancer therapy is limited by its cardiotoxicity, which involves cardiomyocyte apoptosis and oxidative stress. Previously, we showed that general control nonderepressible 2 (GCN2), an eukaryotic initiation factor 2α (eIF2α) kinase, impairs the ventricular adaptation to chronic pressure overload by affecting cardiomyocyte apoptosis. However, the impact of GCN2 on Dox-induced cardiotoxicity has not been investigated. In the present study, we treated wild type (WT) and Gcn2-/- mice with four intraperitoneal injections (5 mg/kg/week) to induce cardiomyopathy. After Dox treatment, Gcn2-/- mice developed less contractile dysfunction, myocardial fibrosis, apoptosis, and oxidative stress compared with WT mice. In the hearts of the Dox-treated mice, GCN2 deficiency attenuated eIF2α phosphorylation and induction of its downstream targets, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), and preserved the expression of anti-apoptotic factor Bcl-2 and mitochondrial uncoupling protein-2(UCP2). Furthermore, we found that GCN2 knockdown attenuated, whereas GCN2 overexpression exacerbated, Dox-induced cell death, oxidative stress and reduction of Bcl-2 and UCP2 expression through the eIF2α-CHOP-dependent pathway in H9C2 cells. Collectively, our data provide solid evidence that GCN2 has a marked effect on Dox induced myocardial apoptosis and oxidative stress. Our findings suggest that strategies to inhibit GCN2 activity in cardiomyocyte may provide a novel approach to attenuate Dox-related cardiotoxicity.