Cinnabarinic Acid Provides Hepatoprotection Against Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition in which excess lipids accumulate in the liver and can lead to a range of progressive liver disorders including non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. While lifestyle and diet modifications have proven to be effective as NAFLD treatments, they are not sustainable in the long-term, and currently no pharmacological therapies are approved to treat NAFLD. Our previous studies demonstrated that cinnabarinic acid (CA), a novel endogenous Aryl hydrocarbon Receptor (AhR) agonist, activates the AhR target gene, Stanniocalcin 2, and confers cytoprotection against a plethora of ER/oxidative stressors. In this study, the hepatoprotective and anti-steatotic properties of CA were examined against free fatty-acid-induced in vitro and high-fat-diet fed in vivo NAFLD models. The results demonstrated that CA treatment significantly lowered weight gain and attenuated hepatic lipotoxicity both before and after the established fatty liver, thereby protecting against steatosis, inflammation, and liver injury. CA mitigated intracellular free fatty acid uptake concomitant with the downregulation of CD36/fatty acid translocase. Genes involved in fatty acid and triglyceride synthesis were also downregulated in response to CA treatment. Additionally, suppressing AhR and Stc2 expression using RNA interference in vitro verified that the hepatoprotective effects of CA were absolutely dependent on both AhR and its target, Stc2. Collectively, our results demonstrate that the endogenous AhR agonist, CA, confers hepatoprotection against NAFLD by regulating hepatic fatty acid uptake and lipogenesis. SIGNIFICANCE STATEMENT: In this study using in vitro and in vivo models, we demonstrate that cinnabarinic acid (CA), an endogenous AhR agonist, provides protection against non-alcoholic fatty liver disease. CA bestows cytoprotection against steatosis and liver injury by controlling expression of several key genes associated with lipid metabolism pathways, limiting the hepatic lipid uptake, and controlling liver inflammation. Moreover, CA-induced hepatoprotection is absolutely dependent on AhR and Stc2 expression.

Maternal Pyrroloquinoline Quinone Supplementation Improves Offspring Liver Bioactive Lipid Profiles throughout the Lifespan and Protects against the Development of Adult NAFLD.

Maternal obesity and consumption of a high-fat diet significantly elevate risk for pediatric nonalcoholic fatty liver disease (NAFLD), affecting 10% of children in the US. Almost half of these children are diagnosed with nonalcoholic steatohepatitis (NASH), a leading etiology for liver transplant. Animal models show that signs of liver injury and perturbed lipid metabolism associated with NAFLD begin in utero; however, safe dietary therapeutics to blunt developmental programming of NAFLD are unavailable. Using a mouse model of maternal Western-style diet (WD), we previously showed that pyrroloquinoline quinone (PQQ), a potent dietary antioxidant, protected offspring of WD-fed dams from development of NAFLD and NASH. Here, we used untargeted mass spectrometry-based lipidomics to delineate lipotoxic effects of WD on offspring liver and identify lipid targets of PQQ. PQQ exposure during pregnancy altered hepatic lipid profiles of WD-exposed offspring, upregulating peroxisome proliferator-activated receptor (PPAR) α signaling and mitochondrial fatty acid oxidation to markedly attenuate triglyceride accumulation beginning in utero. Surprisingly, the abundance of very long-chain ceramides, important in promoting gut barrier and hepatic function, was significantly elevated in PQQ-treated offspring. PQQ exposure reduced the hepatic phosphatidylcholine/phosphatidylethanolamine (PC/PE) ratio in WD-fed offspring and improved glucose tolerance. Notably, levels of protective n - 3 polyunsaturated fatty acids (PUFAs) were elevated in offspring exposed to PQQ, beginning in utero, and the increase in n - 3 PUFAs persisted into adulthood. Our findings suggest that PQQ supplementation during gestation and lactation augments pathways involved in the biosynthesis of long-chain fatty acids and plays a unique role in modifying specific bioactive lipid species critical for protection against NAFLD risk in later life.

Canonical Wnt Signaling in the Pathology of Iron Overload-Induced Oxidative Stress and Age-Related Diseases.

Iron accumulates in the vital organs with aging. This is associated with oxidative stress, inflammation, and mitochondrial dysfunction leading to age-related disorders. Abnormal iron levels are linked to neurodegenerative diseases, liver injury, cancer, and ocular diseases. Canonical Wnt signaling is an evolutionarily conserved signaling pathway that regulates many cellular functions including cell proliferation, apoptosis, cell migration, and stem cell renewal. Recent evidences indicate that iron regulates Wnt signaling, and iron chelators like deferoxamine and deferasirox can inhibit Wnt signaling and cell growth. Canonical Wnt signaling is implicated in the pathogenesis of many diseases, and there are significant efforts ongoing to develop innovative therapies targeting the aberrant Wnt signaling. This review examines how intracellular iron accumulation regulates Wnt signaling in various tissues and their potential contribution in the progression of age-related diseases.

Western diet-induced shifts in the maternal microbiome are associated with altered microRNA expression in baboon placenta and fetal liver.

Maternal consumption of a high-fat, Western-style diet (WD) disrupts the maternal/infant microbiome and contributes to developmental programming of the immune system and nonalcoholic fatty liver disease (NAFLD) in the offspring. Epigenetic changes, including non-coding miRNAs in the fetus and/or placenta may also underlie this risk. We previously showed that obese nonhuman primates fed a WD during pregnancy results in the loss of beneficial maternal gut microbes and dysregulation of cellular metabolism and mitochondrial dysfunction in the fetal liver, leading to a perturbed postnatal immune response with accelerated NAFLD in juvenile offspring. Here, we investigated associations between WD-induced maternal metabolic and microbiome changes, in the absence of obesity, and miRNA and gene expression changes in the placenta and fetal liver. After ~8-11 months of WD feeding, dams were similar in body weight but exhibited mild, systemic inflammation (elevated CRP and neutrophil count) and dyslipidemia (increased triglycerides and cholesterol) compared with dams fed a control diet. The maternal gut microbiome was mainly comprised of Lactobacillales and Clostridiales, with significantly decreased alpha diversity (P = 0.0163) in WD-fed dams but no community-wide differences (P = 0.26). At 0.9 gestation, mRNA expression of IL6 and TNF in maternal WD (mWD) exposed placentas trended higher, while increased triglycerides, expression of pro-inflammatory CCR2, and histological evidence for fibrosis were found in mWD-exposed fetal livers. In the mWD-exposed fetus, hepatic expression levels of miR-204-5p and miR-145-3p were significantly downregulated, whereas in mWD-exposed placentas, miR-182-5p and miR-183-5p were significantly decreased. Notably, miR-1285-3p expression in the liver and miR-183-5p in the placenta were significantly associated with inflammation and lipid synthesis pathway genes, respectively. Blautia and Ruminococcus were significantly associated with miR-122-5p in liver, while Coriobacteriaceae and Prevotellaceae were strongly associated with miR-1285-3p in the placenta; both miRNAs are implicated in pathways mediating postnatal growth and obesity. Our findings demonstrate that mWD shifts the maternal microbiome, lipid metabolism, and inflammation prior to obesity and are associated with epigenetic changes in the placenta and fetal liver. These changes may underlie inflammation, oxidative stress, and fibrosis patterns that drive NAFLD and metabolic disease risk in the next generation.

PPARα agonist fenofibrate attenuates iron-induced liver injury in mice by modulating the Sirt3 and ß-catenin signaling.

Iron accumulation is frequently associated with chronic liver diseases. However, our knowledge on how iron contributes to the liver injury is limited. Aberrant Wnt/ß-catenin signaling is a hallmark of several hepatic pathologies. We recently reported that peroxisome proliferator-activated receptor α (PPARα) agonist, fenofibrate, prevents iron-induced oxidative stress and ß-catenin signaling by chelating the iron. Sirtuin3 (Sirt3), a type of NAD+-dependent deacetylase, that plays a critical role in metabolic regulation was found to prevent ischemia reperfusion injury (IRI) by normalizing the Wnt/ß-catenin pathway. In the present study, we explored if fenofibrate prevents iron-induced liver injury by regulating the Sirt3 and ß-catenin signaling. In vitro and in vivo iron treatment resulted in the downregulation of PPARα, Sirt3, active ß-catenin, and its downstream target gene c-Myc in the mouse liver. Pharmacological activation of Sirt3, both in vitro and in vivo, by Honokiol (HK), a known activator of Sirt3, abrogated the inhibitory effect of iron overload on active ß-catenin expression and prevented the iron-induced upregulation of α smooth muscle actin (αSMA) and TGFß expression. Intrinsically, PPARα knockout mice showed significant downregulation of hepatic Sirt3 levels. In addition, treatment of iron overload mice with PPARα agonist fenofibrate reduced hepatic iron accumulation and prevented iron-induced downregulation of liver Sirt3 and active ß-catenin, mitigating the progression of fibrosis. Thus, our results establish a novel link between hepatic iron and PPARα, Sirt3, and ß-catenin signaling. Further exploration on the mechanisms by which fenofibrate ameliorates iron-induced liver injury likely has significant therapeutic impact on iron-associated chronic liver diseases.NEW & NOTEWORTHY Hepatic intracellular iron accumulation has been implicated in the pathophysiology of chronic liver diseases. In this study, we identified a novel mechanism involved in the progression of fibrosis. Excess iron accumulation in liver caused downregulation of PPARα-Sirt3-Wnt signaling leading to fibrosis. This work has significant translational potential as PPARα agonist fenofibrate could be an attractive therapeutic drug for the treatment of liver disorders associated with iron overload.

Fenofibrate prevents iron induced activation of canonical Wnt/ß-catenin and oxidative stress signaling in the retina.

Accumulating evidence strongly implicates iron in the pathogenesis of aging and disease. Iron levels have been found to increase with age in both the human and mouse retinas. We and others have shown that retinal diseases such as age-related macular degeneration and diabetic retinopathy are associated with disrupted iron homeostasis, resulting in retinal iron accumulation. In addition, hereditary disorders due to mutation in one of the iron regulatory genes lead to age dependent retinal iron overload and degeneration. However, our knowledge on whether iron toxicity contributes to the retinopathy is limited. Recently, we reported that iron accumulation is associated with the upregulation of retinal and renal renin-angiotensin system (RAS). Evidences indicate that multiple genes/components of the RAS are targets of Wnt/ß-catenin signaling. Interestingly, aberrant activation of Wnt/ß-catenin signaling is observed in several degenerative diseases. In the present study, we explored whether iron accumulation regulates canonical Wnt signaling in the retina. We found that in vitro and in vivo iron treatment resulted in the upregulation of Wnt/ß-catenin signaling and its downstream target genes including renin-angiotensin system in the retina. We confirmed further that iron activates canonical Wnt signaling in the retina using TOPFlash T-cell factor/lymphoid enhancer factor promoter assay and Axin2-LacZ reporter mouse. The presence of an iron chelator or an antioxidant reversed the iron-mediated upregulation of Wnt/ß-catenin signaling in retinal pigment epithelial (RPE) cells. In addition, treatment of RPE cells with peroxisome proliferator-activated receptor (PPAR) α-agonist fenofibrate prevented iron-induced activation of oxidative stress and Wnt/ß-catenin signaling by chelating the iron. The role of fenofibrate, an FDA-approved drug for hyperlipidemia, as an iron chelator has potentially significant therapeutic impact on iron associated degenerative diseases.

Pediatric Non-Alcoholic Fatty Liver Disease: Nutritional Origins and Potential Molecular Mechanisms.

Non-alcoholic fatty liver disease (NAFLD) is the number one chronic liver disease worldwide and is estimated to affect nearly 40% of obese youth and up to 10% of the general pediatric population without any obvious signs or symptoms. Although the early stages of NAFLD are reversible with diet and lifestyle modifications, detecting such stages is hindered by a lack of non-invasive methods of risk assessment and diagnosis. This absence of non-invasive means of diagnosis is directly related to the scarcity of long-term prospective studies of pediatric NAFLD in children and adolescents. In the majority of pediatric NAFLD cases, the mechanisms driving the origin and rapid progression of NAFLD remain unknown. The progression from NAFLD to non-alcoholic steatohepatitis (NASH) in youth is associated with unique histological features and possible immune processes and metabolic pathways that may reflect different mechanisms compared with adults. Recent data suggest that circulating microRNAs (miRNAs) are important new biomarkers underlying pathways of liver injury. Several factors may contribute to pediatric NAFLD development, including high-sugar diets, in utero exposures via epigenetic alterations, changes in the neonatal microbiome, and altered immune system development and mitochondrial function. This review focuses on the unique aspects of pediatric NAFLD and how nutritional exposures impact the immune system, mitochondria, and liver/gastrointestinal metabolic health. These factors highlight the need for answers to how NAFLD develops in children and for early stage-specific interventions.

Renal iron accelerates the progression of diabetic nephropathy in the HFE gene knockout mouse model of iron overload.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease associated with high mortality worldwide. Increases in iron levels have been reported in diabetic rat kidneys as well as in human urine of patients with diabetes. In addition, a low-iron diet or iron chelators delay the progression of DN in patients with diabetes and in animal models of diabetes. Possible maladaptive mechanisms of organ damage by tissue iron accumulation have not been well studied. We recently reported that iron induced the retinal renin-angiotensin system (RAS) and accelerated the progression of diabetic retinopathy. However, whether iron regulates the systemic RAS is unknown. To explore if iron alters the expression of intrarenal RAS and its role in the progression of DN, we used the high Fe iron (HFE) knockout mouse, a genetic model of systemic iron overload. We found that diabetes upregulated the expression of iron regulatory proteins and augmented tissue iron accumulation in the kidneys of both type 1 and type 2 diabetic mouse models. Iron accumulation in the kidneys of HFE knockout mice was associated with increase in serum and intrarenal renin expression. Induction of diabetes in HFE knockout mice using streptozotocin caused a much higher accumulation of renal iron and accelerated the progression of nephropathy compared with diabetic wild-type mice. Treatment of diabetic mice with the iron chelator deferiprone reversed the renin upregulation and reduced kidney injury. Thus, our results establish a new link between renal iron and RAS activity. Exploring the mechanisms of iron-induced RAS activation further may have a significant therapeutic impact on hypertension and DN.

Melatonin protects against lipid-induced mitochondrial dysfunction in hepatocytes and inhibits stellate cell activation during hepatic fibrosis in mice.

Lipid generates reactive oxygen species (ROS) in consequence to mitochondrial fission followed by inflammation in propagating hepatic fibrosis. The interaction of SIRT1/Mitofusin2 is critical for maintaining mitochondrial integrity and functioning, which is disrupted upon excess lipid infiltration during the progression of steatohepatitis. The complex interplay between hepatic stellate cells and steatotic hepatocytes is critically regulated by extracellular factors including increased circulating free fatty acids during fibrogenesis. Melatonin, a potent antioxidant, protects against lipid-mediated mitochondrial ROS generation. Lipotoxicity induces disruption of SIRT1 and Mitofusin2 interaction leading to mitochondrial morphological disintegration in hepatocytes. Further, fragmented mitochondria leads to mitochondrial permeability transition pore opening, cell cycle arrest and apoptosis and melatonin protects against all these lipotoxicity-mediated dysfunctions. These impaired mitochondrial dynamics also enhances the cellular glycolytic flux and reduces mitochondrial oxygen consumption rate that potentiates ROS production. High glycolytic flux generates metabolically unfavorable milieu in hepatocytes leading to inflammation, which is abrogated by melatonin. The melatonin-mediated protection against mitochondrial dysfunction was also observed in high-fat diet (HFD)-fed mice through restoration of enzymatic activities associated with respiratory chain and TCA cycle. Subsequently, melatonin reduces hepatic fat deposition and inflammation in HFD-fed mice. Thus, melatonin disrupts the interaction between steatotic hepatocyte and stellate cells, leading to the activation of the latter to abrogate collagen deposition. Altogether, the results of the current study document that the pharmacological intervention with low dose of melatonin could abrogate lipotoxicity-mediated hepatic stellate cell activation and prevent the fibrosis progression.

Dietary fat proportionately enhances oxidative stress and glucose intolerance followed by impaired expression of the genes associated with mitochondrial biogenesis.

Consumption of food that surpasses the metabolic necessity of the body leads to an epidemic condition termed obesity, which causes several metabolic disorders including oxidative damage. Dietary intervention can enlighten the mechanisms and therapeutics associated with these metabolic disorders. The reported studies related to diet include fat of different kinds and from different sources, however they lack dose response aspects. Our study highlighted the importance of dietary fat modification in modulating oxidative stress-induced glucose intolerance. Animals were maintained on a diet with a varied content of fat (30%/45%/60%) for 12 weeks and the 'withdrawal' group was fed a standard diet for another 10 weeks. The diet containing 60 energy% of fat displayed glucose intolerance, high ALT, low GSH levels and tissue-specific modulation of the prooxidant/antioxidant enzymatic activities in the liver/muscles. Prolonged sustenance of the 60 energy% fat containing diet-fed rats on standard diet led to the alteration of antioxidant activities, reversing the oxidative damage. Notably, the 'withdrawal' group displayed an organ-specific response towards dietary modification where the recovery of the antioxidant activities was observed to be much more pronounced in the liver as compared to the muscle. Further, we identified the differential expression of liver/muscle-specific genes associated with oxidative stress and mitochondrial biogenesis in response to the differing fat content. These genes can serve as markers for HFD-induced metabolic complications involving the liver/muscle. Altogether, our study has highlighted the novel area where obesity-induced oxidative stress linked alterations expressed diet and organ specific responses that are recovered by altering the dietary regimen. Future investigation of dietary modulation will open nascent avenues for developing therapeutic modalities addressing obesity-related metabolic complications.

Thioredoxin interacting protein mediates lipid-induced impairment of glucose uptake in skeletal muscle.

Insulin resistance (IR) is an important determinant of type-2 diabetes mellitus (T2DM). Free fatty acids (FFAs) induce IR by various mechanisms. A surfeit of circulating FFA leads to intra-myocellular lipid accumulation that induces mitochondrial ROS generation and worsens IR. However, the molecular mechanisms behind are unclear. We identified thioredoxin interacting protein (TxNIP), which is overexpressed in T2DM, to be a promoter of ROS-induced IR. We observed upregulation of TxNIP upon palmitate treatment in skeletal muscle cells that led to ROS generation and Glut-4 downregulation resulting in impaired glucose-uptake. FFA-induced overexpression of TxNIP gene was mediated through the activation of its bona-fide trans activator, ChREBP. Further, Palmitate-induced impairment in AMPK-SIRT-1 pathway resulted in overexpression of ChREBP. While Fenofibrate, abrogated PA-induced TxNIP expression and ROS generation in skeletal muscle cells, Saroglitazar, a dual PPARα/γ-agonist, not only inhibited PA-induced TXNIP expression but also led to greater improvement in glucose uptake. Taken together, TxNIP appears to be an important factor in FFA-induced ROS generation and IR in skeletal muscle cells, which can be modulated for the management of this complex disorder.

Fenofibrate Reverses Palmitate Induced Impairment in Glucose Uptake in Skeletal Muscle Cells by Preventing Cytosolic Ceramide Accumulation.

BACKGROUNDS/AIMS: The lipid induced insulin resistance is a major pathophysiologic mechanism underlying glucose intolerance of varying severity. PPARα-agonists are proven as effective hypolipidemic agents. The aim of this study was to see if impaired glucose uptake in palmitate treated myotubes is reversed by fenofibrate. METHODS: Palmitate-treated myotubes were used as a model for insulin resistance, impaired glucose uptake, fatty acid oxidation and ceramide synthesis. mRNA levels of CPT1 and CPT2 were determined by PCR array and Q-PCR. RESULTS: The incubation of myotubes with 750 uM palmitate not only reduced glucose uptake but also impaired fatty acid oxidation and cytosolic ceramide accumulation. Palmitate upregulated CPT1b expression in L6 myotubes, while CPT2 expression level remained unchanged. The altered stoichiometric ratio between the two CPT isoforms led to reduced fatty acid oxidation (FAO), ceramide accumulation and impaired glucose uptake, whereas administration of 200 µM fenofibrate significantly reversed the above abnormalities by increasing CPT2 mRNA levels and restoring CPT1b to CPT2 ratio. CONCLUSION: Palmitate-induced alteration in the stoichiometric ratio of mitochondrial CPT isoforms leads to incomplete FAO and enhanced cytosolic ceramide accumulation that lead to insulin resistance. Fenofibrate ameliorated insulin resistance by restoring the altered stoichiometry by upregulating CPT2 and preventing, cytoplasmic ceramide accumulation.

Heterologous priming-boosting with DNA and vaccinia virus expressing kinetoplastid membrane protein-11 induces potent cellular immune response and confers protection against infection with antimony resistant and sensitive strains of Leishmania (Leishmania) donovani.

BACKGROUND: Emergence of resistance against commonly available drugs poses a major threat in the treatment of visceral leishmaniasis (VL), particularly in the Indian subcontinent. Absence of any licensed vaccine against VL emphasizes the urgent need to develop an effective alternative vaccination strategy. METHODOLOGY: We developed a novel heterologous prime boost immunization strategy using kinetoplastid membrane protein-11 (KMP-11) DNA priming followed by boosting with recombinant vaccinia virus (rVV) expressing the same antigen. The efficacy of this vaccination regimen in a murine and hamster model of visceral leishmaniasis caused by both antimony resistant (Sb-R) and sensitive (Sb-S) Leishmania (L.) donovani is examined. RESULT: Heterologous prime-boost (KMP-11 DNA/rVV) vaccination was able to protect mice and hamsters from experimental VL induced by both Sb-S and Sb-R-L. (L.) donovani isolates. Parasite burden is kept significantly low in the vaccinated groups even after 60 days post-infection in hamsters, which are extremely susceptible to VL. Protection in mice is correlated with strong cellular and humoral immune responses. Generation of polyfunctional CD8(+) T cell was observed in vaccinated groups, which is one of the most important prerequisite for successful vaccination against VL. Protection was accompanied with generation of antigen specific CD4(+) and CD8(+) cells that produced effector cytokines such as IFN-γ, IL-2 and TNF-α. KMP-11-DNA/rVV vaccination also developed strong cytotoxic response and reversed T-cell impairment to induce antigen specific T cell proliferation. CONCLUSION: KMP-11 is a unique antigen with high epitope density. Heterologous prime boost vaccination activates CD4(+) and CD8(+) T-cell mediated immunity to confer resistance to VL. This immunization method also produces high quality T-cells secreting multiple effector cytokines thus enhancing durability of the immune response. Thus the vaccination regime as described in the present study could provide a potent strategy for future anti-leishmanial vaccine development.