Effects of acute iron overload on Nrf2-related glutathione metabolism in rat brain.

Iron (Fe) overload triggers free radical production and lipid peroxidation processes that may lead to cell death (ferroptosis). The hypothesis of this work was that acute Fe-dextran treatment triggers Nrf2-mediated antioxidant regulation in rat brain involving glutathione (GSH) metabolism. Over the initial 8 h after Fe-dextran administration (single dose of 500 mg Fe-dextran/kg), total Fe, malondialdehyde (MDA) content, glutathione peroxidase (GPx), GPx-Se dependent (GPx-Se) and glutathione S-transferases (GST) activities were increased in rat whole brain. The content of GSH and the activity of glutathione reductase (GR) showed decreases (p < 0.05) after 6 and 8 h post injection in cortex. A significant increase in nuclear Nrf2 protein levels over control values was achieved after 6 h of Fe-dextran administration, while no significant differences were observed in the cytosolic fraction. Nuclear Nrf2/cytosolic Nrf2 ratios showed enhancement (p < 0.05) after 6 h of Fe overload, suggesting a greater translocation of the factor to the nucleus. No significant differences were observed in the expression of Keap1 in nuclear or cytosolic extracts. It is concluded that acute Fe overload induces oxidative stress in rat brain with the concomitant lipid peroxidation increase and GSH depletion, leading to the elevation of Nrf2-controlled GPx, GPx-Se and GST protein expression as a protective adaptive response. Further studies are required to fully comprehend the complex network of interrelated processes keeping the balance of GSH functions as chelator, antioxidant and redox buffer in the understanding of the ferroptotic and hormetic mechanisms triggered by Fe overload in brain.

Beneficial effects of natural compounds on experimental liver ischemia-reperfusion injury.

Liver ischemia-reperfusion injury (IRI) is a phenomenon inherent to hepatic surgery that severely compromises the organ functionality, whose underlying mechanisms involve cellular and molecular interrelated processes leading to the development of an excessive inflammatory response. Liver resident cells and those recruited in response to injury generate pro-inflammatory signals such as reactive oxygen species, cytokines, chemokines, proteases and lipid mediators that contribute to hepatocellular necrosis and apoptosis. Besides, dying hepatocytes release damage-associated molecular patterns that actívate inflammasomes to further stimulate inflammatory responses leading to massive cell death. Since liver IRI is a complication of hepatic surgery in man, extensive preclinical studies have assessed potential protective strategies, including the supplementation with natural compounds, with the objective to downregulate nuclear factor-κB functioning, the main effector of inflammatory responses. This can be accomplished by either the activation of peroxisome proliferator-activated receptor-α, G protein-coupled receptor 120 or antioxidant signaling pathways, the synthesis of specific pro-resolving mediators, downregulation of Toll-like receptor 4 activity or additional contributory mechanisms that are beginning to be understood. The latter aspect is a crucial issue to be accomplished in preclinical studies, in order to establish adequate conditions for the supplementation with natural products before major liver surgeries in man involving warm IR, such as hepatic trauma or resection of large intrahepatic tumors.

Influence of BDNF Genetic Polymorphisms in the Pathophysiology of Aging-related Diseases.

For the first time in history, most of the population has a life expectancy equal or greater than 60 years. By the year 2050, it is expected that the world population in that age range will reach 2000 million, an increase of 900 million with respect to 2015, which poses new challenges for health systems. In this way, it is relevant to analyze the most common diseases associated with the aging process, namely Alzheimer´s disease, Parkinson Disease and Type II Diabetes, some of which may have a common genetic component that can be detected before manifesting, in order to delay their progress. Genetic inheritance and epigenetics are factors that could be linked in the development of these pathologies. Some researchers indicate that the BDNF gene is a common factor of these diseases, and apparently some of its polymorphisms favor the progression of them. In this regard, alterations in the level of BDNF expression and secretion, due to polymorphisms, could be linked to the development and/or progression of neurodegenerative and metabolic disorders. In this review we will deepen on the different polymorphisms in the BDNF gene and their possible association with age-related pathologies, to open the possibilities of potential therapeutic targets.

The metabolic dysfunction of white adipose tissue induced in mice by a high-fat diet is abrogated by co-administration of docosahexaenoic acid and hydroxytyrosol.

BACKGROUND: Nutritional interventions are promising tools for the prevention of obesity. The n-3 long-chain polyunsaturated fatty acid (n-3 LCPUFA) docosahexaenoic acid (DHA) modulates immune and metabolic responses while the antioxidant hydroxytyrosol (HT) prevents oxidative stress (OS) in white adipose tissue (WAT). OBJECTIVE: The DHA plus HT combined protocol prevents WAT alterations induced by a high-fat diet in mice. Main related mechanisms. METHODS: Male C57BL/6J mice were fed a control diet (CD; 10% fat, 20% protein, and 70% carbohydrates) or a high fat diet (HFD) (60% fat, 20% protein, and 20% carbohydrates) for 12 weeks, without and with supplementation of DHA (50 mg kg-1 day-1), HT (5 mg kg-1 day-1) or both. Measurements of WAT metabolism include morphological parameters, DHA content in phospholipids (gas chromatography), lipogenesis, OS and inflammation markers, mitochondrial activity and gene expression of transcription factors SREBP-1c, PPAR-γ, NF-κB (p65) and Nrf2 (quantitative polymerase chain reaction and enzyme-linked immunosorbent assay). RESULTS: The combined DHA and HT intervention attenuated obesity development, suppressing the HFD-induced inflammatory and lipogenic signals, increasing antioxidant defenses, and maintaining the phospholipid LCPUFA n-3 content and mitochondrial function in WAT. At the systemic level, the combined intervention also improved the regulation of glucose and adipokine homeostasis. CONCLUSION: The combined DHA and HT protocol appears to be an important nutritional strategy for the treatment of metabolic diseases, with abrogation of obesity-driven metabolic inflammation and recovery of a small-healthy adipocyte phenotype.

Protective Effects of Eicosapentaenoic Acid Plus Hydroxytyrosol Supplementation Against White Adipose Tissue Abnormalities in Mice Fed a High-Fat Diet.

OBJECTIVE: Obesity induced by high-fat diet (HFD) elicits white adipose tissue dysfunction. In this study, we have hypothesized that the metabolic modulator eicosapentaenoic acid (EPA) combined with the antioxidant hydroxytyrosol (HT) attenuates HFD-induced white adipose tissue (WAT) alterations. METHODS: C57BL/6J mice were administered with a HFD (60% fat, 20% protein, 20% carbohydrates) or control diet (CD; 10% fat, 20% protein, 70% carbohydrates), with or without EPA (50 mg/kg/day), HT (5 mg/kg/day), or both for 12 weeks. Determinations in WAT include morphological parameters, EPA and docosahexaenoic acid content in phospholipids (gas chromatography), lipogenesis, oxidative stress (OS) and inflammation markers, and gene expression and activities of transcription factors, such as sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-gamma (PPAR-γ), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) (p65 subunit) and nuclear factor erythroid 2-related factor 2 (Nrf2) (quantitative polymerase chain reaction and enzyme linked immunosorbent assay). RESULTS: HFD led to WAT hypertrophy in relation to PPAR-γ downregulation. WAT metabolic dysfunction was characterized by upregulation of lipogenic SREBP-1c system, mitochondrial energy metabolism depression, loss of the antioxidant Nrf2 signaling with OS enhancement, n-3 long-chain polyunsaturated fatty acids depletion and activation of the pro-inflammatory NF-κB system. EPA and HT co-supplementation diminished HFD-dependent effects additively, reaching values close or similar to controls. CONCLUSION: Data presented strengthen the importance of combined protocols such as EPA plus HT to attenuate metabolic-inflammatory states triggered by obesity.

Iron-induced derangement in hepatic Δ-5 and Δ-6 desaturation capacity and fatty acid profile leading to steatosis: Impact on extrahepatic tissues and prevention by antioxidant-rich extra virgin olive oil.

The administration of iron induces liver oxidative stress and depletion of long-chain polyunsaturated fatty acids (LCPUFAs), n-6/n-3 LCPUFA ratio enhancement and fat accumulation, which may be prevented by antioxidant-rich extra virgin olive oil (AR-EVOO) supplementation. Male Wistar rats were subjected to a control diet (50 mg iron/kg diet) or iron-rich diet (IRD; 200 mg/kg diet) with alternate AR-EVOO for 21 days. Liver fatty acid (FA) analysis was performed by gas-liquid chromatography (GLC) after lipid extraction and fractionation, besides Δ-5 desaturase (Δ-5 D) and Δ6-D mRNA expression (qPCR) and activity (GLC) measurements. The IRD significantly (p < 0.05) increased hepatic total fat, triacylglycerols, free FA contents and serum transaminases levels, with diminution in those of n-6 and n-3 LCPUFAs, higher n-6/n-3 ratios, lower unsaturation index and Δ5-D and Δ6-D activities, whereas the mRNA expression of both desaturases was enhanced over control values, changes that were prevented by concomitant AR-EVOO supplementation. N-6 and n-3 LCPUFAs were also decreased by IRD in extrahepatic tissues and normalized by AR-EVOO. In conclusion, AR-EVOO supplementation prevents IRD-induced changes in parameters related to liver FA metabolism and steatosis, an effect that may have a significant impact in the treatment of iron-related pathologies or metabolic disorders such as non-alcoholic fatty liver disease.

Combined administration of docosahexaenoic acid and thyroid hormone synergistically enhances rat liver levels of resolvins RvD1 and RvD2.

Supplementation with omega-3 fatty acids or thyroid hormone (T3) exhibit negative effects on inflammatory reactions in experimental animals. The aim of this work was to assess the hypothesis that docosahexaenoic acid (DHA) plus T3 co-administration enhances liver resolvin (Rv) levels as inflammation resolution mediators. Combined DHA (daily doses of 300 mg/kg for 3 consecutive days)-T3 (0.05 mg/kg at the fourth day) administration significantly increased the content of hepatic RvD1 and RvD2, without changes in that of RvE1 and RvE2, an effect that exhibits synergy when compared to the separate DHA and T3 treatments. Under these conditions, liver DHA levels increased by DHA administration were diminished when combined with T3 (p < 0.05), suggesting enhancement in resolvin D biosynthesis in extrahepatic tissues. It is concluded that co-administration of DHA and T3 rises the capacity of the liver for inflammation resolution by augmenting RvD1(2) availability, which represents an important protocol in hepatoprotection in the clinical setting.

Docosahexaenoic acid-thyroid hormone combined protocol as a novel approach to metabolic stress disorders: Relation to mitochondrial adaptation via liver PGC-1α and sirtuin1 activation.

Docosahexaenoic acid (DHA) and 3,3',5-triiodothyronine (T3 ) combined protocol affords protection against liver injury via AMPK signaling supporting energy requirements. The aim of this work was to test the hypothesis that a DHA + T3 accomplish mitochondrial adaptation through downstream upregulation of PPAR-γ coactivator 1α (PGC-1α). Male Sprague-Dawley rats were given daily oral doses of 300 mg DHA/kg or saline (controls) for three consecutive days, followed by 0.05 mg T3 /kg (or hormone vehicle) ip at the fourth day, or single dose of 0.1 mg T3 /kg alone. Liver mRNA levels were assayed by qPCR, NAD+ /NADH ratios, hepatic proteins, histone 3 acetylation and serum T3 and ß-hydroxybutyrate levels were determined by specific ELISA kits. Combined DHA + T3 protocol led to increased liver AMPK, PGC-1α, NRF-2, COX-IV, and ß-ATP synthase mRNAs, with concomitant higher protein levels of COX-IV and NRF-2, 369% enhancement in the NAD+ /NADH ratio, 47% decrease in histone 3 acetylation and 162% increase in serum levels of ß-hydroxybutyrate over control values. These changes were reproduced by the higher dose of T3 without major alterations by DHA or T3 alone. In conclusion, liver mitochondrial adaptation by DHA + T3 is associated with PGC-1α upregulation involving enhanced transcription of the coactivator, which may be contributed by PGC-1α deacetylation and phosphorylation by SIRT1 and AMPK activation, respectively. This contention is supported by NRF-2-dependent enhancement in COX-1 and ß-ATP synthase induction with higher fatty acid oxidation resulting in a significant ketogenic response, which may represent a suitable strategy for hepatic steatosis with future clinical applications. © 2018 BioFactors, 45(2):271-278, 2019.

Thyroid Hormone-Induced Expression of the Hepatic Scaffold Proteins Sestrin2, ß-Klotho, and FRS2α in Relation to FGF21-AMPK Signaling.

Thyroid hormone (3,3',5-triiodothyronine, T3) accelerates energy metabolism in the liver through mechanisms involving upregulation of AMP-activated protein kinase (AMPK). This study aims to assess the influence of T3 on the expression of the scaffold proteins ß-Klotho, fibroblast growth factor receptor substrate 2α (FRS2α), and Sestrin2 in relation to FGF21-AMPK signaling. Male Sprague-Dawley rats were given 0.1 mg T3/kg or hormone vehicle (controls) and studies were done 24 h after treatment. These include measurements of the mRNA expression (qPCR) of hepatic ß-Klotho, FGF21, FGF21 receptor-1 (FGFR1), extracellular-signal-regulated kinase 1/2 (ERK1/2), FRS2α, ribosomal S6 kinase-1 (RSK1), liver kinase B1 (LKB1), AMPK, and Sestrin2. Also, protein levels of FGF21, FGFR1 (ELISA), and ERK1/2 (Western blot) were measured. T3 elicited a calorigenic response with higher hepatic mRNA expression of ß-Klotho, FRS2α, and FGF21, increased serum FGF21, without changes in liver FGFR1 mRNA and its plasma levels. In addition, T3 enhanced ERK1/2 phosphorylation and the mRNA expression of ERK1/2, RSK1, LKB1, AMPK, and Sestrin2. T3 administration enhances liver FGF21-AMPK signaling involving upregulation of the scaffold proteins ß-Klotho, FRS2α, and Sestrin2. ß-Klotho and FRS2 induction favours the operation of the FGF21-FGFR1-ß-Klotho complex as evidenced by the enhancement in ERK1/2 phosphorylation, whereas that of Sestrin2 recruits LKB1 to achieved AMPK activation, thus supporting a higher energy expenditure condition that may be desirable in some metabolic disorders.

Thyroid hormone suppresses ischemia-reperfusion-induced liver NLRP3 inflammasome activation: Role of AMP-activated protein kinase.

Thyroid hormone (T3) induces liver preconditioning (PC) against ischemia-reperfusion (IR), a response energetically supported by AMP-activated protein kinase (AMPK) upregulation. The aim of this work is to evaluate the influence of T3 on IR-induced liver NLRP3 inflammasome activation and the relevance of AMPK activity on liver injury by the use of the AMPK inhibitor compound C (CC). Male Sprague-Dawley rats were given 0.1mgT3/kg (time zero) and 10mg CC/kg (time zero and 24h) or the respective vehicles, and subjected to 1h ischemia-20h reperfusion 48h after hormone treatment. Measurements included parameters of liver injury, hepatic levels of mRNAs (qPCR) and proteins (Western Blot or ELISA). IR induced substantial distortion of liver architecture, hepatocyte necrosis, and neutrophil infiltration with increased serum aspartate aminotransferase (AST) levels. T3 suppressed IR liver injury and AST enhancement, effects that were reverted by CC. Concomitantly, IR-induced liver mRNA and protein expression of NLRP3 and interleukin-1ß (IL-1ß) were restrained by T3, whereas CC eliminated T3-dependent PC. In conclusion, in vivo T3 administration triggers liver PC against IR injury by suppressing the inflammatory response associated with hepatic NLRP3 and IL-1ß upregulation, with AMPK playing a causal role regulating energy dynamics to upkeep PC.

Upregulation of rat liver PPARα-FGF21 signaling by a docosahexaenoic acid and thyroid hormone combined protocol.

Prevention of ischemia-reperfusion liver injury is achieved by a combined omega-3 and thyroid hormone (T3 ) protocol, which may involve peroxisome-proliferator activated receptor-α (PPAR-α)-fibroblast growth factor 21 (FGF21) signaling supporting energy requirements. Combined docosahexaenoic acid (DHA; daily doses of 300 mg/kg for 3 days) plus 0.05 mg T3 /kg given to fed rats elicited higher hepatic DHA contents and serum T3 levels, increased PPAR-α mRNA and its DNA binding, with higher mRNA expression of the PPAR-α target genes for carnitine-palmitoyl transferase 1α, acyl-CoA oxidase, and 3-hydroxyl-3-methylglutaryl-CoA synthase 2, effects that were mimicked by 0.1 mg T3 /kg given alone or by the PPAR-α agonist WY-14632. Under these conditions, the mRNA expression of retinoic X receptor-α (RXR-α) is also increased, with concomitant elevation of the hepatic mRNA and protein FGF21 levels and those of serum FGF21. It is concluded that PPAR-α-FGF21 induction by DHA combined with T3 may involve ligand activation of PPAR-α by DHA and enhanced expression of PPAR-α by T3 , with consequent upregulation of the FGF21 that is controlled by PPAR-α. Considering the beneficial effects of PPAR-α-FGF21 signaling on carbohydrate and lipid metabolism, further investigations are required to clarify its potential therapeutic applications in human metabolic disorders. © 2016 BioFactors, 42(6):638-646, 2016.

Causal role of oxidative stress in unfolded protein response development in the hyperthyroid state.

L-3,3',5-Triiodothyronine (T3)-induced liver oxidative stress underlies significant protein oxidation, which may trigger the unfolded protein response (UPR). Administration of daily doses of 0.1mg T3 for three consecutive days significantly increased the rectal temperature of rats and liver O2 consumption rate, with higher protein carbonyl and 8-isoprostane levels, glutathione depletion, and absence of morphological changes in liver parenchyma. Concomitantly, liver protein kinase RNA-like endoplasmic reticulum (ER) kinase and eukaryotic translation initiator factor 2α were phosphorylated in T3-treated rats compared to controls, with increased protein levels of binding immunoglobulin protein and activating transcription factor 4. In addition, higher mRNA levels of C/EBP homologous protein, growth arrest and DNA damage 34, protein disulfide isomerase, and ER oxidoreductin 1α were observed, changes that were suppressed by N-acetylcysteine (0.5 g/kg) given before each dose of T3. In conclusion, T3-induced liver oxidative stress involving higher protein oxidation status has a causal role in UPR development, a response that is aimed to alleviate ER stress and promote cell survival.

Thyroid hormone in the frontier of cell protection, survival and functional recovery.

Thyroid hormone (TH) exerts important actions on cellular energy metabolism, accelerating O2 consumption with consequent reactive oxygen species (ROS) generation and redox signalling affording cell protection, a response that is contributed by redox-independent mechanisms. These processes underlie genomic and non-genomic pathways, which are integrated and exhibit hierarchical organisation. ROS production led to the activation of the redox-sensitive transcription factors nuclear factor-κB, signal transducer and activator of transcription 3, activating protein 1 and nuclear factor erythroid 2-related factor 2, promoting cell protection and survival by TH. These features involve enhancement in the homeostatic potential including antioxidant, antiapoptotic, antiinflammatory and cell proliferation responses, besides higher detoxification capabilities and energy supply through AMP-activated protein kinase upregulation. The above aspects constitute the molecular basis for TH-induced preconditioning of the liver that exerts protection against ischemia-reperfusion injury, a strategy also observed in extrahepatic organs of experimental animals and with other types of injury, which awaits application in the clinical setting. Noteworthy, re-adjusting TH to normal levels results in several beneficial effects; for example, it lengthens the cold storage time of organs for transplantation from brain-dead donors; allows a superior neurological outcome in infants of <28 weeks of gestation; reduces the cognitive side-effects of lithium and improves electroconvulsive therapy in patients with bipolar disorders.

T3-induced liver AMP-activated protein kinase signaling: redox dependency and upregulation of downstream targets.

AIM: To investigate the redox dependency and promotion of downstream targets in thyroid hormone (T3)-induced AMP-activated protein kinase (AMPK) signaling as cellular energy sensor to limit metabolic stresses in the liver. METHODS: Fed male Sprague-Dawley rats were given a single ip dose of 0.1 mg T3/kg or T3 vehicle (NaOH 0.1 N; controls) and studied at 8 or 24 h after treatment. Separate groups of animals received 500 mg N-acetylcysteine (NAC)/kg or saline ip 30 min prior T3. Measurements included plasma and liver 8-isoprostane and serum ß-hydroxybutyrate levels (ELISA), hepatic levels of mRNAs (qPCR), proteins (Western blot), and phosphorylated AMPK (ELISA). RESULTS: T3 upregulates AMPK signaling, including the upstream kinases Ca(2+)-calmodulin-dependent protein kinase kinase-ß and transforming growth factor-ß-activated kinase-1, with T3-induced reactive oxygen species having a causal role due to its suppression by pretreatment with the antioxidant NAC. Accordingly, AMPK targets acetyl-CoA carboxylase and cyclic AMP response element binding protein are phosphorylated, with the concomitant carnitine palmitoyltransferase-1α (CPT-1α) activation and higher expression of peroxisome proliferator-activated receptor-γ co-activator-1α and that of the fatty acid oxidation (FAO)-related enzymes CPT-1α, acyl-CoA oxidase 1, and acyl-CoA thioesterase 2. Under these conditions, T3 induced a significant increase in the serum levels of ß-hydroxybutyrate, a surrogate marker for hepatic FAO. CONCLUSION: T3 administration activates liver AMPK signaling in a redox-dependent manner, leading to FAO enhancement as evidenced by the consequent ketogenic response, which may constitute a key molecular mechanism regulating energy dynamics to support T3 preconditioning against ischemia-reperfusion injury.

Nrf2 activation in the liver of rats subjected to a preconditioning sub-chronic iron protocol.

Sub-chronic iron (Fe) administration induces liver oxidative stress upregulating cytoprotective mechanisms that may involve redox-sensitive nuclear factor erythroid 2-related factor 2 (Nrf2). We aimed to investigate whether Fe activates Nrf2, in relation to its negative regulator Kelch-like ECH associated protein 1 (Keap1), with consequent antioxidant enzyme induction. Sprague-Dawley rats received six Fe doses (50 mg kg(-1)) on alternate days or saline (controls), a protocol that abrogates ischemia-reperfusion liver injury. Liver reduced glutathione (GSH) content and Nrf2 (Western blot) were measured 24 h after each Fe dose. Increased hepatic Fe deposition (Perls staining) was paralleled by reversible GSH depletion and enhancements in nuclear Nrf2 content and in nuclear/cytosolic Nrf2 ratios. A similar profile was observed for heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase 1 (NQO-1) contents, antioxidant enzymes that significantly correlated with nuclear/cytosolic Nrf2 ratios. Normalization of Fe-induced oxidative stress status occurred concomitantly with that of Nrf2 and with the Nrf2-dependent HO-1 and NQO-1 expression, which are associated with delayed enhancement in cytosolic Keap1 levels. This is in agreement with the significant inverse correlation of nuclear/cytosolic Nrf2 ratios with those of nuclear Keap1/Nrf2, suggesting a negative feed-back mechanism normalizing Nrf2 signaling. In conclusion, sub-chronic Fe administration leads to transient liver oxidative stress development and Nrf2 activation, as evidenced by early GSH depletion, enhanced nuclear Nrf2 protein levels, and HO-1 and NQO-1 induction, with late normalization of these changes being related to Keap1 upregulation.

Reestablishment of ischemia-reperfusion liver injury by N-acetylcysteine administration prior to a preconditioning iron protocol.

The role of iron (Fe)-induced prooxidant status in Fe preconditioning against ischemia (1 h)-reperfusion (20 h) induced liver injury was assessed using N-acetylcysteine (NAC) (1 g/kg) before Fe (50 mg/kg), given to male Sprague Dawley rats on alternate days during 10 days. IR significantly increased serum aspartate transaminase (AST) and alanine transaminase (ALT) levels, with drastic changes in liver histology, hepatic glutathione depletion, and nuclear factor-κB (NF-κB) p65 diminution (P < 0.05) (ELISA). Fe-induced liver oxidative stress, as evidenced by higher protein carbonyl/glutathione content ratios (P < 0.05) at days 11 and 12 after treatment, was abolished by NAC. Under these conditions, short-term Fe administration exerted significant protection against IR liver injury, as shown by 85% and 60% decreases in IR-induced serum AST and ALT (P < 0.05), respectively, and normalization of hepatic histology, glutathione levels, and NF-κB activation, changes that were suppressed by NAC administration prior to Fe. Results of this study indicate that NAC administration prior to an iron protocol reestablishes IR liver injury, supporting the role of Fe-induced transient oxidative stress in hepatoprotection and its potential clinical application.

Nrf2-regulated phase-II detoxification enzymes and phase-III transporters are induced by thyroid hormone in rat liver.

Thyroid hormone (T3)-induced calorigenesis triggers the hepatic production of reactive oxygen species (ROS) and redox-sensitive nuclear transcription factor erythroid 2-related factor 2 (Nrf2) activation. The aim of this study was to test the hypothesis that in vivo T3 administration upregulates the expression of phase II and III detoxification proteins that is controlled by Nrf2. Male Sprague-Dawley rats were given a single intraperitoneal dose of 0.1 mg T3/kg or T3 vehicle (controls). After treatment, rectal temperature of the animals, liver Nrf2 DNA binding (EMSA), protein levels of epoxide hydrolase 1 (Eh1), NADPH-quinone oxidoreductase 1 (NQO1), glutathione-S-transferases Ya (GST Ya) and Yp (GST Yp), and multidrug resistance-associated proteins 2 (MRP-2) and 4 (MRP-4) (Western blot), and MRP-3 (RT-PCR) were determined at different times. T3 significantly rose the rectal temperature of the animals in the time period studied, concomitantly with increases (P < 0.05) of liver Nrf2 DNA binding at 1 and 2 h after treatment, which was normalized at 4-12 h. Within 1-2 h after T3 treatment, liver phase II enzymes Eh1, NQO1, GST Ya, and GST Yp were enhanced (P < 0.05) as did phase III transporters MRP-2 and MRP-3, whereas MRP-4 remained unchanged. In conclusion, enhancement of liver Nrf2 DNA binding elicited by in vivo T3 administration is associated with upregulation of the expression of detoxification and drug transport proteins. These changes, in addition to antioxidant protein induction previously observed, may represent cytoprotective mechanisms underlying T3 preconditioning against liver injury mediated by ROS and chemical toxicity.

Metabolic basis for thyroid hormone liver preconditioning: upregulation of AMP-activated protein kinase signaling.

The liver is a major organ responsible for most functions of cellular metabolism and a mediator between dietary and endogenous sources of energy for extrahepatic tissues. In this context, adenosine-monophosphate- (AMP-) activated protein kinase (AMPK) constitutes an intrahepatic energy sensor regulating physiological energy dynamics by limiting anabolism and stimulating catabolism, thus increasing ATP availability. This is achieved by mechanisms involving direct allosteric activation and reversible phosphorylation of AMPK, in response to signals such as energy status, serum insulin/glucagon ratio, nutritional stresses, pharmacological and natural compounds, and oxidative stress status. Reactive oxygen species (ROS) lead to cellular AMPK activation and downstream signaling under several experimental conditions. Thyroid hormone (L-3,3',5-triiodothyronine, T(3)) administration, a condition that enhances liver ROS generation, triggers the redox upregulation of cytoprotective proteins affording preconditioning against ischemia-reperfusion (IR) liver injury. Data discussed in this work suggest that T(3)-induced liver activation of AMPK may be of importance in the promotion of metabolic processes favouring energy supply for the induction and operation of preconditioning mechanisms. These include antioxidant, antiapoptotic, and anti-inflammatory mechanisms, repair or resynthesis of altered biomolecules, induction of the homeostatic acute-phase response, and stimulation of liver cell proliferation, which are required to cope with the damaging processes set in by IR.

Thyroid hormone-induced cytosol-to-nuclear translocation of rat liver Nrf2 is dependent on Kupffer cell functioning.

L-3,3',5-triiodothyronine (T(3)) administration upregulates nuclear factor-E2-related factor 2 (Nrf2) in rat liver, which is redox-sensitive transcription factor mediating cytoprotection. In this work, we studied the role of Kupffer cell respiratory burst activity, a process related to reactive oxygen species generation and liver homeostasis, in Nrf2 activation using the macrophage inactivator gadolinium chloride (GdCl(3); 10 mg/kg i.v. 72 h before T(3) [0.1 mg/kg i.p.]) or NADPH oxidase inhibitor apocynin (1.5 mmol/L added to the drinking water for 7 days before T(3)), and determinations were performed 2 h after T(3). T(3) increased nuclear/cytosolic Nrf2 content ratio and levels of heme oxygenase 1 (HO-1), catalytic subunit of glutamate cysteine ligase, and thioredoxin (Western blot) over control values, proteins whose gene transcription is induced by Nrf2. These changes were suppressed by GdCl(3) treatment prior to T(3), an agent-eliciting Kupffer-cell depletion, inhibition of colloidal carbon phagocytosis, and the associated respiratory burst activity, with enhancement in nuclear inhibitor of Nrf2 kelch-like ECH-associated protein 1 (Keap1)/Nrf2 content ratios suggesting Nrf2 degradation. Under these conditions, T(3)-induced tumor necrosis factor-α (TNF-α) response was eliminated by previous GdCl(3) administration. Similar to GdCl(3), apocynin given before T(3) significantly reduced liver Nrf2 activation and HO-1 expression, a NADPH oxidase inhibitor eliciting abolishment of colloidal carbon-induced respiratory burst activity without altering carbon phagocytosis. It is concluded that Kupffer cell functioning is essential for upregulation of liver Nrf2-signaling pathway by T(3). This contention is supported by suppression of the respiratory burst activity of Kupffer cells and the associated reactive oxygen species production by GdCl(3) or apocynin given prior to T(3), thus hindering Nrf2 activation.

Colloidal carbon stimulation of Kupffer cells triggers Nrf2 activation in the isolated perfused rat liver.

Activation of transcription factor Nrf2 was investigated in the isolated perfused rat liver infused with 0.5 mg of colloidal carbon (CC)/ml for 5-15 min to stimulated Kupffer cell function. Infusion of CC enhanced liver O(2) consumption over basal levels, with a time-dependent increase in CC-induced O(2) uptake, at constant rates of CC phagocytosis by Kupffer cells, as assessed histologically, and adequate viability conditions of the livers, as shown by the marginal (0.34 %) total sinusoidal lactate dehydrogenase (LDH) efflux over intrahepatic LDH activity. Under these conditions, cytosolic protein levels of Nrf2 (Western blot) and inhibitor of Nrf2 Keap1 progressively declined by CC infusion, those of nuclear Nrf2 increased, leading to enhancement in the nuclear/cytosolic Nrf2 ratios. It is concluded that the respiratory burst of CC-stimulated Kupffer cells triggers Nrf2 activation in the perfused liver, a response that may afford cellular protection under pro-oxidant conditions underlying Kupffer cell stimulation.