Mechanisms in thyroid hormone calorigenesis and liver oxidative stress
Thyroid hormones play important roles in cell growth, differentiation, and metabolism, through different and complex mechanisms of action. In mammals, major effects are exerted on cellular oxygen consumption (QO2) and metabolic rate, leading to stimulation and maintenance of basal thermogenesis[26,27]. This action of T3 is carried out via thyroid hormone receptors expressed in almost all tissues. These receptors are recognized by specific thyroid hormone response elements across the DNA, leading to ligand-dependent upregulation of the expression of respiratory, metabolic, and uncoupling protein genes (Figure 2A). In addition to the above classical genomic model of T3-dependent calorigenesis, non-genomic mechanisms may also contribute to increase cellular QO2, with the consequent increase of the mitochondrial capacity for oxidative phosphorylation and ROS generation.
Figure 2 Oxidative stress signaling in thyroid hormone (T3) liver preconditioning as mediated by redox-sensitive transcriptional factors NF-κB, AP-1, and STAT3 (A) or Nrf2 (B).
AP-1: Activating protein 1; ARE: Antioxidant responsive element; GdCl3: Gadolinium chloride; IL: Interleukin; iNOS: Inducible nitric oxide synthase; MnSOD: Manganese superoxide dismutase; NF-κB: Nuclear factor-κB; Nrf2: Nuclear factor-erythroid 2-related factor 2; QO2: Rate of oxygen consumption; TNF-α: Tumor necrosis factor-α; STAT3: Signal transducer and activator of transcription 3; TR: Thyroid hormone receptor; UCP: Uncoupling protein.
In addition to T3-induced liver mitochondrial capacity for ROS production, the induction of other enzymatic mechanisms also occurs, namely, (1) higher activity of microsomal NADPH-cytochrome P450 reductase and NADPH oxidase, the latter representing the oxidase activity of cytochrome P450 responsible for the O2•− and H2O2 production related to the T3-mediated induction of the highly pro-oxidant cytochrome P4502E1 isoform; (2) enhancement of cytosolic enzymatic mechanisms, such as the O2•−/H2O2 generator xanthine oxidase and ROS production, possibly coupled to enhanced FA β-oxidation due to liver peroxisomal proliferation; and (3) Kupffer-cell activation with increased respiratory burst activity, due to NADPH oxidase.
T3-induced liver free-radical activity is associated with depletion of antioxidant defences, leading to increased oxidative stress of the liver (Figure 2A)[7,28,36]. However, this pro-oxidant state achieved in the liver by T3-induced calorigenesis can be considered as a mild redox alteration, as suggested by the lack of occurrence of morphological changes in liver parenchyma, except for the significant hyperplasia and hypertrophy of Kupffer cells, the resident macrophages of the liver. The latter effect of T3 might be of importance considering that Kupffer cells play a central role in the homeostatic response to liver injury, through the production and release of a wide array of mediators that provide physiologically diverse and key paracrine effects on all other liver cells[37,38].
T3-induced Kupffer cell-dependent up-regulation of cytokine expression and hepatocyte proteins related to antioxidation, anti-apoptosis, acute-phase response, and cell proliferation
Kupffer cell hyperplasia is a major finding after in vivo T3 administration, an effect that may involve the expansion of Kupffer cell precursors by means of circulating monocyte recruitment, the differentiation of pre-existing local Kupffer cell precursors into mature liver macrophages, or both. Under these conditions, assessment of Kupffer cell function revealed a significant increase in the rate of carbon phagocytosis and the associated carbon-induced O2 uptake, representing the respiratory burst activity of Kupffer cells, a process that is largely dependent on the activity of the ROS-generator NADPH oxidase and abolished by pretreatment with the Kupffer cell inactivator gadolinium chloride (GdCl3) (Figure 2A).
The interdependence between T3-induced calorigenesis, liver QO2, and ROS production is associated with a significant increase in the hepatic DNA binding of the transcription factors NF-κB, STAT3, and AP-1 (Figure 2A). Activation of these transcription factors by T3 administration is suppressed by in vivo pretreatment with GdCl3, whereas NF-κB and STAT3 activation by T3 is also abolished by pretreatment with antioxidants[40,41], thus supporting the view that T3 induces the redox activation of hepatic NF-κB, STAT3, and AP-1 by actions primarily exerted at the Kupffer cell level (Figure 2A). T3 administration involving significant NF-κB and AP-1 activation induced mRNA expression of the NF-κB/AP-1-responsive genes for TNF-α, with increased serum levels of the cytokine that are abolished by pretreatment with the antisense oligonucleotide TJU-2755, targeting the primary RNA transcript of TNF-α. T3 also elicited an increase in the serum levels of IL-6 and in the hepatic mRNA expression and serum levels of IL-1. In addition to NF-κB and AP-1 activation, the enhancement in STAT3 DNA binding by T3 administration may be associated with the proliferation of macrophage precursors and their differentiation into Kupffer cells, considering the central role of STAT3 in gp130-mediated cell growth, differentiation, and survival.
The effects of cytokines released from Kupffer cells are exerted through their interaction with specific surface receptors of liver target cells, mediating the signaling transduction from the cell membrane to the nucleus. In agreement with the above view, the transient TNF-α response elicited by T3 administration correlates with the substantial increase in liver IκB-α phosphorylation[45,46], leading to the activation of the IKK complex that in turn activates NF-κB, after coupling with the TNF-α receptor and associating with different adaptor proteins. T3-induced TNF-α response, liver IKK phosphorylation, and NF-κB activation are abolished by pretreatment with either α-tocopherol or GdCl3, supporting the role of ROS production and Kupffer-cell activation in T3-dependent signaling leading to up-regulation of hepatic gene expression[45,46]. This is shown by the increased expression of the NF-κB-responsive genes encoding for inducible NOS (iNOS), manganese superoxide dismutase (MnSOD), and the anti-apoptotic protein Bcl-2 (Figure 2A). Thus, T3 administration elicits the redox up-regulation of iNOS, MnSOD, and Bcl-2 in the liver, in association with the Kupffer cell-dependent release of TNF-α and activation of the IKK/NF-κB cascade, representing antioxidant and anti-apoptotic responses triggered by the underlying oxidative stress (Figure 2A).
In addition to the above responses, T3 administration up-regulate the acute-phase response (APR) of the liver and the hepatocyte proliferation. The APR is a major pathophysiologic reaction in which normal homeostatic mechanisms are replaced by new set-points, contributing to defensive or adaptive capabilities against inflammation and oxidative stress[48,49]. In fact, T3 induced the Kupffer-cell-dependent release of IL-6 and activation of hepatic STAT3 controlling both type I (haptoglobin) and type II (β-fibrinogen) acute-phase protein (APP) genes. In addition, this response may be contributed by the T3-induced TNF-α/IKK/NF-κB pathway[45,46], which controls type I APP genes, considering that NF-κB activation can synergistically enhance the effects of STAT3 and C/EBPβ upon C-reactive protein induction. Furthermore, the in vivo effects of T3 as a primary hepatic mitogen, leading to hepatocyte proliferation in intact liver, are well established (Figure 2A). This process involves a large number of genes and requires the concurrence of cytokines, growth factors and metabolic networks. Resting hepatocytes, i.e. in the G0 phase of the cell cycle, need to be primed by TNF-α and IL-6 before they can respond to growth factors, with the concomitant activation of NF-κB, STAT3, AP-1, and E/EBPβ, enter the G1 phase and initiate cell cycle progression. T3 administration has been associated with increased liver cyclin-dependent kinase 2 expression and hepatocyte proliferation, as shown by the increase of Ki-67, a nuclear cell proliferation-associated protein expressed in all active parts of the cell cycle, and of the proliferating cell nuclear antigen (PCNA).
Collectively, data reported by our group indicate that T3 triggers cytoprotection in the liver through redox- and Kupffer cell-dependent signaling mechanisms, namely, (1) antioxidant responses (iNOS, MnSOD); (2) anti-apoptosis (Bcl-2); (3) immune, transport, and antioxidant (haptoglobin, ceruloplasmin, ferritin) functions fulfilled by APR induction; and (4) hepatocyte proliferation (Figure 2A), the metabolic demands of which being met by acceleration of energy metabolism due to T3-induced calorigenesis[7,28,52].
Thyroid hormone-induced liver preconditioning
Organ preconditioning, including that involving the liver, consists in strategies protecting the organ from detrimental effects of subsequent noxious events, such as those underlying chemically-induced injury or IR[8,53]. In general terms, IR injury refers to tissue damage produced by blood perfusion to a previously ischemic organ. In the case of the liver, this occurs in the clinical settings of hepatic resection, transplantation, low-blood pressure states, and abdominal surgery requiring hepatic vascular occlusion. IR liver injury assessed in a model involving 1 h of partial ischemia, as induced by vascular clamping, and followed by reperfusion for 20 h, elicited minimal mortality but substantial liver damage, with increased serum transaminase and TNF-α levels as well as metabolic changes, namely, (1) a drastic increase in the oxidative stress status of the liver; (2) loss in the DNA binding of NF-κB and STAT3, implying loss of cytoprotective potential, as shown by the concomitant diminution in the expression of the APR protein haptoglobin, controlled by both these transcription factors; and (3) increase of the hepatic AP-1 DNA binding activity, which may constitute a major determinant of hepatotoxicity under conditions of reduced NF-κB activation and TNF-α response[54,55]. These changes were normalized by T3 treatment given 48 h before the IR protocol, a preconditioning effect that was sensitive to the antioxidant N-acetylcysteine given prior to T3, with enhanced hepatocyte proliferation compensating for liver cells lost due to IR-induced hepatocellular necrosis.
In conclusion, the data discussed above indicate that redox regulation of gene transcription by T3 involves antioxidant-sensitive NF-κB, AP-1, and STAT3 activation and up-regulation of the expression of cytoprotective proteins affording liver preconditioning (Figure 2A). T3 liver preconditioning may also involve the activation of the Nrf2-Keap1 defense pathway, up-regulating antioxidant proteins and phase-2 detoxifying enzymes (Figure 2B), which is currently under study in our laboratory.