Thyroid hormone activates rat liver adenosine 5,-monophosphate-activated protein kinase: relation to CaMKKb, TAK1 and LKB1 expression and energy status.

AMP-activated protein kinase (AMPK) is a sensor of energy status supporting cellular energy homeostasis that may represent the metabolic basis for 3,3,,5-triiodo-L-thyronine (T3) liver preconditioning. Functionally transient hyperthyroid state induced by T3 (single dose of 0.1 mg/kg) in fed rats led to upregulation of mRNA expression (RT-PCR) and protein phosphorylation (Western blot) of hepatic AMPK at 8 to 36 h after treatment. AMPK Thr 172 phosphorylation induced by T3 is associated with enhanced mRNA expression of the upstream kinases Ca2+ -calmodulin-dependent protein kinase kinase-beta (CaMKKbeta) and transforming growth-factor-beta-activated kinase-1 (TAK1), with increased protein levels of CaMKKbeta and higher TAK1 phosphorylation, without changes in those of the liver kinase B1 (LKB1) signaling pathway. Liver contents of AMP and ADP were augmented by 291 percent and 44 percent by T3 compared to control values (p less than 0.05), respectively, whereas those of ATP decreased by 64% (p less than 0.05), with no significant changes in the total content of adenine nucleotides (AMP + ADP + ATP) at 24 h after T3 administration. Consequently, hepatic ATP/ADP content ratios exhibited 64 percent diminution (p less than 0.05) and those of AMP/ATP increased by 425 percent (p less than 0.05) in T3-treated rats over controls. It is concluded that in vivoT3 administration triggers liver AMPK upregulation in association with significant enhancements in AMPK mRNA expression, AMPK phosphorylation coupled to CaMKKbeta and TAK1 activation, and in AMP/ATP ratios, which may promote enhanced AMPK activity to support T3-induced energy consuming processes such as those of liver preconditioning.

Differential levels of Neurofilament Light protein in cerebrospinal fluid in patients with a wide range of neurodegenerative disorders.

Cerebrospinal fluid (CSF) biomarkers are useful in the diagnosis and the prediction of progression of several neurodegenerative diseases. Among them, CSF neurofilament light (NfL) protein has particular interest, as its levels reflect neuroaxonal degeneration, a common feature in various neurodegenerative diseases. In the present study, we analyzed NfL levels in the CSF of 535 participants of the SPIN (Sant Pau Initiative on Neurodegeneration) cohort including cognitively normal participants, patients with Alzheimer disease (AD), Down syndrome (DS), frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS). We evaluated the differences in CSF NfL accross groups and its association with other CSF biomarkers and with cognitive scales. All neurogenerative diseases showed increased levels of CSF NfL, with the highest levels in patients with ALS, FTD, CBS and PSP. Furthermore, we found an association of CSF NfL levels with cognitive impairment in patients within the AD and FTD spectrum and with AD pathology in DLB and DS patients. These results have implications for the use of NfL as a marker in neurodegenerative diseases.

Reduction of high-fat diet-induced liver proinflammatory state by eicosapentaenoic acid plus hydroxytyrosol supplementation: involvement of resolvins RvE1/2 and RvD1/2.

High-fat diet (HFD)-fed mice show obesity with development of liver steatosis and a proinflammatory state without establishing an inflammatory reaction. The aim of this work was to assess the hypothesis that eicosapentaenoic acid (EPA) plus hydroxytyrosol (HT) supplementation prevents the inflammatory reaction through enhancement in the hepatic resolvin content in HFD-fed mice. Male C57BL/6J mice were fed an HFD or a control diet and supplemented with EPA (50 mg/kg/day) and HT (5 mg/kg/day) or their respective vehicles for 12 weeks. Measurements include liver levels of EPA, DHA and palmitate (gas chromatography), liver resolvins and triglyceride (TG) and serum aspartate transaminase (AST) (specific kits) and hepatic and serum inflammatory markers (quantitative polymerase chain reaction and enzyme-linked immunosorbent assay). Compared to CD, HFD induced body weight gain, liver steatosis and TG accumulation, with up-regulation of proinflammatory markers in the absence of histological inflammation or serum AST changes; these results were accompanied by higher hepatic levels of resolvins RvE1, RvE2, RvD1 and RvD2, with decreases in EPA and DHA contents. EPA+HT supplementation in HFD feeding synergistically reduced the steatosis score over individual treatments and increased the hepatic levels of EPA, DHA and resolvins, with attenuation of proinflammatory markers. Lack of progression of HFD-induced proinflammatory state into overt inflammation is associated with resolvin up-regulation, which is further increased by EPA+HT supplementation eliciting steatosis attenuation. These findings point to the importance of combined protocols in hepatoprotection due to the involvement of cross-talk mechanisms, which increase effectiveness and diminish dosages, avoiding undesirable effects.

A combined docosahexaenoic acid-thyroid hormone protocol upregulates rat liver ß-Klotho expression and downstream components of FGF21 signaling as a potential novel approach to metabolic stress conditions.

Liver preconditioning by a docosahexaenoic acid (DHA) and triiodothyronine (T3) combined protocol underlies peroxisome-proliferator activated receptor α (PPARα)-fibroblast growth factor 21 (FGF21) upregulation, the study of the regulatory mechanisms involved being the aim of this work. Combined DHA (daily doses of 300 mg kg-1 for 3 days)-T3 (0.05 mg kg-1 at the fourth day) administration elicited higher levels of liver DHA and serum T3, with enhanced hepatic nuclear/cytosolic PPARα ratios, upregulation of FGF21 and ß-Klotho expression, and a small reduction in that of FGF receptor 1 (FGFR1), compared with the respective controls. Concomitantly, the components of the FGF21 cascade extracellular-signal-regulated kinase 1/2 (ERK1/2), FGF receptor substrate 2α (FRS2α), cFos, ribosomal S6 kinase 1 (RSK1), liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK) were activated. The upregulation of liver PPARα-FGF21-AMPK signaling by the combined DHA-T3 protocol resulted in values significantly higher than those elicited by the addition of the data obtained for DHA and T3 alone. It is concluded that combined DHA-T3 supplementation achieves synergistic effects on liver PPARα-FGF21-AMPK signaling, which may result in significant metabolic changes associated with energy expenditure that are of importance in the treatment of obesity and other metabolic disorders.

Superoxide radical generation, NADPH oxidase activity, and cytochrome P-450 content of rat liver microsomal fractions in an experimental hyperthyroid state: relation to lipid peroxidation.

The effect of thyroid hormone treatment on hepatic microsomal functions related to NADPH-dependent electron transfer reactions was studied in rats given 0.1 mg T3/kg BW for 1, 2, 3, and 7 consecutive days. This treatment resulted in increased rates of O2-. generation by microsomal fractions, concomitantly with an enhancement in NADPH oxidase activity and decreased cytochrome P-450 content, in livers exhibiting increased respiration. Subsequent studies showed elevated levels of malondialdehyde in microsomal fractions and liver homogenates, as well as augmented chemiluminescent response in the latter system. These results indicate that the calorigenic effect of T3 on the liver tissue is accompanied by a stimulation of microsomal functions involving univalent reduction of oxygen. This cellular response might lead to a greater lipid peroxidative rate and cytochrome P-450 loss as secondary events of thyroid hormone action.

Effects of hyperthyroidism on rat liver glutathione metabolism: related enzymes' activities, efflux, and turnover.

The effect of hyperthyroidism on liver glutathione (GSH) metabolism was studied in fed rats after the administration of 0.1 mg T3/kg body wt, for 1-3 consecutive days. T3-calorigenesis resulted in elevated rates of O2 consumption by the liver, together with higher lipid peroxidative processes and GSH depletion, compared to the euthyroid state. The study of the enzymes related to GSH metabolism revealed no significant changes in the activity of glutathione peroxidase and glutathione reductase, with decreases (27-41%) in the activity of glutathione-S-transferases and marked elevation (133%) in that of gamma-glutamyl transferase, 3 days after T3 treatment. At this experimental time, the activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase was enhanced by 84% in the liver of T3-treated rats, compared to that in the controls. In these conditions, the canalicular efflux of GSH was not altered by T3, whereas net and fractional rates of sinusoidal GSH efflux were enhanced by 86% and 288%, respectively. The latter effect of hyperthyroidism was found in parallel with an enhancement in sinusoidal lactate dehydrogenase and protein release, suggesting that loss of GSH might be related to a permeabilization of the hepatocyte plasma membrane. Liver GSH turnover assessed after a pulse of [35S]cysteine resulted in a 209% increase in the fractional turnover rate in hyperthyroid rats over controls, under steady state conditions for both hepatic GSH pools, leading to a 62% enhancement in the respective turnover flux. Data suggest that the elevation in the sinusoidal GSH efflux from the liver and in the hepatic capacity to degrade the tripeptide are major mechanisms leading to GSH depletion in the liver of T3-treated rats. As the increased GSH use is not balanced by the elevation in GSH synthesis, a lower steady state level of GSH is attained in the liver.

Chemically induced antioxidant-sensitive respiration. Relation to glutathione content and lipid peroxidation in the perfused rat liver.

The interrelations between the hepatic chemically induced antioxidant-sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin-free perfused rat liver. Antioxidant-sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t-butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.

On the mechanism of thyroid hormone-induced respiratory burst activity in rat polymorphonuclear leukocytes.

Administration of single doses of 0.1 mg L-triiodothyronine (T3)/kg for 3 consecutive days to fed rats produced a drastic increase in the respiratory burst activity of isolated polymorphonuclear leukocytes (PMN), stimulated with serum-opsonized zymosan. This effect was evidenced by the 3.8-fold increment in the integrated chemiluminescence, and seems to be primarily related to the enhanced activity of NADPH oxidase elicited by T3 treatment, with the observed higher myeloperoxidase activity playing a contributory role. In these conditions, hyperthyroidism determines a net enhancement in the oxidant capacity of PMN, as the increased rate of O2.- generation found occurs in the absence of changes in the activity of superoxide dismutase.

Visible chemiluminescence from rat brain homogenates undergoing autoxidation. II. Kinetics of the luminescence decay.

The visible luminescence emitted in the autoxidation of brain homogenates is only partially quenched when antioxidants are added at concentrations such that further oxidation is prevented. From the time course of the emission after antioxidant addition, it can be estimated that nearly 50% of the light arises from an intermediate that decays with a first order kinetics and with a lifetime of ca. 40 s at 32 degrees C. The remaining light arises from the decomposition of one or several intermediates, and show a kinetics that is independent of the incubation time. From the data obtained it is concluded that bimolecular free radical processes, such as the recombination of peroxy radicals, do not significantly contribute to the observed luminescence.

Potentiation of ischemia-reperfusion liver injury by hyperthyroidism in the rat.

Parameters related to hepatic oxidative stress, cell injury, and liver histology were determined in control rats and in animals treated with 3,3',5-triiodothyronine (T3), after in vitro perfusion under normoxic or ischemia-reperfusion conditions. Thyroid calorigenesis was found concomitantly with higher rates of hepatic O2 consumption and thiobarbituric acid reactive substances (TBARS) formation, glutathione (GSH) depletion, enhanced TBARS/GSH ratio as indicator of oxidative stress, and higher sinusoidal lactate dehydrogenase (LDH) efflux compared to control values, assessed under normoxic conditions. Perfused livers from control animals subjected to ischemia-reperfusion exhibited significant increases in the TBARS/GSH ratio and in the sinusoidal LDH efflux over values obtained under normoxic conditions, concomitantly with the appearance of small foci of necrotic cells in centrilobular and midzonal areas of the liver lobule. These parameters were further modified in the liver of hyperthyroid rats subjected to ischemia-reperfusion, with elevations in the TBARS/GSH ratio and in the sinusoidal LDH efflux largely exceeding the sum of effects elicited by hyperthyroidism or ischemia-reflow alone. In this situation, liver injury was more pronounced than in control rats, being characterized by multifocal areas of necrotic cells, irregularly distributed in the hepatic lobule, with lymphoid and macrophagic reaction. It is concluded that the concurrence of the hepatic mechanisms related to the oxidative stress underlying thyroid calorigenesis and ischemia-reoxygenation exacerbates liver injury, which seems to be mediated by potentiation of the prooxidant state of the organ.

Effects of the Kupffer cell inactivator gadolinium chloride on rat liver oxygen uptake and content of mitochondrial cytochromes.

The effect of gadolinium chloride (GdCl3) on the content of rat liver mitochondrial cytochromes was investigated in relation to the basal rate of O2 uptake and Kupffer cell functioning, assessed in liver perfusion studies. (1) A single dose of GdCl3 (10 mg/kg) produced a significant diminution in Kupffer cell functioning, evidenced by the decreases in colloidal carbon uptake and in carbon-induced O2 uptake observed at 6-24 h after treatment, without changes in the sinusoidal lactate dehydrogenase efflux as index of tissue viability; at 48 h after GdCl3 administration, carbon phagocytosis was recovered to control values, whereas carbon-induced O2 uptake remained lower than control values. (2) GdCl3 also caused a 34% decrease in the basal rate of O2 consumption of the liver at 24 h after treatment, which returned towards control values at 48 h. (3) The content of mitochondrial cytochromes c1 and c at 24 h after GdCl3 treatment was significantly reduced by 40 and 32%, respectively, which returned to control values at 48 h, without changes in that of cytochromes b and a+a3. It is concluded that GdCl3-induced decrease in liver O2 consumption is a reversible phenomenon that seems to be due to a diminution in the content of mitochondrial cytochromes c1 and c.

Antioxidant capacity of diethyldithiocarbamate in a metal independent lipid peroxidative process.

2,2'-Azobis-[2-amidinopropane] initiated lipid peroxidation of egg yolk phosphatydil choline liposomes was measured by oxygen uptake and the emitted visible luminescence. Lipid peroxidation involved a chain process (kinetic chain length = 49 +/- 11) and its rate was independent of added Fe ions. Diethyldithiocarbamate (DDC) behaved as an efficient inhibitor in the microM range, being able to trap 1.05 +/- 0.25 free radicals per added molecule. The efficiency of DDC was also independent of Fe addition to the system. These results indicate that DDC is able to trap the chain carrying free radicals, showing that this compound, besides being a powerful metal chelator, is also an efficient free radical scavenger. It is proposed that the relevant step in this process involves an electron transfer from DDC to the peroxy radical LOO., LOO. + DDC- ----LOO- + DDC. followed by protonation of LOO- and dimerization of the DDC. radical.

Is spontaneous urinary visible chemiluminescence a reflection of in vivo oxidative stress?

Based on the characteristics of the visible spontaneous luminiscence of human urine, we propose that this luminescence is due to the dark decomposition of long-lived luminescent intermediates produced by oxidations at the cellular level, and that it might reflect the development of oxidative stress conditions in vivo. This hypothesis is consistent with the higher emission levels monitored in the urine of hyperthyroid patients and the correlation observed between urinary thiobarbituric acid reactive substances (TBARS) levels and urinary chemiluminescence.

Zymosan-induced luminol-amplified chemiluminescence of whole blood phagocytes in experimental and human hyperthyroidism.

Luminol-amplified CL of whole blood phagocytes was studied in rats given 3 consecutive doses of 0.1 mg L-triiodothyronine T3/kg or in hyperthyroid patients, after stimulation by zymosan. In both cases, CL was significantly increased, an effect which was produced independently of the opsonization of the zymosan particles and markedly inhibited by azide. The in vitro addition of T3 or L-thyroxine (T4) to whole blood phagocytes from normal rats did not modify the opsonized zymosan-dependent CL, when assayed at the concentrations found in euthyroid subjects or in hyperthyroid patients. Administration of propylthiouracil (400 mg/day for 2-3 months) to hyperthyroid patients reduced the CL response observed prior to treatment, to values comparable to those found in the euthyroid group. These data indicate that hyperthyroidism elicits an enhanced respiratory burst activity of whole blood phagocytes, probably related to adaptive changes induced by thyroid hormone on the mieloperoxidase-H2O2 system, rather than to direct actions of the hormone molecule or changes in the opsonic capacity of plasma.

Lindane-induced liver oxidative stress.

The development of an oxidative stress condition in the liver by lindane intoxication is discussed as a possible hepatotoxic mechanism of the insecticide. Lindane is metabolized by liver microsomal enzymes to a variety of metabolites, which are susceptible of conjugation for proper elimination. In addition, the interaction of lindane with the liver tissue results in the induction of the microsomal cytochrome P-450 system, together with enhanced rates of superoxide radical generation and a significant increase in indicators of lipid peroxidation. Concomitantly, lindane intoxication induces a derangement of some antioxidant mechanisms of the liver cell, including decreased superoxide dismutase and catalase activities and alterations in reduced glutathione content leading to depressed GSH/GSSG ratios. The time course study of the changes in hepatic lipid peroxidation and antioxidant parameters are closely interrelated and coincide with the onset and progression of morphological lesions.

Enhanced urinary spontaneous luminescence in Duchenne muscular dystrophy.

Urinary spontaneous visible luminescence is enhanced in children with Duchenne Muscular Dystrophy (DMD). This result is indicative of systemic oxidative stress in DMD patients. It is proposed that measurement of the urinary luminescence could be employed to follow the progress of the disease, as well as the response of the patients to antioxidant therapy.

Steroidogenic capacity and oxidative stress-related parameters in human luteal cell regression.

The steroidogenic capacity and oxidative stress-related parameters of the human corpus luteum (CL) at different stages of the luteal phase were studied under basal and human chorionic gonadotropin (hCG)-stimulated conditions. Mid CL exhibited the maximal steroidogenic capacity, together with lower levels of glutathione and higher thiobarbituric acid reactants content, macrophage count, and superoxide dismutase (SOD) activity, compared to the late CL. Addition of hCG to luteal cell cultures led to a preferential increase in progesterone synthesis in the late CL compared to the mid CL, without changes in the oxidative stress-related parameters, except for the increased SOD activity found in the late CL. It is concluded that an oxidative stress condition is established in the mid CL, coinciding with the maximal steroidogenic capacity and macrophage infiltration of the organ, which may be of relevance as one of the major mechanisms initiating CL involution in the human.

Transdermal estrogens do not appear to modify the extent of lesional areas of aortic atherosclerosis in oophorectomized rabbits on a cholesterol-rich diet.

Cardiovascular disease (CVD) is the leading cause of death in older women in industrialised countries. It has been suggested that it is the cessation of estrogen production by the ovaries that puts postmenopausal women at increased risk of CVD. Estrogen therapy has demonstrated a protective effect against CVD and several reports suggest that diverse mechanisms may be involved. Oral estrogen appears to be associated with a better lipid profile than the use of transdermal estrogens; however, it is assumed that estrogens, oral and non-oral, have direct actions on the blood vessels that may exert an important role in cardiovascular disease prevention. To investigate the effect of transdermal estrogen therapy on aorta atherogenesis, we studied 20 cholesterol-fed New Zealand White rabbits for 4 months. The rabbits were oophorectomized and randomly assigned to two groups. Ten rabbits underwent bilateral ovariectomy followed by treatment with transdermal 17-beta-estradiol (group E) and the other 10 received placebo after sterilization (Group C). After diet total levels of cholesterol increase in group C from 50. 0+/-12.5 to 820.9+/-186.0 mg/dl, and in group E from 52.6+/-9.4 to 811.4+/-213.0 mg/dl (no significant differences were observed between groups). Estrogen therapy increased twofold the total reactive antioxidant potential (TRAP group C: 22.5+/-16.7 mmol of Trolox/l vs. TRAP group E: 43.4+/-22.4 mmol of Trolox/l; P<0.04). At 4 months, the cholesterol-rich diet caused atherosclerotic lesions in both treated and untreated rabbits affecting 18.7+/-14.5 and 21. 6+/-9.7% of the aortic surface respectively. In summary, the principal result from this study was that although treatment with transdermal 17-beta-estradiol in cholesterol-fed ovariectomized rabbits increases the TRAP to pre-surgery values, it does not inhibit aortic cholesterol accumulation.

Functional luteolysis in response to hydrogen peroxide in human luteal cells.

To evaluate the effect of reactive oxygen species in human corpus luteum function, we investigated whether hydrogen peroxide (H2O2) affects the in vitro luteal cell production of steroids. H2O2 treatment (1.0-100 microM) of mid and late luteal cell cultures elicited a dose-dependent decrease in basal progesterone production. However, treatment of mid luteal cells with a low concentration of H2O2 (0.01 microM) significantly stimulated progesterone secretion (P < 0.05). In addition, H2O2 (100 microM) markedly inhibited human chorionic gonadotropin (hCG)-stimulated progesterone and estradiol secretion. cAMP production was enhanced (2.4-fold, P < 0.05) by hCG treatment of luteal cells. The addition of H2O2 (0.1-100 microM) to hCG-stimulated luteal cell cultures elicited a decrease in cAMP concentration (P < 0.05) and in the specific binding of radiolabeled hCG by luteal cells. Progesterone and estradiol production stimulated by dibutyryl cAMP were significantly inhibited by H2O2 (P < 0.05). These findings suggest that H2O2 interferes with basal steroid production and, in hCG-stimulated conditions, it may inactivate the gonadotropin-receptor complex. The anti-steroidogenic action of H2O2 therefore raises the possibility of a modulatory role of H2O2 in human luteal steroidogenesis.

Influence of thyroid hormone administration on hepatic glutathione content and basolateral gamma-glutamyltransferase ectoactivity in the isolated perfused rat liver.

The effect of thyroid hormone administration on liver glutathione (GSH) content and gamma-glutamyltransferase activity in the isolated perfused liver was studied for a period of 1-7 days in fed rats following a single dose of 0.1 mg 3,5,3'-L-triiodothyronine (T3)/kg. T3 elicited an early and transient calorigenic response, together with GSH depletion at 1 day after treatment. Recovery of hepatic GSH content and enhancement in total basolateral gamma-glutamyltransferase activity occurred in parallel 2-3 days after T3 treatment, parameters that were normalized in the 4- to 7-day time interval studied. The increase in total basolateral gamma-glutamyltransferase activity by T3 at early times after treatment was due mainly to increments in its transpeptidation mechanism, and was characterized by increments in the apparent maximum velocities without changes in the apparent Michaelis constant (Km) for the substrate gamma-glutamyl-p-nitroanilide. Data presented suggest that the elevation in sinusoidal gamma-glutamyltransferase activity could be related to the recovery of hepatic GSH content after depletion by T3 treatment, by supplying the precursors for intracellular GSH synthesis, an effect that seems to be mediated by enhanced synthesis of the enzyme.