Dental caries prevalence and severity positively associate with AMY1 gene copy number.

OBJECTIVE: To establish the possible relation between total caries (TC) and caries severity (CS) with the AMY1 gene copy number (AMY1GCN). MATERIALS AND METHODS: This was an observational, cross-sectional, population-based, and association study with 303 participants. Each participant underwent a complete anamnesis and stomatological check-up, and peripheral blood was obtained to extract gDNA. TC and CS were determined as the number of caries at the dental exploration and the number of dental surfaces affected by caries, respectively, and AMY1GCN was determined by qPCR. RESULTS: We found an elevated caries prevalence (92.7%); TC and CS were 8 ± 10 and 10 ± 13 (median ± IR). There were higher TC and CS in those participants with AMY1GCN above the mean value (0.02 and 0.01 p values, respectively). A positive correlation between TC and CS with AMY1GCN (0.11 and 0.125 r values, 0.03 and 0.01 p values, respectively) was found, in addition to an association between TC and CS with AMY1GCN (1.5 and 1.6 OR values, 0.48 and 0.26 p values, respectively). CONCLUSION: TC and CS were positively related to the AMY1GCN. CLINICAL RELEVANCE: Dental caries has a high prevalence and a multifactorial etiology and has been related to a genetic component. Indeed, the salivary enzyme alpha-amylase could play a significant role in caries susceptibility, considering that its codifying gene (AMY1) can show variation in its gene copy number. This can be considered an important factor for the development of caries at a genetic level.

Cerebellar spongiform degeneration is accompanied by metabolic, cellular, and motor disruption in male rats with portacaval anastomosis.

The episodes of cerebral dysfunction, known as encephalopathy, are usually coincident with liver failure. The primary metabolic marker of liver diseases is the increase in blood ammonium, which promotes neuronal damage. In the present project, we used an experimental model of hepatic encephalopathy in male rats by portacaval anastomosis (PCA) surgery. Sham rats had a false operation. After 13 weeks of surgery, the most distinctive finding was vacuolar/spongiform neurodegeneration exclusively in the molecular layer of the cerebellum. This cerebellar damage was further characterized by metabolic, histopathological, and behavioral approaches. The results were as follows: (a) Cellular alterations, namely loss of Purkinje cells, morphological changes, such as swelling of astrocytes and Bergmann glia, and activation of microglia; (b) Cytotoxic edema, shown by an increase in aquaporin-4 and N-acetylaspartate and a reduction in taurine and choline-derivate osmolytes; (c) Metabolic adjustments, noted by the elevation of circulating ammonium, enhanced presence of glutamine synthetase, and increase in glutamine and creatine/phosphocreatine; (d) Inflammasome activation, detected by the elevation of the marker NLRP3 and microglial activation; (e) Locomotor deficits in PCA rats as assessed by the Rotarod and open field tests. These results lead us to suggest that metabolic disturbances associated with PCA can generate the cerebellar damage that is similar to morphophysiological modifications observed in amyloidogenic disorders. In conclusion, we have characterized a distinctive cerebellar multi-disruption accompanied by high levels of ammonium and associated with spongiform neurodegeneration in a model of hepatic hypofunctioning.

Increased Purinergic Responses Dependent on P2Y2 Receptors in Hepatocytes from CCl4-Treated Fibrotic Mice.

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.

Neuroprotective Effects of Growth Hormone (GH) and Insulin-Like Growth Factor Type 1 (IGF-1) after Hypoxic-Ischemic Injury in Chicken Cerebellar Cell Cultures.

It has been reported that growth hormone (GH) and insulin-like growth factor 1 (IGF-1) exert protective and regenerative actions in response to neural damage. It is also known that these peptides are expressed locally in nervous tissues. When the central nervous system (CNS) is exposed to hypoxia-ischemia (HI), both GH and IGF-1 are upregulated in several brain areas. In this study, we explored the neuroprotective effects of GH and IGF-1 administration as well as the involvement of these endogenously expressed hormones in embryonic chicken cerebellar cell cultures exposed to an acute HI injury. To induce neural damage, primary cultures were first incubated under hypoxic-ischemic (<5% O2, 1g/L glucose) conditions for 12 h (HI), and then incubated under normal oxygenation and glucose conditions (HI + Ox) for another 24 h. GH and IGF-1 were added either during or after HI, and their effect upon cell viability, apoptosis, or necrosis was evaluated. In comparison with normal controls (Nx, 100%), a significant decrease of cell viability (54.1 ± 2.1%) and substantial increases in caspase-3 activity (178.6 ± 8.7%) and LDH release (538.7 ± 87.8%) were observed in the HI + Ox group. On the other hand, both GH and IGF-1 treatments after injury (HI + Ox) significantly increased cell viability (77.2 ± 4.3% and 72.3 ± 3.9%, respectively) and decreased both caspase-3 activity (118.2 ± 3.8% and 127.5 ± 6.6%, respectively) and LDH release (180.3 ± 21.8% and 261.6 ± 33.9%, respectively). Incubation under HI + Ox conditions provoked an important increase in the local expression of GH (3.2-fold) and IGF-1 (2.5-fold) mRNAs. However, GH gene silencing with a specific small-interfering RNAs (siRNAs) decreased both GH and IGF-1 mRNA expression (1.7-fold and 0.9-fold, respectively) in the HI + Ox group, indicating that GH regulates IGF-1 expression under these incubation conditions. In addition, GH knockdown significantly reduced cell viability (35.9 ± 2.1%) and substantially increased necrosis, as determined by LDH release (1011 ± 276.6%). In contrast, treatments with GH and IGF-1 stimulated a partial recovery of cell viability (45.2 ± 3.7% and 53.7 ± 3.2%) and significantly diminished the release of LDH (320.1 ± 25.4% and 421.7 ± 62.2%), respectively. Our results show that GH, either exogenously administered and/or locally expressed, can act as a neuroprotective factor in response to hypoxic-ischemic injury, and that this effect may be mediated, at least partially, through IGF-1 expression.

Changes in amino acid profiles and liver alterations in pregnant rats with a high carbohydrate/low protein diet.

INTRODUCTION AND AIM: Intake of a high-carbohydrate, low-protein diet (HCD/LPD) during pregnancy promotes metabolic disturbances. It has been suggested that liver function during pregnancy contributes to the synthesis of proteins necessary for fetal development during this stage. The liver is a site of response to the synthesis of macronutrients such as proteins. However, it is unknown how HCD/LPD is associated with modifications to the amino acid profiles and hepatic alterations in the maternal environment during pregnancy. MATERIALS AND METHODS: A transverse longitudinal study was done in primiparous mothers during gestation (G) (G1 day 1, G5 day 5, G15 day 15, and G20 day 20). Histological analysis was used to assess hepatic alterations, and amino acid profiles in the liver were analyzed with high performance liquid chromatography (HPLC). Food and water intake was quantified, and peripheral biochemical indicators in serum were measured. RESULTS: Mothers with HCD/LPD had increased micro and macro vesicles of fat, necrosis, and inflammation in the liver on G5. The total concentration of hepatic amino acids increased by 40% on G1, 17% on G5, and 25% on G15; and, there was a 12% decrease on G20. The following increases were observed in the liver on G1: arginine 68%, histidine 75%, alanine 18%, methionine 71%, and phenylalanine 51% (p>0.05); on G5: arginine 12%, methionine 34%, and phenylalanine 83% (p>0.05); on G15: arginine and phenylalanine 66%, tryptophan 81% and histidine 60.4% (p>0.05); and on G20: arginine 32% (p>0.05). No weight loss, changes in food consumption, or hepatomegaly occurred. CONCLUSIONS: HCD/LPD during pregnancy in primiparous mothers may promote development of fat vesicles. Possibly, this condition causes metabolic adaptations and nitrogen management reflected in decreased levels of serum urea and altered amino acid profiles in the liver.

Time-caloric restriction inhibits the neoplastic transformation of cirrhotic liver in rats treated with diethylnitrosamine.

Hepatocellular cancer is the most common type of primary liver cancer. Cirrhosis is the main risk factor that generates this malady. It has been proven that caloric restriction protocols and restricted feeding schedules are protective in experimental carcinogenic models. We tested the influence of a time-caloric restriction protocol (2 h of food access during the daytime for 18 weeks) in an experimental model of cirrhosis-hepatocarcinoma produced by weekly administration of diethylnitrosamine. Our results indicate that time-caloric restriction reduced hepatomegaly and prevented the increase in blood leukocytes promoted by diethylnitrosamine. Strikingly, time-caloric restriction preserved functional and histological characteristics of the liver in fibrotic areas compared to the cirrhotic areas of the Ad Libitum-fed group. Tumoural masses in the restricted group were well differentiated; consider a neoplastic or early stage of HCC. However, time-caloric restriction enhanced collagen deposits. With regard to the cancerous process, food restriction prevented systemic inflammation and an increase in carcinoembryonic antigen, and it favoured the occurrence of diffuse multinodular tumours. Histologically, it prevented hepatocyte inflammation response, the regenerative process, and neoplastic transformation. Time-caloric restriction stimulated circadian synchronization in fibrotic and cancerous liver sections, and it increased BMAL1 clock protein levels. We conclude that time-caloric restriction prevents fibrosis from progressing into cirrhosis, thus avoiding chronic inflammation and regenerative processes. It also prevents, probably through circadian entrainment and caloric restriction, the neoplastic transformation of tumoural lesions induced by diethylnitrosamine.

Interactive effects of chronic stress and a high-sucrose diet on nonalcoholic fatty liver in young adult male rats.

Glucocorticoids have been implicated in nonalcoholic fatty liver diseases (NAFLD). The influence of a palatable diet on the response to stress is controversial. This study explored whether a high-sucrose diet could protect from hepatic steatosis induced by chronic restraint stress in young adult rats. Male Wistar rats aged 21 days were allocated into four groups (n = 6-8 per group): control, chronic restraint stress, 30% sucrose diet, and 30% sucrose diet plus chronic restraint stress. After being exposed to either tap water or sucrose solution during eight weeks, half of the rats belonging to each group were subject or not to repeated restraint stress (1 h per day, 5 days per week) during four weeks. Triacylglycerol (TAG), oxidative stress, activity of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1), infiltration of immune cells, and glycogen amount in the liver were quantified. Serum concentrations of corticosterone and testosterone were also measured. The stressed group showed normal serum concentrations of corticosterone and did not have hepatic steatosis. However, this group showed increased glycogen, inflammation, mild fibrosis, oxidative stress, and a high activity of 11ß-HSD-1 in the liver. The group exposed to the high-sucrose diet had lower concentrations of corticosterone, hepatic steatosis and moderate fibrosis. The group subject to high-sucrose diet plus chronic restraint stress showed low concentrations of corticosterone, hepatic steatosis, oxidative stress, and high concentrations of testosterone. Thus, restraint stress and a high-sucrose diet each generate different components of nonalcoholic fatty liver in young adult rats. The combination of both the factors could promote a faster development of NAFLD.

Molecular and biochemical modifications of liver glutamine synthetase elicited by daytime restricted feeding.

BACKGROUND & AIMS: The circadian clock system in the liver plays important roles in regulating metabolism and energy homeostasis. Restricted feeding schedules (RFS) become an entraining stimulus that promotes adaptations that form part of an alternative circadian clock known as the food entrained oscillator (FEO). The aim of this study was to evaluate the daily variations of glutamine synthetase (GS) in liver under a daytime RFS. METHODS: Hepatic GS properties were analysed at 3-h intervals over a 24-h period in adult male Wistar rats maintained in a 12:12 h light­dark cycle. RFS group: food access for 2-h in light phase, during 3 weeks. AL group: feeding ad libitum. Fa group: acute fast (21 h). Fa­Re group: acute fast followed by refed 2 h.mRNA expression was measured by RT-qPCR, protein presence by Western-blot and immunohistochemistry, enzyme activity by a spectrophotometric assay, and glutamine by high pressure liquid chromatography. RESULTS AND CONCLUSIONS: Restricted feeding schedule induced circadian rhythmicity inmRNA levels of GS and the loss of the rhythmic pattern in mitochondrial GS activity. GS activity in liver homogenates displayed a robust rhythmic pattern in AL that was not modified by RFS. The presence of GS and its zonal distribution did not show rhythmic pattern in both groups. However, acute Fa and Fa­Re diminished GS protein and activity in liver homogenates. Hepatic glutamine concentrations showed a 24-h rhythmic pattern in both groups, in an antiphasic pattern. In conclusion, daytime RFS influences the liver GS system at different levels, that could be part of rheostatic adaptations associated to the FEO, and highlight the plasticity of this system.

Diurnal and nutritional adjustments of intracellular Ca2+ release channels and Ca2+ ATPases associated with restricted feeding schedules in the rat liver.

BACKGROUND: Intracellular calcium is a biochemical messenger that regulates part of the metabolic adaptations in the daily fed-fast cycle. The aim of this study was to characterize the 24-h variations of the liver ryanodine and IP3 receptors (RyR and IP3R) as well as of the endoplasmic-reticulum and plasma-membrane Ca2+-ATPases (SERCA and PMCA) in daytime restricted feeding protocol. METHODS: A biochemical and immunohistochemical approach was implemented in this study: specific ligand-binding for RyR and IP3R, enzymatic activity (SERCA and PMCA), and protein levels and zonational hepatic-distribution were determined by immunoblot and immunohistochemistry respectively under conditions of fasting, feeding, and temporal food-restriction. RESULTS: Binding assays and immunoblots for IP3R1 and 2 showed a peak at the light/dark transition in the ad-libitum (AL) group, whereas in the restricted-feeding (RF) group the peak shifted towards the food-access time. In the case of RyR binding experiments, both AL and RF groups showed a modest elevation during the dark period, with the RF rats exhibiting increased binding in response to feeding. The AL group showed 24-h rhythmicity in SERCA level; in contrast, RF group showed a pronounced amplitude elevation and a peak phase-shift during the light-period in SERCA level and activity. The activity of PMCA was constant along day in both groups; PMCA1 levels showed a 24-h rhythmicity in the RF rats (with a peak in the light period), meanwhile PMCA4 protein levels showed rhythmicity in both groups. The fasted condition promoted an increase in IP3R binding and protein level; re-feeding increased the amount of RyR; neither the activity nor expression of SERCA and PMCA protein was affected by fasting-re-feeding conditions. Histochemical experiments showed that the distribution of the Ca2+-handling proteins, between periportal and pericentral zones of the liver, varied with the time of day and the feeding protocol. CONCLUSIONS: Our findings show that RF influences mainly the phase and amplitude of hepatic IP3R and SERCA rhythms as well as discrete zonational distribution for RyR, IP3Rs, SERCA, and PMCA within the liver acinus, suggesting that intracellular calcium dynamics could be part of the rheostatic adaptation of the liver due to diurnal meal entrainment/food entrained oscillator expression.

Mitochondrial Oxidation of the Cytoplasmic Reducing Equivalents at the Onset of Oxidant Stress in the Isoproterenol-Induced Rat Myocardial Infarction.

We have developed and characterized a model of isoproterenol (ISO)-induced myocardial necrosis, identifying three stages of cardiac damage: a pre-infarction (0-12 h), infarction (24 h), and post-infarction period (48-96 h). Using this model, we have previously found alterations in calcium homeostasis and their relationship with oxidant stress in mitochondria, which showed deficient oxygen consumption and coupled ATP synthesis. Therefore, the present study was aimed at assessing the mitochondrial ability to transport and oxidize cytoplasmic reducing equivalents (NADH), correlating the kinetic parameters of the malate-aspartate shuttle, oxidant stress, and mitochondrial functionality. Our results showed only discreet effects during the cardiotoxic ISO action on the endogenous malate-aspartate shuttle activity, suggesting that endogenous mitochondrial NADH oxidation capacity (Nohl dehydrogenase) was not affected by the cellular stress. On the contrary, the reconstituted system showed significant enhancement in maximal capacity of the malate-aspartate shuttle activity only at later times (post-infarction period), probably as a compensatory part of cardiomyocytes' response to the metabolic and functional consequences of the infarcted tissue. Therefore, these findings support the notion that heart damage associated with myocardial infarction suffers a set of sequential biochemical and metabolic modifications within cardiomyocytes, where mitochondrial activity, controlling the redox state, could play a relevant role.

Daytime food restriction alters liver glycogen, triacylglycerols, and cell size. A histochemical, morphometric, and ultrastructural study.

BACKGROUND: Temporal restriction of food availability entrains circadian behavioral and physiological rhythms in mammals by resetting peripheral oscillators. This entrainment underlies the activity of a timing system, different from the suprachiasmatic nuclei (SCN), known as the food entrainable oscillator (FEO). So far, the precise anatomical location of the FEO is unknown. The expression of this oscillator is associated with an enhanced arousal prior to the food presentation that is called food anticipatory activity (FAA). We have focused on the study of the role played by the liver as a probable component of the FEO. The aim of this work was to identify metabolic and structural adaptations in the liver during the expression of the FEO, as revealed by histochemical assessment of hepatic glycogen and triacylglycerol contents, morphometry, and ultrastructure in rats under restricted feeding schedules (RFS). RESULTS: RFS promoted a decrease in the liver/body weight ratio prior to food access, a reduction of hepatic water content, an increase in cross-sectional area of the hepatocytes, a moderate reduction in glycogen content, and a striking decrease in triacylglyceride levels. Although these adaptation effects were also observed when the animal displayed FAA, they were reversed upon feeding. Mitochondria observed by electron microscopy showed a notorious opacity in the hepatocytes from rats during FAA (11:00 h). Twenty four hour fasting rats did not show any of the modifications observed in the animals expressing the FEO. CONCLUSIONS: Our results demonstrate that FEO expression is associated with modified liver handling of glycogen and triacylglycerides accompanied by morphometric and ultrastructural adaptations in the hepatocytes. Because the cellular changes detected in the liver cannot be attributed to a simple alternation between feeding and fasting conditions, they also strengthen the notion that RFS promotes a rheostatic adjustment in liver physiology during FEO expression.

Molecular iodine synergized and sensitized neuroblastoma cells to the antineoplastic effect of ATRA.

Neuroblastoma (NB) is the most common solid childhood tumor, and all-trans retinoic acid (ATRA) is used as a treatment to decrease minimal residual disease. Molecular iodine (I2) induces differentiation and/or apoptosis in several neoplastic cells through activation of PPARγ nuclear receptors. Here, we analyzed whether the coadministration of I2 and ATRA increases the efficacy of NB treatment. ATRA-sensitive (SH-SY5Y), partially-sensitive (SK-N-BE(2)), and non-sensitive (SK-N-AS) NB cells were used to analyze the effect of I2 and ATRA in vitro and in xenografts (Foxn1 nu/nu mice), exploring actions on cellular viability, differentiation, and molecular responses. In the SH-SY5Y cells, 200 µM I2 caused a 100-fold (0.01 µM) reduction in the antiproliferative dose of ATRA and promoted neurite extension and neural marker expression (tyrosine hydroxylase (TH) and tyrosine kinase receptor alpha (Trk-A)). In SK-N-AS, the I2 supplement sensitized these cells to 0.1 µM ATRA, increasing the ATRA-receptor (RARα) and PPARγ expression, and decreasing the Survivin expression. The I2 supplement increased the mitochondrial membrane potential in SK-N-AS suggesting the participation of mitochondrial-mediated mechanisms involved in the sensibilization to ATRA. In vivo, oral I2 supplementation (0.025%) synergized the antitumor effect of ATRA (1.5 mg/kg BW) and prevented side effects (body weight loss and diarrhea episodes). The immunohistochemical analysis showed that I2 supplementation decreased the intratumoral vasculature (CD34). We suggest that the I2 + ATRA combination should be studied in preclinical and clinical trials to evaluate its potential adjuvant effect in addition to conventional treatments.

Reduced Liver Lipid Peroxidation in Subcellular Fractions Is Associated with a Hypometabolic State in Rats with Portacaval Anastomosis.

A surgical connection between portal and inferior cava veins was performed to generate an experimental model of high circulating ammonium and hepatic hypofunctioning. After 13 weeks of portacaval anastomosis (PCA), hyperammonemia and shrinkage in the liver were observed. Low glycemic levels accompanied by elevated levels of serum alanine aminotransferase were recorded. However, the activity of serum aspartate aminotransferase was reduced, without change in circulating urea. Histological and ultrastructural observations revealed ongoing vascularization and alterations in the hepatocyte nucleus (reduced diameter with indentations), fewer mitochondria, and numerous ribosomes in the endoplasmic reticulum. High activity of hepatic caspase-3 suggested apoptosis. PCA promoted a marked reduction in lipid peroxidation determined by TBARs in liver homogenate but specially in the mitochondrial and microsomal fractions. The reduced lipoperoxidative activity was also detected in assays supplemented with Fe2+. Only discreet changes were observed in conjugated dienes. Fluorescent probes showed significant attenuation in mitochondrial membrane potential, reactive oxygen species (ROS), and calcium content. Rats with PCA also showed reduced food intake and decreased energy expenditure through indirect calorimetry by measuring oxygen consumption with an open-flow respirometric system. We conclude that experimental PCA promotes an angiogenic state in the liver to confront the altered blood flow by reducing the prooxidant reactions associated with lower metabolic rate, along with significant reduction of mitochondrial content, but without a clear hepatic dysfunction.

Restricted feeding modulates the daily variations of liver glutamate dehydrogenase activity, expression, and histological location.

Glutamate dehydrogenase is an important enzyme in the hepatic regulation of nitrogen and energy metabolism. It catalyzes one of the most relevant anaplerotic reactions. Although its relevance in liver homeostasis has been widely described, its daily pattern and responsiveness to restricted feeding protocols has not been studied. We explored the daily variations of liver glutamate dehydrogenase transcription, protein, activity, and histochemical and subcellular location in a protocol of daytime food synchronization in rats. Restricted feeding involved food access for 2 h each day for three weeks. Control groups included food ad libitum as well as acute fasting (21 h fasting) and refeeding (22 h fasting followed by 2 h of food access). Glutamate dehydrogenase mRNA, protein, activity, and histological location were measured every 3 h by qPCR, Western blot, spectrophotometry, and immunohistochemistry, respectively, to generate 24-h profiles. Restricted feeding promoted higher levels of mitochondrial glutamate dehydrogenase protein and activity, as well as a loss of 24-h rhythmicity, in comparison to ad libitum conditions. The rhythmicity of glutamate dehydrogenase activity detected in serum was changed. The data demonstrated that daytime restricted feeding enhanced glutamate dehydrogenase protein and activity levels in liver mitochondria, changed the rhythmicity of its mRNA and serum activity, but without effect in its expression in hepatocytes surrounding central and portal veins. These results could be related to the adaptation in nitrogen and energy metabolism that occurs in the liver during restricted feeding and the concomitant expression of the food entrainable oscillator. Impact statement For the first time, we are reporting the changes in daily rhythmicity of glutamate dehydrogenase (GDH) mRNA, protein and activity that occur in the liver during the expression of the food entrained oscillator (FEO). These results are part of the metabolic adaptations that modulate the hepatic timing system when the protocol of daytime restricted feeding is applied. As highlight, it was demonstrated higher GDH protein and activity in the mitochondrial fraction. These results contribute to a better understanding of the influence of the FEO in the energy and nitrogen handling in the liver. They could also be significant in the pathophysiology of hepatic diseases related with circadian abnormalities.

Redox regulation and pro-oxidant reactions in the physiology of circadian systems.

Rhythms of approximately 24 h are pervasive in most organisms and are known as circadian. There is a molecular circadian clock in each cell sustained by a feedback system of interconnected "clock" genes and transcription factors. In mammals, the timing system is formed by a central pacemaker, the suprachiasmatic nucleus, in coordination with a collection of peripheral oscillators. Recently, an extensive interconnection has been recognized between the molecular circadian clock and the set of biochemical pathways that underlie the bioenergetics of the cell. A principle regulator of metabolic networks is the flow of electrons between electron donors and acceptors. The concomitant reduction and oxidation (redox) reactions directly influence the balance between anabolic and catabolic processes. This review summarizes and discusses recent findings concerning the mutual and dynamic interactions between the molecular circadian clock, redox reactions, and redox signaling. The scope includes the regulatory role played by redox coenzymes (NAD(P)+/NAD(P)H, GSH/GSSG), reactive oxygen species (superoxide anion, hydrogen peroxide), antioxidants (melatonin), and physiological events that modulate the redox state (feeding condition, circadian rhythms) in determining the timing capacity of the molecular circadian clock. In addition, we discuss a purely metabolic circadian clock, which is based on the redox enzymes known as peroxiredoxins and is present in mammalian red blood cells and in other biological systems. Both the timing system and the metabolic network are key to a better understanding of widespread pathological conditions such as the metabolic syndrome, obesity, and diabetes.

Inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis by molecular iodine (I2) but not by iodide (I-) treatment Evidence that I2 prevents cancer promotion.

We analyzed the effect of molecular iodine (I2), potassium iodide (KI) and a subclinical concentration of thyroxine (T4) on the induction and promotion of mammary cancer induced by N-methyl-N-nitrosourea. Virgin Sprague-Dawley rats received short or continuous treatment. Continuous I2 treated rats exhibited a strong and persistent reduction in mammary cancer incidence (30%) compared to controls (72.7%). Interruption of short or long term treatments resulted in a higher incidence in mammary cancer compared to the control groups. The protective effect of I2 was correlated with the highest expression of the I-/Cl- transporter pendrin and with the lowest levels of lipoperoxidation expression in mammary glands. Triiodothyronine serum levels and Na+/I- symporter, lactoperoxidase, or p53 expression did not show any changes. In conclusion continuous I2 treatment has a potent antineoplastic effect on the progression of mammary cancer and its effect may be related to a decrease in the oxidative cell environment.

Synchronization by food access modifies the daily variations in expression and activity of liver GABA transaminase.

Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks. γ-Aminobutyric acid (GABA) is a signaling molecule in the liver, and able to modulate the cell cycle and apoptosis. This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO.

Time course of retinal degeneration associated with the absence of 1, 4, 5-inositol trisphosphate receptor in Drosophila melanogaster.

The absence of the inositol trisphosphate receptor is associated with a gradual retinal degeneration in Drosophila melanogaster. To characterize the time-course profile of this process, mosaic flies expressing a null allele of the itp gene in the eye were studied by electroretinograms and electronic microscopy. Membrane contour alterations, disrupted mitochondria, altered morphology and even loss of photoreceptors were increased progressively starting 5 d after hatching, were more evident during days 10-15 and promoted highly disorganized structures thereafter. Comparison between electroretinograms recorded in wild type and mutant tissues showed progressive differences in the on and off transients as well as in the magnitude of the summed receptor potentials of photoreceptor cells from day 5 of hatching, [corrected] and the functional defects became progressively more severe. Unexpectedly, these alterations were detected not only in the non-pigmented mutant ommatidia, but also in the pigmented ommatidia, including heterozygous and twin clones expressing 1, 4, 5-inositol trisphosphate receptor (IP(3)R). To explore the mechanism underlying this degenerative process, the progression of pro-oxidant and apoptotic reactions was characterized by immunohistochemical techniques. Mutant ommatidia showed intermittent episodes of increased pro-oxidant reactions (detected as adducts of 4-hydroxy-nonenal) throughout the fly's life. Similarly, several episodes of active caspase 3, an apoptotic effector, were evident with the same time pattern. Episodes of enhanced lipid peroxidation and apoptosis were also observed in the pigmented ommatidia of the mosaic eyes. The results indicate that photoreceptors lacking IP(3)R suffer episodes of increased lipid peroxidation, which eventually perturb the retinal subcellular organization and disrupt the phototransduction process and cell viability. Pigmented ommatidia also showed a similar pattern of damage, indicating that the degenerative process is non-autonomous and is so intense that it propagated to the non-mutant retinal cells in the mosaic eyes. In conclusion, ommatidia with a null mutation of IP(3)R degenerate by a process associated with intermittent lipid peroxidation and apoptotic activities.

Food restricted schedules promote differential lipoperoxidative activity in rat hepatic subcellular fractions.

Restricted access to food (from 12:00 to 14:00 h) produces a behavioral activation known as food anticipatory activity (FAA), which is a manifestation of the food entrained oscillator (FEO). Peripheral oscillators, especially in the liver, are thought to be part of the FEO. A variety of metabolic adaptations have been detected in the liver during the expression of this oscillator, including activation of mitochondrial respiration and changes in the cytoplasmic and mitochondrial redox states. Biological clocks are regulated by redox-sensitive factors. The present study explored the lipoperoxidative activity (LP) in the liver during the activity of the FEO. Conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS), with and without Fe2+-supplementation, were quantified in six subcellular fractions: whole homogenate, plasma membrane, mitochondria, microsomes, nucleus, and cytosol. The experimental protocol involved control groups of ad libitum fed and 24-h fasted rats, and groups under the restricted food schedule (RFS) which were sampled before FAA (08:00 h), during FAA (11:00 h) and after feeding (14:00 h). Clear differences in pro-oxidant activity was observed between ad libitum fed and 24-h fasted rats in almost all the subcellular fractions studied. RFS rats presented: CD levels more similar to the fasted rats, even at 14:00 h, after food presentation, and basal and Fe2+-supplemented TBARS levels tended to be lower than both controls, suggesting an increased antioxidant capacity associated with food restriction. In addition, a microarray analysis showed that several isoforms of peroxiredoxins, a family of antioxidant and hydrogen peroxide-catabolizing enzymes, were consistently up-regulated in each and every condition in which RFS was applied. Together, these data indicate a rheostatic adaptation of the liver in the handling of pro-oxidant reactions during the activity of the FEO.

Metabolic adaptations of liver mitochondria during restricted feeding schedules.

Food anticipatory activity (FAA) is an output of the food-entrained oscillator (FEO), a conspicuous biological clock that expresses when experimental animals are under a restricted food schedule (RFS). We have shown that the liver is entrained by RFS and exhibits an anticipatory response before meal time in its oxidative and energetic state. The present study was designed to determine the mitochondrial oxidative and phosphorylating capacity in the liver of rats under RFS to further support the biochemical anticipatory role that this organ plays during the food entrainment (9). Metabolic and functional parameters of liver mitochondria were characterized before (0800 h), during (1100 h), and after (1400 h) FAA. The main results were as follows. First, there was an enhancement during FAA (1100 h) in 1) oxidative capacity (site I of the electron transport chain), 2) phosphorylating ability (estimated by ATP synthesis), and 3) activities of NADH shuttles. Second, after rats were fed (1400 h), the phosphorylating capacity remained high, but this was not the case for the respiratory control ratio for site I. Finally, in the three experimental conditions before, during, and after FAA, an increment was detected in the H(+) electrochemical potential, due to an elevation in mitochondrial membrane potential, and in mitochondrial yield. Most of the changes in mitochondrial properties related to RFS were also present when results were compared with those from the 24-h fasted group. In conclusion, the results support the notion that a distinctive rheostatic state is installed in the metabolic activity of the liver when FEO is being expressed.