Purinergic Signaling in Non-Parenchymal Liver Cells.

Purinergic signaling has emerged as an important paracrine-autocrine intercellular system that regulates physiological and pathological processes in practically all organs of the body. Although this system has been thoroughly defined since the nineties, recent research has made substantial advances regarding its role in aspects of liver physiology. However, most studies have mainly targeted the entire organ, 70% of which is made up of parenchymal cells or hepatocytes. Because of its physiological role, the liver is exposed to toxic metabolites, such as xenobiotics, drugs, and fatty acids, as well as to pathogens such as viruses and bacteria. Under injury conditions, all cell types within the liver undergo adaptive changes. In this context, the concentration of extracellular ATP has the potential to increase dramatically. Indeed, this purinergic response has not been studied in sufficient detail in non-parenchymal liver cells. In the present review, we systematize the physiopathological adaptations related to the purinergic system in chronic liver diseases of non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, and cholangiocytes. The role played by non-parenchymal liver cells in these circumstances will undoubtedly be strategic in understanding the regenerative activities that support the viability of this organ under stressful conditions.

Adenosine Receptor A2B Negatively Regulates Cell Migration in Ovarian Carcinoma Cells.

The purinergic system is fundamental in the tumor microenvironment, since it regulates tumor cell interactions with the immune system, as well as growth and differentiation in autocrine-paracrine responses. Here, we investigated the role of the adenosine A2B receptor (A2BR) in ovarian carcinoma-derived cells' (OCDC) properties. From public databases, we documented that high A2BR expression is associated with a better prognostic outcome in ovarian cancer patients. In vitro experiments were performed on SKOV-3 cell line to understand how A2BR regulates the carcinoma cell phenotype associated with cell migration. RT-PCR and Western blotting revealed that the ADORA2B transcript (coding for A2BR) and A2BR were expressed in SKOV-3 cells. Stimulation with BAY-606583, an A2BR agonist, induced ERK1/2 phosphorylation, which was abolished by the antagonist PSB-603. Pharmacological activation of A2BR reduced cell migration and actin stress fibers; in agreement, A2BR knockdown increased migration and enhanced actin stress fiber expression. Furthermore, the expression of E-cadherin, an epithelial marker, increased in BAY-606583-treated cells. Finally, cDNA microarrays revealed the pathways mediating the effects of A2BR activation on SKOV-3 cells. Our results showed that A2BR contributed to maintaining an epithelial-like phenotype in OCDC and highlighted this purinergic receptor as a potential biomarker.

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.

Functional expression of P2Y2 receptors in mouse ovarian surface epithelium (OSE).

Ovarian surface epithelium (OSE) is a cell monolayer surrounding the ovary; it is involved in the regulation of the ovulatory process and the genesis of ovarian carcinoma. However, intercellular messengers regulating signaling events, like proliferation in the OSE, have not been completely described. Purines have emerged as novel intercellular messengers in the ovary, in which expression of purinergic receptors has been reported in different cell types. In the present work, we described the functional expression of P2Y2 receptor (P2Y2R), a purinergic receptor widely associated with cell proliferation, in the OSE. The expression of P2Y2R by immunofluorescence and RT-PCR, and its functionality by Ca2+ recording was demonstrated in primary cultured OSE. Functional expression of P2Y2R was also exhibited in situ, by recording of intracellular Ca2+ release and detection of ERK phosphorylation after injection of a selective agonist into the ovarian bursa. Furthermore, P2Y2R activation with UTPγS, in situ, induced cell proliferation at 24 h, whereas continuous stimulation of P2Y2R during a complete estrous cycle significantly modified the size distribution of the follicular population. This is the first evidence of the functional expression of purinergic P2Y2R in the OSE and opens new perspectives on the roles played by purines in ovarian physiology.

Changes in 24 h Rhythmicity of Spontaneous Locomotor Activity in the Triple Transgenic Mouse for Alzheimer's Disease (3xTg-AD) in a Jet Lag Protocol: Correlations with Retinal Sensitivity.

The progression of amyloid plaques and neurofibrillary tangles in different brain areas is associated with the effects of Alzheimer's disease (AD). In addition to cognitive impairment, circadian alterations in locomotor activity have also been detected, but they have not been characterized in a jet lag protocol. Therefore, the present study aimed to compare 3xTg-AD and non-transgenic mice in changes of 24 h cycles of spontaneous locomotor activity in a jet lag protocol, in an environment without a running wheel, at 3 different states of neuronal damage: early, intermediate and advanced (3, 8 and 13 months, respectively). The 3xTg-AD mice at 3 months presented differences in phase angle and acrophase, and differentially increased activity after advances more than after delays. At 13 months, a shortening of the free-running period in constant darkness was also noted. 3xTg-AD mice showed a significant increase (123%) in global activity at 8 to 13 months and in nighttime activity (153%) at 13 months. In the advance protocol (ADV), 3xTg-AD mice displayed a significant increase in global activity (171%) at 8 and 13 months. The differences in masking effect were evident at 8 months. To assess a possible retinal dysfunction that could interfere with photic entrainment as part of the neurodegenerative process, we compared electroretinogram recordings. The results showed early deterioration in the retinal response to light flashes in mesopic conditions, observed in the B-wave latency and amplitude. Thus, our study presents new behavioral and pathological characteristics of 3xTg-AD mice and reveals the usefulness of non-invasive tools in early diagnosis.

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.

Experimental polycystic ovarian syndrome is associated with reduced expression and function of P2Y2 receptors in rat theca cells.

Extracellular purines through specific receptors have been recognized as new regulators of ovarian function. It is known that P2Y2 receptor activity induces theca cell proliferation, we hypothesized that purinergic signaling controls the changes related to hyperthecosis in polycystic ovarian syndrome (PCOS). The aim of this study was to analyze the expression of UTP-sensitive P2Y receptors and their role in theca cells (TC) proliferation in experimentally-induced PCOS (EI-PCOS). In primary cultures of TC from intact rats, all the transcripts of P2Y receptors were detected by polymerase chain reaction; in these cells, UTP (10 µM) induced extracellular signal-regulated kinases (ERK) phosphorylation. Rats with EI-PCOS showed a reduced expression of P2Y2R in TC whereas P2Y4R did not change. By analyzing ERK phosphorylation, it was determined that P2Y2R is the most relevant receptor in TC. UTP promoted cell proliferation in TC from control but not from EI-PCOS rats. The in silico analysis of P2yr2 promoter indicated the presence of androgen response elements; the stimulation of TC primary cultures with testosterone promoted a significant reduction in the expression of the P2yr2 transcript. We concluded that P2Y2R participates in controlling the proliferative rate of TCs from healthy ovaries, but this regulation is lost during EI-PCOS.

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.

Effects of lead and lead-melatonin exposure on protein and gene expression of metal transporters, proteins and the copper/zinc ratio in rats.

Human lead (Pb) exposure induces many adverse health effects, including some related to lead accumulation in organs. Although lead bio-distribution in the body has been described, the molecular mechanism underlying distribution and excretion is not well understood. The transport of essential and toxic metals is principally mediated by proteins. How lead affects the expression of metal transporter proteins in the principal metal excretory organs, i.e., the liver and kidney, is unknown. Considering that co-administration of melatonin and lead reduces the toxic effects of lead and lead levels in the blood in vivo, we examined how lead and co-administration of lead and melatonin affect the gene and protein expression of metal transporter proteins (ZIP8, ZIP14, CTR1 and DMT1) in these organs. Rats were exposed intraperitoneally to lead or lead-melatonin. Our results show that Pb exposure induces changes in the protein and gene expression of ZIP8, ZIP14 and CTR1. Alterations in the copper/zinc ratio found in the blood, liver and kidney were likely related to these changes. With DMT1 expression (gene and protein), a positive correlation was found with lead levels in the kidney. Co-administration of melatonin and lead reduced lead-induced DMT1 expression through an unknown mechanism. This effect of melatonin relates to reduced lead levels in the blood and kidney. The metal transport protein function and our results suggest that DMT1 likely contributes to lead accumulation in organs. These data further elucidate the effects of lead on Cu and Zn and the molecular mechanism underlying lead bio-distribution in animals.

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.

Differences in Photic Entrainment of Circadian Locomotor Activity Between Lean and Obese Volcano Mice (Neotomodon alstoni).

Obesity is a growing problem worldwide with a clear impact on health status. It is also a condition that negatively affects circadian rhythms. When the mouse Neotomodon alstoni is fed a regular rodent chow, some individuals develop obesity, representing an opportunity to compare the effects of spontaneous obesity upon the circadian organization in this species with that observed in other rodents with induced obesity. We report differences in the free running circadian locomotor activity rhythm and in the effects of light pulses between lean and obese mice. Also, the photo-induced expression of the c-Fos protein and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN) were examined at circadian time (CT) 14 and 22. We show that obese mice have a larger dispersion of the period of circadian locomotor rhythm in constant darkness. Photic induced phase shifts are nearly 50% shorter at CT 14, and 50% larger at CT 22 than in lean mice. The photoinduction of VIP in the SCN at CT 22 was larger in obese mice, which may be related to the differences observed in photic phase shifting. Our work indicates that the obesity in Neotomodon has effects on the neural mechanisms that regulate the circadian system.

Ryanodine-sensitive intracellular Ca2+ channels are involved in the output from the SCN circadian clock.

The suprachiasmatic nuclei (SCN) contain the major circadian clock responsible for generation of circadian rhythms in mammals. The time measured by the molecular circadian clock must eventually be translated into a neuronal firing rate pattern to transmit a meaningful signal to other tissues and organs in the animal. Previous observations suggest that circadian modulation of ryanodine receptors (RyR) is a key element of the output pathway from the molecular circadian clock. To directly test this hypothesis, we studied the effects of RyR activation and inhibition on real time expression of PERIOD2::LUCIFERASE, intracellular calcium levels and spontaneous firing frequency in mouse SCN neurons. Furthermore, we determined whether the RyR-2 mRNA is expressed with a daily variation in SCN neurons. We provide evidence that pharmacological manipulation of RyR in mice SCN neurons alters the free [Ca2+ ]i in the cytoplasm and the spontaneous firing without affecting the molecular clock mechanism. Our data also show a daily variation in RyR-2 mRNA from single mouse SCN neurons with highest levels during the day. Together, these results confirm the hypothesis that RyR-2 is a key element of the circadian clock output from SCN neurons.

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.

Hypothalamic expression of anorexigenic and orexigenic hormone receptors in obese females Neotomodon alstoni: effect of fasting.

Obesity is a world problem that requires a better understanding of its physiological and genetic basis, as well as the mechanisms by which the hypothalamus controls feeding behavior. The volcano mouse Neotomodon alstoni develops obesity in captivity when fed with regular chow diet, providing a novel model for the study of obesity. Females develop obesity more often than males; therefore, in this study, we analysed in females, in proestrous lean and obese, the differences in hypothalamus expression of receptors for leptin, ghrelin (growth hormone secretagogue receptor GHS-R), and VPAC, and correlates for plasma levels of total ghrelin. The main comparisons are between mice fed ad libitum and mice after 24 hours of fasting. Mice above 65 g body weight were considered obese, based on behavioral and physiological parameters such as food intake, plasma free fatty acids, and glucose tolerance. Hypothalamic tissue from obese and lean mice was analysed by western blot. Our results indicate that after ad libitum food access, obese mice show no significant differences in hypothalamic leptin receptors, but a significant increase of 60% in the GHS-R, and a nearly 62% decrease in VPAC2 was noted. After a 24-hour fast, plasma ghrelin increased nearly two fold in both lean and obese mice; increases of hypothalamic leptin receptors and GHS-R were also noted, while VPAC2 did not change significantly; levels of plasma free fatty acids were 50% less after fasting in obese than in lean animals. Our results indicate that in obese N. alstoni mice, the levels of orexigenic receptors in the hypothalamus correlate with overfeeding, and the fact that lean and obese females respond in different ways to a metabolic demand such as a 24-hour fast.

A maternal low-protein diet results in sex-specific differences in synaptophysin expression and milk fatty acid profiles in neonatal rats.

The developmental origins of health and disease hypothesis have highlighted the link between early life environment and long-term health outcomes in offspring. For example, maternal protein restriction during pregnancy and lactation can result in adverse metabolic and cognitive outcomes in offspring postnatal. Hence, in the present study, we assess whether an isocaloric low-protein diet (ILPD) affects the fatty acid profile in breast milk, the hippocampal synaptophysin (Syn) ratio, and the oxidative stress markers in the neonatal stage of male and female offspring. The aim of this work was to assess the effect of an ILPD on the fatty acid profile in breast milk, quantified the hippocampal synaptophysin (Syn) ratio and oxidative stress markers in neonatal stage of male and female offspring. Female Wistar rats were fed with either a control diet or an ILPD during gestation to day 10 of lactation. Oxidative stress markers were assessed in serum and liver. All quantifications were done at postnatal day 10. The results showed: ILPD led to decreases of 38.5% and 17.4% in breast milk volume and polyunsaturated fatty acids content. Significant decreases of hippocampal Syn ratio in male offspring (decreases of 98% in hippocampal CA1 pyramidal and CA1 oriens, 83%, stratum pyramidal in CA3, 80%, stratum lucidum in CA3, and 81% stratum oriens in CA3). Male offspring showed an increase in pro-oxidant status in serum and liver. Thus, the data suggest that male offspring are more vulnerable than females to an ILPD during gestation and lactation.

Extracellular ATP/P2X7 receptor, a regulatory axis of migration in ovarian carcinoma-derived cells.

ATP is actively maintained at high concentrations in cancerous tissues, where it promotes a malignant phenotype through P2 receptors. In this study, we first evaluated the effect of extracellular ATP depletion with apyrase in SKOV-3, a cell line derived from metastatic ovarian carcinoma. We observed a decrease in cell migration and an increase in transepithelial electrical resistance and cell markers, suggesting a role in maintaining a mesenchymal phenotype. To identify the P2 receptor that mediated the effects of ATP, we compared the transcript levels of some P2 receptors and found that P2RX7 is three-fold higher in SKOV-3 cells than in a healthy cell line, namely HOSE6-3 (from human ovarian surface epithelium). Through bioinformatic analysis, we identified a higher expression of the P2RX7 transcript in metastatic tissues than in primary tumors; thus, P2X7 seems to be a promising effector for the malignant phenotype. Subsequently, we demonstrated the presence and functionality of the P2X7 receptor in SKOV-3 cells and showed through pharmacological approaches that its activity promotes cell migration and contributes to maintaining a mesenchymal phenotype. P2X7 activation using BzATP increased cell migration and abolished E-cadherin expression. On the other hand, a series of P2X7 receptor antagonists (A438079, BBG and OxATP) decreased cell migration. We used a CRISPR-based knock-out system directed to P2RX7. According to the results of our wound-healing assay, SKOV3-P2X7KO cells lacked receptor-mediated calcium mobilization and decreased migration. Altogether, these data let us propose that P2X7 receptor is a regulator for cancer cell migration and thus a potential drug target.

Hepatocyte ballooning and steatosis in early and late gestation without liver malfunction: Effects of low protein/high carbohydrate diet.

Pregnancy is a challenging metabolic and physiological condition. The aim of this study was to include a second demanding situation as a low protein/high carbohydrate diet (LPHCD) to characterize the histological and functional responses of the maternal liver. It is unknown how the maternal liver responds during early and late pregnancy to LPHCD intake. We explored early pregnancy (3 and 8 gestational age, G) and late pregnancy (15 and 20 G). The results indicated that pregnant rats under control diet showed an evident presence of ballooned hepatocytes, lipid vesicles and edema at late pregnancy (15G); in contrast, pregnant rats under LPHCD showed similar pattern of histological modification but at early pregnancy (3G). Unexpectedly, the serum biomarkers didn't display functional alterations in either group, despite of the evident histological changes no liver malfunction was detected. We conclude that pregnant rats fed with control diet and experimental LPHCD, are subjected to metabolic and physiological conditions that impact the histopathological condition of the maternal liver. Control diet promoted the histological modifications during late pregnancy whereas LPCHCD advanced the onset of these changes. Further experiments are needed to explore the biochemical mechanisms that underlie these histological modifications. Our results are also an example of the resilience associated with the pregnancy: since no functional hepatic alterations accompanied the histopathological changes, another conclusion is that no evident pathological condition was detected in this nutritional protocol.

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.