Predominant periportal expression of the phosphoenolpyruvate carboxykinase and tyrosine aminotransferase genes in rat liver

Summary The zonal distribution of phosphoenolpyruvate carboxykinase (PCK) and tyrosine aminotransferase (TAT) mRNA in liver was studied by in situ hybridization with radiolabelled cRNA probes and the abundance of PCK and TAT mRNA was quantified by Northern blot analysis of total RNA with biotinylated cRNA probes. Livers were taken from rats during a normal 12 h day/night rhythm, when they had access to food only during the dark period from 7 pm to 7 am, or during refeeding, when they had access to food after having been starved for 60 h. 1. Daily feeding rhythm: High levels of PCK mRNA were distributed mainly in the periportal and intermediate zone during the fasting period at noon and 6 pm. Feeding caused a rapid decrease in PCK mRNA level and a restriction of PCK mRNA localization to the periportal area within the first 2 h. No further alterations were observed during the following hours of the feeding period. TAT mRNA was distributed also in the periportal and intermediate zone during the fasting period. Feeding first reduced the mRNA level without changing the distribution pattern. Then towards the end of the feeding period TAt mRNA increased again to half-maximal levels and became restricted mainly to the periportal area. 2. Starvation-refeeding cycle: High amounts of PCK mRNA as well as of TAT mRNA were localized predominantly in the periportal and intermediate zone after 60 h of starvation. PCK and TAT mRNA both decreased markedly during the first 2 h of refeeding and then remained almost constant. Whereas the alterations in the overall abundance of the two mRNAs were similar, the distribution patterns of both mRNAs differed. While PCK mRNA became more and more restricted to a small area of periportal cells towards the end of refeeding, TAT mRNA was first evenly distributed in the periportal and perivenous area with higher amounts in the intermediate zone and then again was predominantly located in the periportal area. The present data indicate that the predominant periportal localization of PCK and TAT activity and enzyme protein is regulated mainly at the pretranslational level.     

Predominant periportal expression of the phosphoenolpyruvate carboxykinase and tyrosine aminotransferase genes in rat liver. Dynamics during the daily feeding rhythm and starvation-refeeding cycle demonstrated by in situ hybridization

The zonal distribution of phosphoenolpyruvate carboxykinase (PCK) and tyrosine aminotransferase (TAT) mRNA in liver was studied by in situ hybridization with radiolabelled cRNA probes and the abundance of PCK and TAT mRNA was quantified by Northern blot analysis of total RNA with biotinylated cRNA probes. Livers were taken from rats during a normal 12 h day/night rhythm, when they had access to food only during the dark period from 7 pm to 7 am, or during refeeding, when they had access to food after having been starved for 60 h. 1. Daily feeding rhythm: High levels of PCK mRNA were distributed mainly in the periportal and intermediate zone during the fasting period at noon and 6 pm. Feeding caused a rapid decrease in PCK mRNA level and a restriction of PCK mRNA localization to the periportal area within the first 2 h. No further alterations were observed during the following hours of the feeding period. TAT mRNA was distributed also in the periportal and intermediate zone during the fasting period. Feeding first reduced the mRNA level without changing the distribution pattern. Then towards the end of the feeding period TAT mRNA increased again to half-maximal levels and became restricted mainly to the periportal area. 2. Starvation-refeeding cycle: High amounts of PCK mRNA as well as of TAT mRNA were localized predominantly in the periportal and intermediate zone after 60 h of starvation. PCK and TAT mRNA both decreased markedly during the first 2 h of refeeding and then remained almost constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian rhythms of feeding, oviposition, and emergence of the boll weevil （Coleoptera： Curculionidae）

Circadian rhythm of feeding, oviposition, and emergence of boll weevil adults were determined at five different photophases （24, 14, 12, 10, and 0 hours） and a constant 27℃ temperature, 65% RH in the laboratory. Squares from Petri dishes, where they were exposed to boll weevil females, were removed and examined for feeding and oviposition punctures every 4 hours during daylight （0700-1900 h） and every 12 h at night （1900-0700 h） over eight consecutive days. Cohorts of randomly selected egg-punctured squares were sampled from ovipositing females at 0700, 1100, 1500, and 1900 during 24 hours and under different photophase treatments, and maintained in Petri dishes at 27 ＋ I℃, 65% RH. Dishes were observed twice daily （1900 and 0700 h） for adults emerging at day or night. Circadian rhythm of oviposition was not affected by the length of the photophase. The boll weevil has round-the-clock circadian rhythm of oviposition, with a daytime preference. We observed that 82.4%-86.0% of the boll weevil eggs were deposited between 0700 and 1900 h, and 14.0%-17.6% between 1900 and 0700 h during a 24-h period. Feeding of boll weevil females in photoperiods 24：0 h （complete light） and 0：24 h （complete darkness） did not significantly change between 0700-1900 h versus 1900-0700 h, while the d .ally cycle of light and darkness in other photoperiods significantly increased the feeding punctures from 0700-1900 compared with 1900-0700 h. The circadian rhythm of emergence depended significantly on the time of oviposition and the length of the photophase. Investigation of boll weevil circadian rhythm provides a better understanding of boll weevil ecology and reveals potential weak links for improving control technologies targeting their reproductive strategies.     

Predominant localization of phosphoenolpyruvate carboxykinase mRNA in the periportal zone of rat liver parenchyma demonstrated by in situ hybridization

In rat liver parenchyma, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was studied by Northern blot analysis with a biotinylated cRNA probe and the zonal localization of PEPCK mRNA was demonstrated by in situ hybridization with a radiolabelled cRNA probe. During the feeding period at night, overall PEPCK mRNA levels were low and PEPCK mRNA was detected only in small areas of the periportal zone. At the beginning of the light period (7 am) the overall PEPCK mRNA level began increasing and the periportal areas containing PEPCK mRNA broadened. The maximum of the total abundance and of the area with high levels of PEPCK mRNA was reached at noon. Fasting for 24-72 h did not cause further significant alterations in the level or localization of PEPCK mRNA. The present data are in line with previous findings of the predominant localization of PEPCK activity and enzyme protein in periportal hepatocytes. They suggest that the heterogeneous expression of the PEPCK gene in rat liver is regulated at the pretranslational level.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient. Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Unequal Day-Night Terbutaline I.V. Dosing in Acute Severe Asthma: Effect on Nocturnal Bronchial Patency, Heart Rate, and Arterial Pressure

Our study investigated the differential effects of continuous or unequal day-night terbutaline dosing on circadian bronchial patency, heart rate, and arterial pressure in severe acute asthma. Forty-five hospitalized asthmatic patients (19 women and 26 men, mean age 45.4 years, mean weight 63.5 kg) were included in this multicenter study. Three groups of patients (corresponding to three dosing schedules) were randomized; the three groups were comparable, since no statistically significant difference was detected in the age, weight, or peak expiratory flow values at the beginning of the study. In order to reach immediately the concentrations of terbutaline corresponding to the desired unequal day-night concentrations, a theoretical pharmacokinetic simulation was done to predict the outcome in terms of the plasma concentrations after the three dosing regimens; the results of this simulation allowed us to calculate the initial bolus dose to be given over 5 min to groups A, B, and C, i.e., 1.47, 2.94, and 4.41 Mg/kg, respectively. This bolus was given to all patients at 0700 h, the beginning of the study. The patients were randomly divided into three groups (A, B, C) receiving one of these treatments: 0.0111 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate delivered by an electrical pump and 0.0222 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (A) (one third the total daily dose during the day and the remaining two thirds at night), 0.0166 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0166 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (B) (one half the total daily dose during the day and the remaining one half at night), or 0.0222 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0111 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (C) (two thirds the total daily dose during the day and the remaining one third at night). Since acute severe asthma could not be treated without steroids, a 40 mg dose of SoluMedrol was injected into all patients at 0700. Peak expiratory flow rate, heart rate, systolic arterial pressure, and possible side effects were recorded at different times during the 24-h scale: 0700, 1000, 1300, 1600, 1900, 2300, 0300, and 0700 h. Our results have shown a significant therapeutic effect of terbutaline i.v. dosing in severe acute asthma whatever the unequal daynight dosing, but did not demonstrate the efficacy of one of the three dosing schedules over the others.     

Zonal distribution of fatty acid synthase in liver parenchyma of male and female rats

1. A sensitive radiochemical assay was established to determine the activity of fatty acid synthase in microdissected liver tissue of less than 1 microgram dry mass. 2. In female rats, the enzyme activity in perivenous tissue was twice that in periportal liver tissue while it was homogeneously distributed in livers of male animals. The overall activity was higher in female than in male animals. 3. The absolute activity, as well as the perivenous/periportal ratio, was reduced during starvation and in diabetes. They were greatly increased after refeeding to values above those observed in animals during normal feeding. 4. Ovariectomy or administration of testosterone to female rats resulted in a significant reduction of the zonal heterogeneity. 5. Castration or administration of estradiol to male animals was followed by an increase in the enzyme activity exclusively in the perivenous tissue, resulting in a zonal heterogeneity as observed in female rats.     

Unequal Day-Night Terbutaline I.V. Dosing in Acute Severe Asthma: Effect on Nocturnal Bronchial Patency,Heart Rate,and Arterial Pressure

Our study investigated the differential effects of continuous or unequal day-night terbutaline dosing on circadian bronchial patency, heart rate, and arterial pressure in severe acute asthma. Forty-five hospitalized asthmatic patients (19 women and 26 men, mean age 45.4 years, mean weight 63.5 kg) were included in this multicenter study. Three groups of patients (corresponding to three dosing schedules) were randomized; the three groups were comparable, since no statistically significant difference was detected in the age, weight, or peak expiratory flow values at the beginning of the study. In order to reach immediately the concentrations of terbutaline corresponding to the desired unequal day-night concentrations, a theoretical pharmacokinetic simulation was done to predict the outcome in terms of the plasma concentrations after the three dosing regimens; the results of this simulation allowed us to calculate the initial bolus dose to be given over 5 min to groups A, B, and C, i.e., 1.47, 2.94, and 4.41 Mg/kg, respectively. This bolus was given to all patients at 0700 h, the beginning of the study. The patients were randomly divided into three groups (A, B, C) receiving one of these treatments: 0.0111 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate delivered by an electrical pump and 0.0222 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (A) (one third the total daily dose during the day and the remaining two thirds at night), 0.0166 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0166 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (B) (one half the total daily dose during the day and the remaining one half at night), or 0.0222 mg/kg of terbutaline i.v. from 0700 to 1900 h at a constant rate and 0.0111 mg/kg of terbutaline i.v. from 1900 to 0700 h at a constant rate (C) (two thirds the total daily dose during the day and the remaining one third at night). Since acute severe asthma could not be treated without steroids, a 40 mg dose of SoluMedrol was injected into all patients at 0700. Peak expiratory flow rate, heart rate, systolic arterial pressure, and possible side effects were recorded at different times during the 24-h scale: 0700, 1000, 1300, 1600, 1900, 2300, 0300, and 0700 h. Our results have shown a significant therapeutic effect of terbutaline i.v. dosing in severe acute asthma whatever the unequal daynight dosing, but did not demonstrate the efficacy of one of the three dosing schedules over the others.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Hourly distribution of time of parturition in the domestic goat

Data on 2589 records collected during 12 years in one breed of dairy goats that were maintained in stables were analysed to examine the influence of various factors on the time of parturition. Parturitions were observed from mid-January to the end of April. Hourly frequencies of birth showed an unimodal distribution with maximum births at midday and minimum births around midnight with 90.6% of deliveries occurring between 0600 and 2000 h. The distribution showed a noticeable homogeneity when the following parameters were taken into account: litter-size, litters with live or stillborn kids, time of the year and length of gestation. In two other herds, 117 and 166 records obtained after 4 and 3 years of observation were studied. As in the first herd, births were more frequent between 0700 to 1900 h (day; 72.3 and 70.2 %, respectively) than between 1900 and 0700 h (night; P<0.001). These observations on the time of birth of the domestic goat suggest a regulation of the birth process by circadian events which themselves remain unknown.     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Nutritional and gonadal effects on the intra-acinar profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Total and low-Km aldehyde dehydrogenase (ALDH) activity was measured in 50-150 ng microdissected liver tissue samples of the entire sinusoidal length. High-Km ALDH activity was calculated by subtracting the low-Km ALDH values from the total ALDH activity. Enzyme activity was measured by a microchemical assay, using the oil-well technique with luminometric determination of NADH. The intra-acinar profiles of high-Km and low-Km ALDH activity could be demonstrated graphically for both male and female rats after 84 h of starvation, and after starvation and refeeding for 6 nights. In addition, the ALDH distribution patterns of juvenile, castrated, and castrated and testosterone-treated rats were determined. It could be demonstrated that starvation, and starvation followed by refeeding, lead to changes in enzyme activity which parallel the loss and regain of liver- and body-weight. The nutritional factors do not essentially alter the normal intra-acinar profiles. In juvenile rats, ALDH is lower by 30% in comparison with the controls, but sex-differences in the distribution profiles are not yet present. Castration has no effect on the amount of enzyme activity but the sex specific distribution profiles are less marked. The main effect of testosterone treatment is an elevation of low-Km ALDH in the perivenous zone. The characteristics of the intra-acinar profiles of high-Km and low-Km ALDH activity are discussed with respect to hepatic acetaldehyde oxidation and alcoholic liver damage.     

The effects of starvation and refeeding on intestinal cell proliferation in the mouse

The effects of starvation and refeeding on intestinal cell proliferation were studied in four sites of the mouse intestine. Control mice were studied at different times of day in order to compensate for any circadian variations in proliferation. A circadian rhythm in crypt cell production rate was observed in all the sites of the small intestine and colon, and this rhythm appeared to be entrained to the food intake. The fractional crypt cell production rate decreased in all sites of the intestine after 24 h starvation, and remained low until 9 h after refeeding, when there was a marked increase in the crypt cell production rate of all the small intestinal sites, especially the proximal sites. There was little change in colonic crypt cell production rate until 12 h after refeeding, when there was a large increase in cell production. The crypt cell production rate of all sites then returned to control values for the remainder of the investigation. Crypt cell number decreased after refeeding and villus cell number increased, however a similar effect was observed in the control animals, nevertheless the changes in villus cell population of the refed mice occurred before any increase in crypt cell production, suggesting that cell migration from crypt to villi is not immediately dependent on cell proliferation.     

Effects of food deprivation and refeeding on neuropeptide Y (NPY) mRNA levels in goldfish

In mammals, NPY is a key factor in the regulation of feeding behavior. In the present study, the effects of refeeding for 1-3 h in 72-75-h food deprived (FD) goldfish on the levels of NPY mRNA in telencephalon-preoptic (TEL-POA), hypothalamus (HYP) and optic tectum-thalamus (OT-THAL) were examined, using Northern blot analysis. Goldfish FD for 72 h exhibited a significant increase in NPY mRNA levels in all brain regions. At 1 h after 72-h FD (73-h FD), NPY mRNA was significantly increased in TEL-POA and OT-THAL, but remained the same as 72-h FD fish in HYP. At 3 h after 72-h FD (75 h), all brain regions exhibited a significant increase in NPY mRNA levels. However, subsequent refeeding for 1-3 h rapidly and completely reversed the effects of FD in all brain regions, reaching fed levels within 1-3 h of refeeding. Serum GH levels were highest in 72-h FD fish, but decreased significantly over 1-3 h after 72-h FD; whereas, refeeding reversed the increase in serum GH levels only at 3 h after refeeding. Taken together, these results further support that NPY is a physiological brain transducer involved in the regulation of daily appetite and feeding in goldfish.     

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.     

Diurnal variations in response of rat liver tyrosine aminotransferase activity to food intake

Effects of fasting and refeeding on the hepatic tyrosine aminotransferase activity were examined in rats that had been fed during the night. The tyrosine aminotransferase activity showed clear diurnal variations, with a maximal activity after the feeding time. The tyrosine aminotransferase rhythm persisted even under starvation, though the amplitude decreased remarkably. When the starved rats were refed at night, the tyrosine aminotransferase activity increased rapidly to a high level, but it increased slowly to a rather lower level when they were refed in daytime.     

Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding

The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase in hepatic glucose metabolism.