Zonal expression of the glucokinase gene in rat liver. Dynamics during the daily feeding rhythm and starvation-refeeding cycle demonstrated by in situ hybridization

The abundance and zonal distribution of glucokinase (GK) mRNA were studied in rat liver during a normal 12 h day/12 h night rhythm (dark from 1900 to 0700 hours) and during refeeding after 60 h of starvation. Zonation of GK gene expression was examined by in situ hybridization with a radiolabelled cRNA probe and GK mRNA abundance was determined by Northern blot analysis with a digoxigenin-labelled cRNA probe. GK mRNA appeared to be almost homogeneously distributed throughout the whole daily feeding cycle; yet it was predominantly localized in the perivenous and intermediate zone during refeeding after 60 h of starvation. During the daily feeding rhythm, the total amount of GK mRNA increased quickly with the beginning of the feeding period at 1900 hours reaching a maximum at midnight and then decreased continuously to a basal level at noon. Virtually no GK mRNA was detected after 60 h of starvation. Refeeding caused a rapid increase in GK mRNA to a maximum at 2400 hours followed by a decrease to approximately two-thirds of the maximum value at 0700 hours. If the homogeneous distribution of GK mRNA during the daily feeding rhythm was real rather than apparent because of too low a sensitivity of the cRNA probe, the present results suggest that during the normal circadian cycle the mainly perivenous distribution of GK enzyme activity and protein is regulated preferentially at a translational level. The findings clearly show that during refeeding after 60 h of starvation the GK distribution is controlled predominantly at a pretranslational level.     

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.     

Periportal expression of the serine dehydratase gene in rat liver

Summary The mRNA for rat liver serine dehydratase, a gluconeogenic enzyme, exhibits a circadian rhythm with a maximum at the onset of darkness marking the end of the fasting period and a minimum at the onset of light that marks the end of the feeding period, when rats have free access to food and water.In situ hybridization with an antisense cRNA probe revealed that serine dehydratase mRNA was localized in the periportal area of rat liver parenchyma in the evening, whereas it was scarce in the liver in the morning. The predominant localization of serine dehydratase mRNA in the periportal area also occurred in livers of rats that underwent laparotomy, glucagon and dexamethasone administration, and streptozotocin-induced diabetes mellitus, all of which are known to induce serine dehydratase mRNA levels remarkably. Immunostaining revealed that the localization of serine dehydratase protein agreed with that of succinate dehydrogenase, another enzyme known to be predominant in the periportal zone. Thus, the periportal serine dehydratase gene expression strongly supports the idea of metabolic zonation that gluconeogenesis from amino acids occurs preferentially in the periportal parenchyma of rat liver.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

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.     

Effect of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus

We evaluated the effects of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus. Fish were divided into four feeding groups (mean mass 1.68 ± 0.12 g). The control group was fed to satiation twice a day throughout the experiment with formulated diet (SFK). The other three groups were deprived of feed for 1(S1), 2(S2), and 3(S3) weeks, respectively, and then fed to satiation during the refeeding period. The results showed that trypsin specific activity was not affected significantly either by starvation or refeeding, in all experimental groups. Chymotrypsin specific activity did not change significantly in S1 fish during the experimental period. In S2 and S3 fish no significant changes were observed during the starvation period. Upon refeeding, the activity increased in S2 fish, while it decreased in S3 fish. Amylase specific activity decreased significantly during the starvation period in all experimental groups. Upon refeeding, the activity increased. Alkaline phosphatase specific activity did not change significantly during the experiment period in S3 fish, while it showed significant changes during the starvation and refeeding period in the S1 and S2 fish. Starvation also had a significant effect on the structure of the intestine.     

Impact of interleukin-6 on the glucose metabolic capacity in rat liver

The actute phase reaction mediated by the proinflammatory cytokine IL6 initiates a number of metabolic changes in the liver, which may contribute to the pathogenesis of the septic shock during prolonged exposition. Here, the impact of IL6 on the hepatic glucose providing capacity was studied by monitoring glycogen degradation and the expression of the gluconeogenic phosphoenolpyruvate carboxykinase (PCK1) in rat livers during the daily feeding rhythm. Eight hours after i.p. injection of IL6, mRNA levels of α2-macroglobulin, a prominent acute phase reactant in rat liver, were elevated as shown by Northern blot analysis and in situ hybridization (ISH). PCK1 mRNA levels were decreased by IL6 to 50% of levels in untreated animals due to the reduction of PCK1 mRNA in the periportal zone of the liver as shown by ISH. PCK1 enzyme activity was not affected by IL6. Glycogen degradation was accelerated by IL6, which led to nearly complete depletion of glycogen pools in periportal areas 8 h after IL6 injection. This was very likely due to inhibition of glycogen pool replenishment. Thus, the depletion of glycogen stores in the liver might contribute to the impairment of hepatic glucose production during prolonged acute phase challenge.     

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.     

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.     

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.     

Metabolic responses to food deprivation and refeeding in juveniles of Rutilus rutilus (Teleostei: Cyprinidae)

Synopsis Effect of food deprivation and refeeding on metabolic parameters were studied in juvenile Rutilus rutilus, weighing 280–460 mg. Tissue hydration increased with the length of the starvation period, reaching a new steady state after 4–5 weeks. Total protein concentration remained constant at about 60% of dry body mass. The concentration of glycogen decreased during food deprivation, a new steady state being reached at about 30% of control values after 4 weeks. Refeeding caused a dramatic increase of glycogen concentration which exceeded the value in fed controls by 6- to 9-fold. This is seen as a tactic for rapid storage of food energy, to be used later for the synthesis of body materials. With respect to their responses to food deprivation the 12 enzymes investigated formed four groups: (1) activity unaffected by food deprivation or refeeding (COX, THIOL, CK, GOT); (2) activity drops to about 60% of control value during the initial phase of food deprivation but remains constant thereafter (PK, LDH, Pase); (3) slow but continuous decrease in activity during the whole period of starvation, i.e. up to 7 weeks (PFK, OGDH, CS, FBPase); (4) activity increases during food deprivation, decreases again upon refeeding (GPT). A model is discussed which distinguishes between four phases in the general response of young fish to food deprivation and refeeding: stress, transition, adaptation, and recovery.     

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.     

Refeeding after fasting elicits insulin-dependent regulation of Per2 and Rev-erbα with shifts in the liver clock

The mammalian circadian clock is known to be entrained by both a daily light-dark cycle and daily feeding cycle. However, the mechanisms of feeding-induced entrainment are not as fully understood as those of light entrainment. To elucidate the first step of entrainment of the liver clock, we identified the circadian clock gene(s) that show both phase advance and acute change of gene expression during the early term of the daytime refeeding schedule in mice. The expressions of liver Per2 and Rev-erbα genes were phase-advanced within 1 day of refeeding. Additionally, the upregulation of Per2 mRNA and down-regulation of Rev-erbα mRNA were induced within 2 hours, not only by food intake but also by insulin injection in intact mice. These expression changes by food intake were not revealed in streptozotocin-treated insulin-deficient mice, but insulin injection was able to recover the impairment of Per2 and Rev-erbα gene expression. Furthermore, we demonstrated using an ex vivo luciferase monitoring system that insulin injection during the daytime causes a phase advance of liver Per2 expression rhythm in Per2::luciferase knock-in mice. In embryonic fibroblasts from Per2::luciferase knock-in mice, insulin infusion caused an acute increase of Per2 gene expression and a similar phase advance of Per2 expression rhythm. Our results indicate that an acute change of Per2 and Rev-erbα gene expression mediated by refeeding-induced insulin secretion is a critical step mediating the early phase of feeding-induced entrainment of the liver clock.     

Induction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase mRNA by refeeding and insulin

The effects of fasting/refeeding and untreated or insulin-treated diabetes on the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its mRNA in rat liver were determined. Both enzymatic activities fell to 20% of control values with fasting or streptozotocin-induced diabetes and were coordinately restored to normal within 48 h of refeeding or 24 h of insulin administration. These alterations in enzymatic activities were always mirrored by corresponding changes in amount of enzyme as determined by phosphoenzyme formation and immunoblotting. In contrast, mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase did not decrease during starvation or in diabetes, but there was a 3-6-fold increase upon refeeding a high carbohydrate diet to starved rats or insulin treatment of diabetic rats. The decrease of the enzyme in starved or diabetic rats without associated changes in mRNA levels suggests a decrease in the rate of mRNA translation, an increase in enzyme degradation, or both. The rise in enzyme amount and mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with refeeding and insulin treatment suggests an insulin-dependent stimulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression. Northern blots of RNA from heart, brain, kidney, and skeletal muscle probed with restriction fragments of a full-length cDNA from liver showed that only skeletal muscle contained an RNA species that hybridized to any of the probes. Skeletal muscle mRNA for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was 2.0 kilobase pairs but in contrast to the liver message (2.2 kilobase pairs) was not regulated by refeeding.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats.

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: 14C-labeled tryptamine, dopamine and kynuramine. With dopamine as substrate, the enzyme activity showed decline during 24 and 48 h of starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decrease after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.