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.     

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.     

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.     

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.     

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.     

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.     

Increase of the gluconeogenic and decrease of the glycolytic capacity of rat liver with a change of the metabolic zonation after partial hepatectomy.

During the first 72 h after 67% partial hepatectomy of female Wistar rats (160 g) the specific activities [mumol X min-1 X (g liver)-1] of the glucogenic glucose-6-phosphatase and fructose-bisphosphatase and of the glycolytic hexokinase and 6-phosphofructokinase remained essentially constant. However, the activity of the glycolytic pyruvate kinase (L- plus M2-type) was decreased slightly and that of glucokinase was decreased markedly to below 30%, while the glucogenic phosphoenolpyruvate carboxykinase was increased to over 200%. Between 10 and 40 h after partial hepatectomy, when the proliferation started in the periportal area, a shift of the glucogenic glucose-6-phosphatase-rich zone from its normal periportal to an intermediate or even perivenous position was observed histochemically. After 48 h, when the proliferation was no longer restricted to the periportal zone, the normal glucose-6-phosphatase zonation (as before partial hepatectomy) was restored. Glycogen was degraded rapidly during the first 4 h after operation; it was later repeatedly resynthesized and degraded in correlation with the feeding rhythm of the animals. The zonation of glycogen metabolism was in accord with the observed zonation of glucose-6-phosphatase.     

Neuropeptide Y content in the hypothalamic paraventricular nucleus responds to fasting and refeeding in broiler chickens

To examine the neural mechanism by which hypothalamic neuropeptide Y (NPY) regulates energy homeostasis and feeding behavior in commercial broilers, we measured NPY content in several hypothalamic regions of birds that were fasted and then refed. After fasting for 48 and 72 h, body weight significantly decreased, and food intake significantly increased during the subsequent refeeding. The lost body weight was not restored to ad libitum feeding levels even after 3 days of refeeding. Plasma glucose concentration and body fat content significantly decreased and plasma non-esterified fatty acid (NEFA) concentration significantly increased after 48- and 72-h fasting. Refeeding for 24 h restored plasma metabolites and body fat content to pre-fasting levels. NPY content in the paraventricular nucleus (PVN) and infundibular nucleus significantly increased during fasting, and NPY content of the PVN was restored to pre-fasting levels after 24-h refeeding. However, there was no significant change in the NPY content of the lateral hypothalamic area during fasting or refeeding. The present results of changes in the hypothalamic NPY content during fasting and refeeding support the hypothesis that NPY plays a central role in regulation of energy homeostasis, with especially important effect on feeding behavior and body weight in broiler chickens.     

Feeding activates protein synthesis in mouse pancreas at the translational level without increase in mRNA

To determine the mechanism of meal-regulated synthesis of pancreatic digestive enzymes, we studied the effect of fasting and refeeding on pancreatic protein synthesis, relative mRNA levels of digestive enzymes, and activation of the translational machinery. With the use of the flooding dose technique with L-[3H]phenylalanine, morning protein synthesis in the pancreas of Institute for Cancer Research mice fed ad libitum was 7.9 +/- 0.3 nmol phenylalanine.10 min(-1).mg protein(-1). Prior fasting for 18 h reduced total protein synthesis to 70 +/- 1.4% of this value. Refeeding for 2 h, during which the mice consumed 29% of their daily food intake, increased protein synthesis to 117.3 +/- 4.9% of the control level. Pancreatic mRNA levels of amylase, lipases, trypsins, chymotrypsin, elastases, as well as those for several housekeeping genes tested were not significantly changed after refeeding compared with fasted mice. By contrast, the major translational control pathway involving Akt, mTOR, and S6K was strongly regulated by fasting and refeeding. Fasting for 18 h decreased phosphorylation of ribosomal protein S6 to almost undetectable levels, and refeeding highly increased it. The most highly phosphorylated form of the eIF4E binding protein (4E-BP1) made up the 14.6% of total 4E-BP1 in normally fed animals, was only 2.8% after fasting, and was increased to 21.4% after refeeding. This was correlated with an increase in the formation of the eIF4E-eIF4G complex after refeeding. By contrast, feeding did not affect eIF2B activity. Thus food intake stimulates pancreatic protein synthesis and translational effectors without increasing digestive enzyme mRNA levels.     

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.     

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.     

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.     

NADP-dependent dehydrogenases in rat liver parenchyma

Summary The activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and isocitrate dehydrogenase (ICDH) were investigated with optimized histochemical methods (Rieder et al. 1978), and the activity of 3-hydroxybutyrate dehydrogenase (3HBDH) and neutral fat content with conventional techniques in the liver of male rats under the following experimental dietary conditions: (A) Fasting for 0, 12 and 84 h; (B) 84-h fasting followed by refeeding with a low-fat, high-carbohydrate diet for 6 h and for 2, 3, 5, 7, 11 and 14 nights; (C) refeeding with standard diet for 5 nights; (D) low-fat, high-carbohydrate diet for 7 and 14 nights.The activities of G6PDH, 6PGDH and ME decreased slightly during fasting primarily in zone 1 and increased dramatically on refeeding with a low-fat, high-carbohydrate diet. This activity increase was confined mainly to zone 3 during the first 3 days and was accompanied by a deposition of neutral fats that began in zone 3 and progressed to zone 1. Neutral fat accumulation was maximal after 3 nights, with a uniform accumulation of large droplets in all the hepatocytes; this was followed by a release that started in zone 3 and proceeded in a periportal direction. On the other hand, G6PDH, 6PGDH and ME attained their maximum activities after 5 and 7 nights of the low-fat diet, the activities being nearly homogeneously distributed over the liver acinus in a few cases. Subsequently the activities fell mainly in zone 1, causing the activity patterns and levels to approach those of the animals in group (D). In contrast to this, the activity of ICDH increased during fasting principally in zone 1, so that the otherwise steep activity gradient in favor of zone 3 lessened. Refeeding led at first to a fall of activity below the initial value, but later the normal distribution pattern was restored. The activity of 3HBDH showed a behavior similar to that of ICDH. The findings are discussed with reference to the functional heterogeneity of the liver perenchyma, and the existence of a liponeogenic area in zone 3 is proposed.Essential parts of this study have been presented to the Medical Faculty of the University of Freiburg/Br. as an inaugural dissertationSupported by grants from the Deutsche Forschungsgemeinschaft (Sa 127/7) and SFB 46     

Glycemic index of a meal fed before exercise alters substrate use and glucose flux in exercising horses.

In a randomized, balanced, crossover study each of six fit, adult horses ran on a treadmill at 50% of maximal rate of oxygen consumption for 60 min after being denied access to food for 18 h and then 1) fed corn (51.4 kJ/kg digestible energy), or 2) fed an isocaloric amount of alfalfa 2-3 h before exercise, or 3) not fed before exercise. Feeding corn, compared with fasting, resulted in higher plasma glucose and serum insulin and lower serum nonesterified fatty acid concentrations before exercise (P < 0.05) and in lower plasma glucose, serum glycerol, and serum nonesterified fatty acid concentrations and higher skeletal muscle utilization of blood-borne glucose during exercise (P < 0.05). Feeding corn, compared with feeding alfalfa, resulted in higher carbohydrate oxidation and lower lipid oxidation during exercise (P < 0.05). Feeding a soluble carbohydrate-rich meal (corn) to horses before exercise results in increased muscle utilization of blood-borne glucose and carbohydrate oxidation and in decreased lipid oxidation compared with a meal of insoluble carbohydrate (alfalfa) or not feeding. Carbohydrate feedings did not produce a sparing of muscle glycogen compared with fasting.     

Daily Changes of Plasma Corticosterone by an 8-h Daytime Feeding Occur Without Body Weight Loss or Severe Food Restriction in the Rat

Different studies have reported that daytime feeding entrains the circadian rhythm of corticosterone secretion in rats. However, it remained unclear whether calorie restriction or daytime feeding access have an effect. The aim of our study is to evaluate the effect of an 8-h daytime feeding access on the circadian rhythm of plasma corticosterone. Eleven adult male Wistar rats were assigned to two different conditions of access to food: ad lib feeding for one week and daytime feeding for the following two weeks. On the 7th, 14th and 21st day, blood samples were collected every 4 h from 08:00 to 04:00. Food intake and body weight were recorded daily. During daytime feeding, rats ingested 88% of the amount of food ingested over 24 h in the ad lib feeding period. However, body weight increased significantly from the first day to the end of experiment. Peak plasma corticosterone was 12 h shifted during daytime feeding period compared to the ad lib condition. This study showed that an 8-h daytime feeding entrained the circadian rhythm of plasma corticosterone without body weight loss or severe food restriction.     

Daily Changes of Plasma Corticosterone by an 8-h Daytime Feeding Occur Without Body Weight Loss or Severe Food Restriction in the Rat

Different studies have reported that daytime feeding entrains the circadian rhythm of corticosterone secretion in rats. However, it remained unclear whether calorie restriction or daytime feeding access have an effect. The aim of our study is to evaluate the effect of an 8-h daytime feeding access on the circadian rhythm of plasma corticosterone. Eleven adult male Wistar rats were assigned to two different conditions of access to food: ad lib feeding for one week and daytime feeding for the following two weeks. On the 7th, 14th and 21st day, blood samples were collected every 4 h from 08:00 to 04:00. Food intake and body weight were recorded daily. During daytime feeding, rats ingested 88% of the amount of food ingested over 24 h in the ad lib feeding period. However, body weight increased significantly from the first day to the end of experiment. Peak plasma corticosterone was 12 h shifted during daytime feeding period compared to the ad lib condition. This study showed that an 8-h daytime feeding entrained the circadian rhythm of plasma corticosterone without body weight loss or severe food restriction.     

Complex interaction between circadian rhythm and diet on bile acid homeostasis in male rats

Desynchronization between the master clock in the brain, which is entrained by (day) light, and peripheral organ clocks, which are mainly entrained by food intake, may have negative effects on energy metabolism. Bile acid metabolism follows a clear day/night rhythm. We investigated whether in rats on a normal chow diet the daily rhythm of plasma bile acids and hepatic expression of bile acid metabolic genes is controlled by the light/dark cycle or the feeding/fasting rhythm. In addition, we investigated the effects of high caloric diets and time-restricted feeding on daily rhythms of plasma bile acids and hepatic genes involved in bile acid synthesis. In experiment 1 male Wistar rats were fed according to three different feeding paradigms: food was available ad libitum for 24 h (ad lib) or time-restricted for 10 h during the dark period (dark fed) or 10 h during the light period (light fed). To allow further metabolic phenotyping, we manipulated dietary macronutrient intake by providing rats with a chow diet, a free choice high-fat-high-sugar diet or a free choice high-fat (HF) diet. In experiment 2 rats were fed a normal chow diet, but food was either available in a 6-meals-a-day (6M) scheme or ad lib. During both experiments, we measured plasma bile acid levels and hepatic mRNA expression of genes involved in bile acid metabolism at eight different time points during 24 h. Time-restricted feeding enhanced the daily rhythm in plasma bile acid concentrations. Plasma bile acid concentrations are highest during fasting and dropped during the period of food intake with all diets. An HF-containing diet changed bile acid pool composition, but not the daily rhythmicity of plasma bile acid levels. Daily rhythms of hepatic Cyp7a1 and Cyp8b1 mRNA expression followed the hepatic molecular clock, whereas for Shp expression food intake was leading. Combining an HF diet with feeding in the light/inactive period annulled CYp7a1 and Cyp8b1 gene expression rhythms, whilst keeping that of Shp intact. In conclusion, plasma bile acids and key genes in bile acid biosynthesis are entrained by food intake as well as the hepatic molecular clock. Eating during the inactivity period induced changes in the plasma bile acid pool composition similar to those induced by HF feeding.