Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata-reticularis cells by aging

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata-reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3-month) and old (24-month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37 degrees C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)-, forskolin-, 3-isobutyl-l-methylxanthine (IBMX)-, 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP)-, and ovine prolactin (oPRL)-stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin-, or IBMX-induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11 beta-hydroxylase that converts deoxycorticosterone (DOC, 10(-9) or 10(-8) M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10(-7) M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11 beta-hydroxylase.     

DNA repair and aging in mouse liver: 8-oxodG glycosylase activity increase in mitochondrial but not in nuclear extracts

8-oxo-deoxyguanosine (8-oxodG) is one of the major DNA lesions formed upon oxidative attack of DNA. It is a mutagenic adduct that has been associated with pathological states such as cancer and aging. Base excision repair (BER) is the main pathway for the repair of 8-oxodG. There is a great deal of interest in the question about age-associated accumulation of this DNA lesion and its intracellular distribution, particularly with respect to mitochondrial or nuclear localization. We have previously shown that 8-oxodG-incision activity increases with age in rat mitochondria obtained from both liver and heart. In this study, we have investigated the age-associated changes in DNA repair activities in both mitochondrial and nuclear extracts obtained from mouse liver. We observed that 8-oxodG incision activity of mitochondrial extracts increases significantly with age, from 13.4 + or - 2.2 fmoles of oligomer/100 microg of protein/16 h at 6 to 18.6 + or - 4.9 at 14 and 23.7 + or - 3.8 at 23 months of age. In contrast, the nuclear 8-oxodG incision activity showed no significant change with age, and in fact slightly decreased from 11.8 + or - 3 fmoles/50 microg of protein/2 h at 6 months to 9.7 + or - 0.8 at 14 months. Uracil DNA glycosylase and endonuclease G activities did not change with age in nucleus or mitochondria. Our results show that the repair of 8-oxodG is regulated differently in nucleus and mitochondria during the aging process. The specific increase in 8-oxodG-incision activity in mitochondria, rather than a general up-regulation of DNA metabolizing enzymes in those organelles, suggests that this pathway may be up regulated during aging in mice.     

Bioenergetics in aging: mitochondrial proton leak in aging rat liver, kidney and heart

Aging is associated with a decline in performance in many organs and loss of physiological performance can be due to free radicals. Mitochondria are incompletely coupled: during oxidative phosphorylation some of the redox energy is dissipated as natural proton leak across the inner membrane. To verify whether proton leak occurs in mitochondria during aging, we measured the mitochondrial respiratory chain activity, membrane potential and proton leak in liver, kidneys and heart of young and old rats. Mitochondria from old rats showed normal rates of Complex I and Complex II respiration. However, they had a lower membrane potential compared to mitochondria from younger rats. In addition, they exhibited an increased rate of proton conductance which partially dissipated the mitochondrial membrane potential when the rate of electron transport was suppressed. This could compromise energy homeostasis in aging cells in conditions that require additional energy supply and could minimize oxidative damage to DNA.     

Contractions but not AICAR increase FABPpm content in rat muscle sarcolemma

In the present study, it was investigated whether acute muscle contractions in rat skeletal muscle increased the protein content of FABPpm in the plasma membrane. Furthermore, the effect of AICAR stimulation on FAT/CD36 and FABPpm protein content in sarcolemma of rat skeletal muscle was evaluated. Methods Male wistar rats (150 g) were anesthetized and either subjected to in situ electrically induced contractions (hindlimb muscles: 20 min, 10–20 V, 200 ms trains, 100 Hz) or stimulated with the pharmacological activator of AMPK, AICAR. To investigate changes in the content of FABPpm and FAT/CD36 in the plasma membrane by these stimuli, the giant sarcolemma vesicle (GSV) technique was applied. The hindlimb muscles were removed and used for the production of GSV and lysates. All samples were analyzed using the western blotting technique. Results Electrical stimulation of rat hindlimb muscle resulted in an increase in FABPpm protein content in the GSV of 61% (P < 0.05) and in FAT/CD36 protein content in the GSV of 33% (P < 0.05). AICAR stimulation increased FAT/CD36 protein content in GSV by 22% (P < 0.05), whereas FABPpm protein content in GSV was unaffected by AICAR treatment. There was no change in total FAT/CD36 and FABPpm protein expression, measured in lysates with western blotting, by either stimulus. AMPK thr172 and ERK1/2 thr202/204 phosphorylation were significantly increased with muscle contractions (P < 0.05), whereas only AMPK thr172 phosphorylation was increased with AICAR stimulation (P < 0.05). Conclusion These data show that contractions increase both FAT/CD36 and FABPpm protein content in skeletal muscle plasma membrane, whereas only FAT/CD36 protein content is increased when muscle are stimulated with AICAR. This suggests that AMPK is involved in regulation of FAT/CD36, but not FABPpm in skeletal muscle. However, since both ERK1/2 thr202/204 and AMPK thr172 phosphorylation are increased during muscle contractions, the present study cannot rule out that both could play a significant role in regulation of FAT/CD36 and FABPpm during muscle contractions.     

Age-related increase in prostacyclin production in the rat aorta

Normal Sprague-Dawley rats convert a substantial percentage of exogenous arachidonic acid to prostacyclin. This conversion can be quantitated by an aqueous sampling technique utilizing thin layer chromatography and liquid scintillation counting. There is a clear age-related increase in this conversion that can be demonstrated in aortas from rats of 3 weeks to 20 weeks of age.     

Retinol-binding protein mRNA is induced by estrogen in the kidney but not in the liver

Vitamin A is mobilized from the liver and transported in plasma as retinol bound to retinol-binding protein (RBP). In addition to the liver, several extrahepatic tissues including the kidney have been shown to contain RBP mRNA. A study was conducted to explore the role of sex hormones in the regulation of RBP mRNA levels in the kidney compared to those in the liver. Treatment of female rats with a single dose of testosterone or chronic treatment with testosterone had only a slight effect on the steady-state level of RBP mRNA in the kidney and the liver. However, treatment of male rats with estrogen caused an increase in the steady-state level of RBP mRNA in the kidney but not in the liver. A single injection of 17 beta-estradiol, either 1.0 or 0.1 micrograms/g body weight, resulted in a rapid rise in the level of RBP mRNA in the kidney which was maximal at 3-6 h (fivefold induction) after treatment. In addition, treatment of ovariectomized female rats with estrogen also resulted in a rapid rise in the accumulated level of RBP mRNA in the kidney while having no influence in the liver. Finally, studies using the anti-estrogen drug, hydroxytamoxifen, resulted in blockage of the estrogen-related induction of RBP mRNA in the kidney, suggesting that the induction of RBP mRNA in the kidney by estrogen may be mediated by the nuclear estrogen receptor. Taken together these data suggest that the regulation of RBP mRNA, levels in the liver and kidney, at least with respect to estrogen, is different.     

Contrasting cascades: insectivorous birds increase pine but not parasitic mistletoe growth

1. Intraguild predation occurs when top predators feed upon both intermediate predators and herbivores. Intraguild predators may thus have little net impact on herbivore abundance. Variation among communities in the strength of trophic cascades (the indirect effects of predators on plants) may be due to differing frequencies of intraguild predation. Less is known about the influence of variation within communities in predator-predator interactions upon trophic cascade strength. 2. We compared the effects of a single predator community between two sympatric plants and two herbivore guilds. We excluded insectivorous birds with cages from ponderosa pine Pinus ponderosa trees parasitized by dwarf mistletoe Arceuthobium vaginatum. For 3 years we monitored caged and control trees for predatory arthropods that moved between the two plants, foliage-feeding caterpillars and sap-feeding hemipterans that were host-specific, and plant damage and growth. 3. Excluding birds increased the abundance of ant-tended aphids on pine and resulted in an 11% reduction in pine woody growth. Mutualist ants protected pine-feeding aphids from predatory arthropods, allowing aphid populations to burgeon in cages even though predatory arthropods also increased in cages. By protecting pine-feeding aphids from predatory arthropods but not birds, mutualist ants created a three-tiered linear food chain where bird effects cascaded to pine growth via aphids. 4. In contrast to the results for tended aphids on pine, bird exclusion had no net effects on untended pine herbivores, the proportion of pine foliage damaged by pine-feeding caterpillars, or the proportion of mistletoe plants damaged by mistletoe-feeding caterpillars. These results suggest that arthropod predators, which were more abundant in cages as compared with control trees, compensated for bird predation of untended pine and mistletoe herbivores. 5. These contrasting effects of bird exclusion support food web theory: where birds were connected to pine by a linear food chain, a trophic cascade occurred. Where birds fed as intraguild predators, the reticulate food webs linking birds to pine and mistletoe resulted in no net effects on herbivores or plant biomass. Our study shows that this variation in food web structure occurred between sympatric plants and within plants between differing herbivore guilds.     

Changes in glutaminase activities of rat liver and kidney during pre- and post-natal development

The activities of two phosphate-dependent glutaminase reactions characteristic of adult rat liver and kidney were determined in these organs from 2(1/2) days before to 7 days after birth and compared with the activities in the adult tissues. In the kidney, before and after birth, only the kidney type of activity was detected, and it increased in concentration in parallel with the steady growth of that organ throughout the period examined. In the liver, however, the kidney type of activity was the only one present 2(1/2) days before birth, and its concentration decreased to barely significant values by the end of the first week after birth. In contrast, the liver type of activity appeared only just before birth and increased to 60% of adult values over the next 4 days. There was no obvious relation between these changes in glutaminase type and changes in liver weight, protein content and total cell number that occurred during this time. But there was a very close correlation between the fall in kidney-type activity and the estimated fall in haematopoietic-cell number in liver.     

Trigonelline and curcumin alone,but not in combination,counteract oxidative stress and inflammation and increase glycation product detoxification in the liver and kidney of mice with high-fat diet-induced obesity

The development of obesity-associated complications is related to various pathogenic events including chronic inflammation, oxidative stress and generation of advanced glycation end products (AGEs). Due to their antioxidant, anti-inflammatory and antiglycation properties, trigonelline and curcumin are interesting candidates to counteract complications of obesity and diabetes mellitus. The current study aimed to investigate the effects of treatment with curcumin or trigonelline mixed into yoghurt, alone or in combination, on mice fed high-fat diet (HFD); the focus was mainly on the potential of these phytochemicals to counteract oxidative and glycative stress. Yoghurt alone improved glucose tolerance and reduced proinflammatory cytokine levels in HFD mice; however, it did not affect the antioxidant status. Trigonelline-enriched yoghurt prevented fat accumulation in adipose tissue, improved both insulin sensitivity and glucose tolerance and exerted anti-inflammatory and antiglycation activities (reduced AGEs and AGE receptor levels and increased the levels of components related to AGE detoxification) in liver and kidney of HFD mice. Curcumin-enriched yoghurt exerted anti-inflammatory and potent antioxidant properties (increased antioxidant enzyme activities and decreased lipid peroxidation) in liver and kidney of HFD mice. However, several beneficial effects were nullified when trigonelline and curcumin were administered in combination. Trigonelline and curcumin have emerged as promising complementary therapy candidates for liver and kidney complications associated with obesity. However, the administration of these phytochemicals in combination, at least in HFD mice, was not effective; inhibition of biotransformation processes and/or the reaching of toxic doses during combined treatment may be prevailing over the individual pharmacodynamic actions of these phytochemicals.     

Peroxynitrite but not nitric oxide donors destroys epinephrine: HPLC measurement and rat aorta contractility

Nitric oxide (NO) and peroxynitrite (ONOO) have been reported to destroy catecholamines. We compared the ability of NO donors and peroxynitrite to decompose epinephrine in both chemical and pharmacological experiments. Epinephrine (1 microM) was incubated with NO donors (SNAP and MAHMA NONOate) and ONOO at a concentration of 0.1 mM in phosphate buffer (pH 7.4; 0.1 M) or Krebs solution for 10 minutes at 37 degrees C. HPLC revealed that the concentration of epinephrine in the presence of NO donors was unaltered. In contrast, peroxynitrite decreased epinephrine concentration more than 20 fold. Similar relationships were obtained in the study of rat thoracic aorta ring contraction. The contractile activity (EC50) of epinephrine in control solutions and after incubation of NE with NO donors did not change. EC50 was measured at 8-10 nM in control solutions and after preincubation with NO donors. However when epinephrine was preincubated with peroxynitrite, no contractile effect was evoked. Therefore, under these experimental conditions peroxynitrite, but not NO donors, was capable of destroying epinephrine.     

Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes.

Basolateral membrane vesicles were isolated from rat kidney cortex and small intestinal enterocytes. Both membrane preparations show ATP-dependent calcium uptake and cytochalasin B-sensitive D-glucose transport. In renal membranes, sodium influx is stimulated by bicarbonate; bicarbonate-dependent sodium flux is membrane-potential-dependent and inhibited by 4,4'-di-isothiocyanato-2, 2'-stilbenedisulphanic acid ('DIDS'). Small intestinal basolateral membranes do not show bicarbonate-dependent sodium fluxes.     

Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by alpha-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.     

Dietary betaine modifies hepatic metabolism but not renal injury in rat polycystic kidney disease

We undertook a morphometric and proton nuclear magnetic resonance ((1)H-NMR) study to test the hypothesis that 1% dietary betaine supplementation would ameliorate renal disease in the heterozygous Han:SPRD-cy rat, a model of polycystic kidney disease (PKD) and progressive chronic renal failure. After 8 wk of pair feeding, betaine had no effect on renal cystic change, renal interstitial fibrosis, serum creatinine, serum cholesterol, or serum triglycerides. (1)H-NMR spectroscopy of renal tissue revealed no change in renal osmolytes, including betaine, or renal content of other organic anions in response to diet. (1)H-NMR spectroscopy of hepatic tissue performed to explore the metabolic fate of ingested betaine revealed that heterozygous animals fed the control diet had elevated hepatic levels of gluconeogenic amino acids, increased beta-hydroxybutyrate, and increased levels of some citric acid cycle metabolites compared with animals without renal disease. Betaine supplementation eliminated these changes. Chronic renal failure in the Han:SPRD-cy rat is associated with disturbances of hepatic metabolism that can be corrected with betaine therapy, suggesting the presence of a reversible methylation defect in this form of chronic renal failure.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

Role of adrenals in the vitamin A-mediated increase in the activities of gluconeogenic enzymes of rat liver.

Oral administration of large doses of vitamin A to rats even for two days was found to cause marked increase in the activities of PEP-carboxykinase, fructose-1, 6-diphosphatase, glucose-6-phosphatase and alanine aminotransferase in liver. However, overdosage of this vitamin failed to enhance the activities of these enzymes in the livers of bilaterally adrenalectomized rats. The adrenalectomy was also found to abolish the vitamin A-mediated increase in the levels of glucose and lactic acid in the blood. Thus, it is concluded that stimulation of gluconeogenesis in hypervitaminosis is, perhaps, caused by the increase in the activities of the key gluconeogenic enzymes of the liver, and that adrenal hormones are directly or indirectly involved in this process.     

Zonation of glycogen and glucose syntheses, but not glycolysis, in rat liver.

We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the methods of Lindros & Penttila [Biochem. J. (1985) 228, 757-760] and of Quistorff [Biochem. J. (1985) 229, 221-226]. A modified procedure which yields hepatocytes capable of consistent rates of glycogen synthesis is described, and the rates of glucose and glycogen syntheses and of glycolysis in hepatocytes from the two zones are compared. Glycogen synthesis in cells was greatly impaired by very low concentrations (0.01-0.05 mg/ml) of digitonin, which had little effect on glucose and protein syntheses and Trypan Blue exclusion. Cells exposed to such low concentrations of digitonin lose all their synthetic capacity and ability to exclude Trypan Blue when incubated with EGTA, which does not affect cells not exposed to digitonin. With a modified procedure based on this phenomenon, our study reveals that hepatocyte preparations enriched with cells from the periportal zone synthesized glucose from lactate and alanine at rates twice those by cells from the perivenous zone, whereas the rate of glycogen synthesis from C3 precursors in periportal cells was 4 times that in the perivenous preparations. With substrates entering the pathway at the triose phosphate level, gluconeogenesis in periportal-cell preparations was 20% higher, and glycogen synthesis was twice that in perivenous preparations. Glycolysis was studied by the formation of 3HOH from [2-3H]glucose, the yield of lactate, and the conversion of [14C]glucose into [14C]lactate. In cell preparations from both zones glycolysis by all criteria was negligible at 10 mM-glucose, but was substantial at higher concentrations. However, there was no difference between the zones. We confirm that the capacities for glucose and glycogen syntheses in periportal cells are higher than in perivenous cells, but that at physiological glucose concentrations there is negligible glycolysis in liver parenchyma in both zones. The metabolic pattern in the perivenous cells is not glycolytic.