Triiodothyronine regulation of multiple rat hepatic genes: requirement for ongoing protein synthesis

We have examined the role of rapidly turning over proteins in the T3 regulation of multiple rat hepatic genes. T3 induction of the rapidly responsive mRNA-S14 was markedly inhibited by cycloheximide (1 mg/100 g BW) or emetine (3 mg/100 g) injected ip 30 min before T3 (mRNA-S14 concentration was only 35% of that in T3-treated controls 8.5 h after administration of either protein synthesis inhibitor, P less than 0.01). Cycloheximide exhibited a similar effect on each of five other more slowly responsive T3 regulated genes. When cycloheximide was given 10 h after T3, the expected T3-induced rise of mRNA-S7 activity was completely prevented, and for mRNA-S4 activity the anticipated rise was blunted to 40% of T3-treated control (P less than 0.05). Cycloheximide caused sharp declines in the activity of two other mRNAs, S6 and S8, which because of shorter lag times of response to T3, had already risen when the drug was given. Values for both these mRNAs returned to the baseline hypothyroid level within 6 h of injection of the drug and remained low for a further 8 h (P less than 0.05). The expected deinduction of mRNA-S10 by T3 was also markedly modified. T3 lowered this mRNA to 11% of the hypothyroid control after 8 h, whereas cycloheximide given 30 min before the hormone blunted this fall to only 72% of control (P less than 0.01). Thus there appeared to be a 70% reduction in the rate of T3 induced fall of mRNA-S10. We did not find that cycloheximide caused a generalized decrease in poly (A)+ RNA mass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of glucagon-stimulated cAMP accumulation and fatty acid oxidation by E-series prostaglandins in isolated rat hepatocytes

E-series prostaglandins have been shown to inhibit hepatic glucagon-stimulated glycogenolysis without inhibiting glycogenolysis stimulated by cAMP analogs. In the present studies, prostaglandin E2 and 16,16-dimethylprostaglandin E2 inhibited glucagon-stimulated cAMP accumulation in isolated rat hepatocytes by 25% and 46%, respectively, without affecting basal cAMP levels. Half-maximal inhibition of glucagon-stimulated cAMP accumulation occurred at approx. 10(-7) M 16,16-dimethylprostaglandin E2. 16,16-Dimethylprostaglandin E2 inhibited glucagon-stimulated palmitate oxidation in intact hepatocytes without affecting basal rates of palmitate oxidation. 16,16-Dimethylprostaglandin E2 had no effect on palmitate oxidation in a liver homogenate system. These studies demonstrate that prostaglandin E antagonizes the effects of glucagon on hepatic metabolism by inhibiting glucagon-stimulated cAMP accumulation.     

Thyroid hormone and circadian regulation of the binding activity of a liver-specific protein associated with the 5'-flanking region of the S14 gene

Recent studies have described a DNase I hypersensitive site in the 5'-flanking region of the rat hepatic S14 gene that is closely associated with its expression. A 111-base pair subfragment (-389 to -279) of this region interacts specifically in a gel shift assay with a protein present in hepatic nuclear protein extracts. This protein, designated P1, was not present in extracts of other tissues, even those in which the gene is expressed and hormonally regulated. The binding activity of P1 is exceedingly low in extracts from hypothyroid rats and is markedly increased by administration of thyroid hormone. However, the slow accumulation of P1 after thyroid hormone administration indicates that increased levels of P1 are not necessary for the acute hormonal induction of S14 gene expression. The level of P1 binding activity increases in the evening, synchronous with circadian variation of hepatic mRNA S14. Since neither P1 binding activity nor circadian variation in mRNA-S14 levels are observed in the other tissues expressing the S14 gene, P1 may function to modulate the circadian rhythm observed in hepatic S14 gene expression. DNase I footprinting analysis revealed that P1 binds to a defined nucleotide sequence, 5'-AAAAGAGCTATTGATTGCCTGCA-3', located between -310 and -288 in the S14 gene.     

Decreased cAMP responsiveness to glucagon in livers from ethynyl estradiol treated rats.

The effects of glucagon on the concentration and output of cAMP were studied in liver slices and in perfused livers from female rats and from animals treated with ethynyl estradiol (15 μg/kg daily for 14 days). The basal content of cAMP in liver slices, or of cAMP released into the perfusion medium in the absence of glucagon, was unaffected by prior treatment of the animal with estrogen. When glucagon was added to the medium, the concentration of cAMP in liver slices was 2.29 ± 0.32 and 1.10 ± 0.11 pmol cAMP/mg wet weight from control and ethynyl estradiol treated rats, respectively. When glucagon was added, the output of cAMP by perfused livers was maximal at 20 minutes with livers from either control or ethynyl estradiol treated rats. Output of cAMP by the perfused liver, when glucagon was added to the medium, was 8.76 ± 0.69 and 1.84 ± 0.08 nmol/g by livers from control and ethynyl estradiol treated rats, respectively. This effect was the same whether animals had been fasted for 12 hours previously, or were allowed free access to food until sacrifice. Clearly, as measured by cAMP accumulation, prior treatment of the rat with ethynyl estradiol reduced the sensitivity of the hepatic cAMP response to glucagon.     

Hepatic cyclic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the pre-weanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic beta 2 receptors by the selective antagonist ICI 118,551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of beta 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor RO20-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. RO20-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or beta 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.     

Angiotensin II inhibits hepatic cAMP accumulation induced by glucagon and epinephrine and their metabolic effects

Incubation of isolated hepatocytes containing normal Ca2+ levels with angiotensin II, vasopressin or A23187 caused significant inhibition of the cAMP response to glucagon. Angiotensin II also inhibited cAMP accumulation induced by either glucagon or epinephrine in Ca2+-depleted hepatocytes. When submaximal doses of hormone were employed such that cell cAMP was elevated only 3-4-fold (approximately 2 pmol cAMP/mg wet wt cells) inhibition by angiotensin II was correlated with a decrease in phosphorylase activation. The data demonstrate that inhibition of hepatic cAMP accumulation results in reduced metabolic responses to glucagon and epinephrine and do not support the contention that the hepatic actions of glucagon are independent of cAMP.     

The effects of time of day-specific resistance training on adaptations in skeletal muscle hypertrophy and muscle strength: A systematic review and meta-analysis

The present paper endeavored to elucidate the topic on the effects of morning versus evening resistance training on muscle strength and hypertrophy by conducting a systematic review and a meta-analysis of studies that examined time of day-specific resistance training. This systematic review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines with searches conducted through PubMed/MEDLINE, Scopus, and SPORTDiscus databases. The Downs and Black checklist was used for the assessment of the methodological quality of the included studies. Studies that examined the effects of time of day-specific resistance training (while equating all other training variables, such as training frequency and volume, between the groups) on muscle strength and/or muscle size were included in the present review. The random effects model was used for the meta-analysis. Meta-analyses explored (1) the differences in strength expression between morning and evening hours at baseline; (2) the differences in strength within the groups training in the morning and evening by using their post-intervention strength data from the morning and evening strength assessments; (3) the overall differences between the effects of morning and evening resistance training (with subgroup analyses conducted for studies that assessed strength in the morning hours and for the studies that assessed strength in the evening hours). Finally, a meta-analysis was also conducted for studies that assessed muscle hypertrophy. Eleven studies of moderate and good methodological quality were included in the present review. The primary findings of the review are as follows: (1) at baseline, a significant difference in strength between morning and evening is evident, with greater strength observed in the evening hours; (2) resistance training in the morning hours may increase strength assessed in the morning to similar levels as strength assessed in the evening; (3) training in the evening hours, however, maintains the general difference in strength across the day, with greater strength observed in the evening hours; (4) when comparing the effects between the groups training in the morning versus in the evening hours, increases in strength are similar in both groups, regardless of the time of day at which strength assessment is conducted; and (5) increases in muscle size are similar irrespective of the time of day at which the training is performed.     

Acute Glucagon Induces Postprandial Peripheral Insulin Resistance

Glucagon levels are often moderately elevated in diabetes. It is known that glucagon leads to a decrease in hepatic glutathione (GSH) synthesis that in turn is associated with decreased postprandial insulin sensitivity. Given that cAMP pathway controls GSH levels we tested whether insulin sensitivity decreases after intraportal (ipv) administration of a cAMP analog (DBcAMP), and investigated whether glucagon promotes insulin resistance through decreasing hepatic GSH levels.Insulin sensitivity was determined in fed male Sprague-Dawley rats using a modified euglycemic hyperinsulinemic clamp in the postprandial state upon ipv administration of DBcAMP as well as glucagon infusion. Glucagon effects on insulin sensitivity was assessed in the presence or absence of postprandial insulin sensitivity inhibition by administration of L-NMMA. Hepatic GSH and NO content and plasma levels of NO were measured after acute ipv glucagon infusion. Insulin sensitivity was assessed in the fed state and after ipv glucagon infusion in the presence of GSH-E. We founf that DBcAMP and glucagon produce a decrease of insulin sensitivity, in a dose-dependent manner. Glucagon-induced decrease of postprandial insulin sensitivity correlated with decreased hepatic GSH content and was restored by administration of GSH-E. Furthermore, inhibition of postprandial decrease of insulin sensitivity L-NMMA was not overcome by glucagon, but glucagon did not affect hepatic and plasma levels of NO. These results show that glucagon decreases postprandial insulin sensitivity through reducing hepatic GSH levels, an effect that is mimicked by increasing cAMP hepatic levels and requires physiological NO levels. These observations support the hypothesis that glucagon acts via adenylate cyclase to decrease hepatic GSH levels and induce insulin resistance. We suggest that the glucagon-cAMP-GSH axis is a potential therapeutic target to address insulin resistance in pathological conditions.     

Exercise elicits phase shifts and acute alterations of melatonin that vary with circadian phase

To examine the immediate phase-shifting effects of high-intensity exercise of a practical duration (1 h) on human circadian phase, five groups of healthy men 20-30 yr of age participated in studies involving no exercise or exposure to morning, afternoon, evening, or nocturnal exercise. Except during scheduled sleep/dark and exercise periods, subjects remained under modified constant routine conditions allowing a sleep period and including constant posture, knowledge of clock time, and exposure to dim light intensities averaging (+/-SD) 42 +/- 19 lx. The nocturnal onset of plasma melatonin secretion was used as a marker of circadian phase. A phase response curve was used to summarize the phase-shifting effects of exercise as a function of the timing of exercise. A significant effect of time of day on circadian phase shifts was observed (P < 0.004). Over the interval from the melatonin onset before exercise to the first onset after exercise, circadian phase was significantly advanced in the evening exercise group by 30 +/- 15 min (SE) compared with the phase delays observed in the no-exercise group (-25 +/- 14 min, P < 0.05). Phase shifts in response to evening exercise exposure were attenuated on the second day after exercise exposure and no longer significantly different from phase shifts observed in the absence of exercise. Unanticipated transient elevations of melatonin levels were observed in response to nocturnal exercise and in some evening exercise subjects. Taken together with the results from previous studies in humans and diurnal rodents, the current results suggest that 1) a longer duration of exercise exposure and/or repeated daily exposure to exercise may be necessary for reliable phase-shifting of the human circadian system and that 2) early evening exercise of high intensity may induce phase advances relevant for nonphotic entrainment of the human circadian system.     

Interactive effects of high stocking density and triiodothyronine-administration on aspects of the in vivo intermediary metabolism and in vitro hepatic response to catecholamine and pancreatic hormone stimulation in brook charr, Salvelinus fontinalis.

Brook charr (Salvelinus fontinalis) were maintained at one of two stocking densities (SD) (30 or 120 kg/m3) and fed either a control or a T3-supplemented (20 mg/kg) diet for 30 days in order to investigate possible interactive effects of SD and T3-administration on growth, feeding rate, food conversion efficiency, and hepatic and dark muscle enzyme activity. In addition, liver slices were incubated in vitro for 6 h with epinephrine, norepinephrine, isoproterenol, propranolol, insulin, glucagon, or somatostatin to evaluate possible SD-T3 interactive effects on hepatic responses to hormonal stimulation. Maintaining the fish at high SD appeared to increase the clearance rate of T3 from the T3-supplemented group. There was no clear evidence of SD-T3 interactive effects on growth rate, feeding rate, or food conversion efficiency, although T3-administration decreased food conversion efficiency, and high SD decreased growth and feeding rates. Of the hepatic enzymes studied, HOAD, malic enzyme, G6PDH, CS, PFK, HK, and GDH activities all showed changes suggestive of interactive SD-T3 effects. Although hepatic FBPase was stimulated by both high SD and T3-administration, there was no evidence of interactive SD-T3 effects. Dark muscle HOAD, CS, and PFK also showed SD-T3-related responses; dark muscle malic enzyme, G6PDH, HK, and GDH were unaffected by either altered SD or T3-administration. Prior treatment of the fish with T3 and high SD had significant effects on free fatty acid (ffa) release to the medium and on hepatic lipid content, but had no effect on the responses to the various endocrine agents used. Glucose release from liver slices of fish stocked at high density (both T3-supplemented and controls) was higher than that of the fish stocked at low density; with the exception of insulin and glucagon, glucose release was similar in all pre-treatment groups. The insulin- and glucagon-stimulated changes in glucose release seen in the fish fed non-supplemented diets were not found in the two groups of fish fed the T3-supplemented diets. High SD and/or T3-administration induced significant lowering of hepatic glycogen content, but there was no effect of pre-treatment on the response to any of the endocrine agents used. The data show a marked effect of SD on energy partitioning processes in brook charr and the animal's ability to respond to T3-stimulation, but provided no evidence of such effects on the liver response to the various agents used.     

Mice with a deletion in the gene for CCAAT/enhancer-binding protein beta have an attenuated response to cAMP and impaired carbohydrate metabolism

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.     

Studies of the interaction between glucagon and alpha-adrenergic agonists in the control of hepatic glucose output

alpha-Adrenergic stimulation of hepatocytes prevented, in a dose-dependent manner, the stimulation of [U-14C]lactate conversion to [14C]glucose by glucagon and exogenously added cAMP and Bt2cAMP. The inhibition was referable to an interaction with adrenergic receptors which resulted in a small decrease in hepatic cAMP levels. Low concentrations of epinephrine (10 nM) were able to inhibit phosphorylase activation and glucose output elicited by low doses of glucagon (5 X 10(-11) M to 2 X 10(-10) M). The ability of epinephrine (acting via alpha 1-adrenergic receptors), vasopressin, and angiotensin II to elicit calcium efflux was inhibited by glucagon, suggesting that intracellular redistributions of Ca2+ are importantly involved in the gluconeogenic process. It is proposed that vasopressin, angiotensin II, and catecholamines, acting primarily via alpha 1-adrenergic receptors, are responsible for inhibition of glucagon mediated stimulation of gluconeogenesis by altering subcellular calcium redistribution and decreasing cAMP levels.     

Effects of estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate on catalytic activity, amount, and rate of de novo synthesis of hepatic histidase.

The mechanisms by which estrogen, glucocorticoid, glucagon, and adenosine 3':5'-monophosphate (cAMP), regulators which participate in the postnatal development of rat liver histidase, elevate the catalytic activity of this enzyme have been explored. A monospecific antibody against homogeneously purified preparations of rat liver histidase has been elaborated in the goat. Employing this antibody in immunotitration experiments, it has been demonstrated that the elevations of hepatic histidase activity elicited by administration in vivo of estradiol-17beta, cortisol acetate, glucagon, and N6,O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cAMP) are paralleled, in each instance, by equivalent increments in immunoprecipitable histidase protein. Following administration of each of the three hormones and dibutyryl cAMP, rates of [14C]leucine incorporation in vivo into rat liver histidase, isolated by immunoprecipitation, relative to incorporation rates into total soluble hepatic protein, increase in magnitudes which are comparable to increases in enzyme amount and catalytic activity. It is thus inferred that estrogen, glucocorticoids, and glucagon, via cAMP, each regulate rat liver histidase development at specific postnatal stages by inducing increases in histidase biosynthetic rates.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.