Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver

S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.     

Possible growth hormone regulation of rat liver glutamine synthetase activity.

Hypophysectomy results in a marked decrease in glutamine synthetase activity of rat liver homogenates. The enzyme is affected to a lesser extent in the kidneys and is not influenced in the brain. Bovine growth hormone treatment of hypophysectomized rats elevates the diminished glutamine synthetase activity in liver and kidneys but has no effect on the brain enzyme. Adrenalectomy also results in decreased liver glutamine synthetase activity although less than the decline seen with hypophysectomy. Cortisol treatment has no effect on glutamine synthetase activity in hypophysectomized animals. Our results suggest that growth hormone is involved in the regulation of liver glutamine synthetase activity. This regulation may be important in the utilization of α-amino nitrogen from glucogenic amino acids associated with growth hormone enhanced glucose production.     

Paradoxical sleep deprivation increases glutamine synthetase in rat brain.

The modifications of glutamine synthetase (GS) level, an enzyme mainly located in astrocytes, were investigated in rat after paradoxical sleep deprivation and during recovery. An immunotitration method was used to evaluate the relative level of GS in brain tissue. At the end of a 24 hrs. paradoxical sleep deprivation, a significant increase in GS level was observed both in the frontoparietal cortex and the locus coeruleus area. 4 hrs. later, during recovery, the GS level returned to control level in the cortex but was lower in the locus coeruleus area.     

Heterogeneous distribution of glutamine synthetase during rat liver development

Two days before birth, immunohistochemical detection of glutamine synthetase already reveals a heterogeneous distribution pattern related to the vascular architecture of the liver. Only a small number of hepatocytes in the vicinity of the efferent venules show relatively high staining intensity. Before that age, only megakaryocytes show intense staining, while liver parenchyma is only faintly stained. The developmental profile of glutamine synthetase activity shows two periods of increasing enzyme activity: one in the perinatal period and one in the second and third postnatal week. Both periods are correlated with high levels of circulating corticosteroid hormones. Although the relative number of intensely stained hepatocytes increases during the first rise in enzyme activity, the second rise is correlated with a decreasing number of glutamine synthetase-positive hepatocytes which, however, show a considerable increase in staining intensity. Carbamoylphosphate synthetase shows a homogeneous distribution pattern in the perinatal period. Conditions that lead during development to a relatively high level of glutamine synthetase expression in the pericentral compartment apparently originate before the appearance of conditions that lead to a relatively high level of carbamoylphosphate synthetase gene expression in the periportal compartment. Our results indicate that downstream localization of glutamine synthetase in liver acinus is essential from the perinatal period onwards, whereas reciprocal distribution of glutamine synthetase and carbamoylphosphate synthetase gene expression (that is found in adult rat liver) is not.     

Over-expression of cytosolic glutamine synthetase increases photosynthesis and growth at low nitrogen concentrations.

Nitrogen, which is a major limiting nutrient for plant growth, is assimilated as ammonium by the concerted action of glutamine synthetase (GS) and glutamate synthase (GOGAT). GS catalyses the critical incorporation of inorganic ammonium into the amino acid glutamine. Two types of GS isozymes, located in the cytosol (GS1) and in the chloroplast (GS2) have been identified in plants. Tobacco (Nicotiana tabacum) transformants, over-expressing GS1 driven by the constitutive CaMV 35S promoter were analysed. GS in leaves of GS-5 and GS-8 plants was up-regulated, at the level of RNA and proteins. These transgenic plants had six times higher leaf GS activity than controls. Under optimum nitrogen fertilization conditions there was no effect of GS over-expression on photosynthesis or growth. However, under nitrogen starvation the GS transgenics had c. 70% higher shoot and c. 100% greater root dry weight as well as 50% more leaf area than low nitrogen controls. This was achieved by the maintenance of photosynthesis at rates indistinguishable from plants under high nitrogen, while photosynthesis in control plants was inhibited by 40-50% by nitrogen deprivation. It was demonstrated that manipulation of GS activity has the potential to maintain crop photosynthetic productivity while reducing nitrogen fertilization and the concomitant pollution.     

Regulation of flux through glutaminase and glutamine synthetase in isolated perfused rat liver

1. Glutaminase and glutamine synthetase are simultaneously active in the intact liver, resulting in an energy consuming cycling of glutamine at a rate up to 0.2 mumol per g per min. 2. An increase in portal glutamine concentration was followed by an increased flux through glutaminase, but flux through glutamine synthetase remained unchanged. Glutaminase flux was also increased by ammonium ions or glucagon; these effects were additive. 3. Glutamine synthetase flux was increased by ammonium ions, but this activation was partly overcome by increasing portal glutamine concentrations. Glutamine synthetase flux was slightly increased by glucagon at portal glutamine concentrations of about 0.2-0.3 mM, but was strongly inhibited above 0.6 mMs. 4. During experimental metabolic acidosis there was an increased net release of glutamine by the liver, being due to opposing changes of flux through glutaminase and glutamine synthetase. Conversely, an increased glutamine uptake by the liver during metabolic alkalosis was observed due to an inhibition of glutamine synthetase and an activation of glutaminase. However, the two enzyme activities respond differently depending on whether glucagon or ammonium ions are present.     

Effect of dietary proteins on the turnover of rat liver argininosuccinate synthetase.

The rates of synthesis and degradation of arginosuccinate synthetase in rat liver under various dietary conditions were determined. The relative rate of the enzyme synthesis in the livers of rats fed on 70% casein diet was 4.0 times greater than that for rats fed on 5% casein diet. The rate constants of degradation (Kd of argininosuccinate synthetase were estimated to be 0.15 and 0.16 day-1 under 70% and 5% casein feeding, respectively. When the dietary conditions were changed acutely from 70% to 5% casein diet or vice versa, the rates of the enzyme synthesis decreased or increased, respectively, and the rates of enzyme degradation were also affected. The change from 5% to 70% casein diet caused a transient decrease in the rate of degradation. After the enzyme activity had achieved a new steady-state level, the enzyme degradation proceeded at the normal steady rate. On the other hand, the change from 70% to 5% casein diet caused a transient increase in the rate of degradation. Thus, the only factor regulating the amount of enzyme in rat liver is the rate of enzyme synthesis under the steady-state conditions. However, the rates of both enzyme synthesis and degradation are involved in the regulation of the amount of enzyme during dietary transition.     

Haploinsufficiency of glutamine synthetase increases susceptibility to experimental febrile seizures

Glutamine synthetase (GS) is a pivotal glial enzyme in the glutamate–glutamine cycle. GS is important in maintaining low extracellular glutamate concentrations and is downregulated in the hippocampus of temporal lobe epilepsy patients with mesial–temporal sclerosis, an epilepsy syndrome that is frequently associated with early life febrile seizures (FS). Human congenital loss of GS activity has been shown to result in brain malformations, seizures and death within days after birth. Recently, we showed that GS knockout mice die during embryonic development and that haploinsufficient GS mice have no obvious abnormalities or behavioral seizures. In the present study, we investigated whether reduced expression/activity of GS in haploinsufficient GS mice increased the susceptibility to experimentally induced FS. FS were elicited by warm-air-induced hyperthermia in 14-day-old mice and resulted in seizures in most animals. FS susceptibility was measured as latencies to four behavioral FS characteristics. Our phenotypic data show that haploinsufficient mice are more susceptible to experimentally induced FS ( P  < 0.005) than littermate controls. Haploinsufficient animals did not differ from controls in hippocampal amino acid content, structure (Nissl and calbindin), glial properties ( glial fibrillary acidic protein and vimentin) or expression of other components of the glutamate–glutamine cycle (excitatory amino acid transporter-2 and vesicular glutamate transporter-1). Thus, we identified GS as a FS susceptibility gene. GS activity-disrupting mutations have been described in the human population, but heterozygote mutations were not clearly associated with seizures or epilepsy. Our results indicate that individuals with reduced GS activity may have reduced FS seizure thresholds. Genetic association studies will be required to test this hypothesis.     

Characterization of glutamine synthetase from avian liver mitochondria

1. Glutamine synthetase has been purified to homogeneity from chicken liver mitochondria. 2. The native enzyme is an octamer composed of identical subunits with monomeric mol. wt of 42,000 dalton. 3. Apparent Kms for NH4+, ATP and glutamate were 0.5, 0.9 and 6 mM, respectively. D-Glutamate and L-alpha-hydroxyglutarate were utilized as substrates with activities approx. 40% those obtained with glutamate. Of several nucleotides tested, none were effective replacements for ATP. 4. Heavy metal ions were inhibitory as were Mn2+, Ca2+ and lanthanide ions. 5. Despite its different subcellular localization and physiological function, avian glutamine synthetase is markedly similar to the weakly-bound microsomal rat liver enzyme with respect to a number of physical and chemical properties.     

Growth hormone increases phosphoinositide turnover in rat adipocytes that are sensitive to the insulin-like action of the hormone

Summary The effect of pituitary human growth hormone (hGH) on the ³²P-labelling of phosphoinositides and phosphatidic acid was studied in noradrenaline-stimulated rat adipocytes which were either responsive or non-responsive to the antilipolytic (insulin-like) effect of hGH. In cells responsive to the insulin-like effect of hGH, hormone treatment resulted in a marked increase of the ³²P-labelling of phosphatidic acid and phosphatidyl inositol in the plasma membrane, high density microsomal, and low density microsomal fractions. The increased ³²P-labelling most likely reflects an activation of phosphoinositide phospholipase C.Abbreviations IP3 Myo-Inositol 1,4,5-Triphosphate - BSA Bovine Serum Albumin - HEPES N-2Hydroxyethylpiperazine-N-2-Ethanesulphonic Acid - hGH Human Pituitary Growth Hormone - LPC Lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - LPI Lysophosphatidylinositol - PA Phosphatidic Acid - PC Phosphatidylcholine - PE Phosphatidylethanolamine - PI Phosphatidylinositol - PI Phosphatidylinositol-4-Phosphate - PIPZ Phosphatidylinositol-4,5-Diphosphate - PS Phosphatidylserine     

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).     

Selection of a rat glutamine synthetase cDNA clone

We have selected a glutamine synthetase clone (pGSRK-1) from a rat kidney cDNA library. A partial restriction map has been constructed for the 1.65 kilobase pair (kbp) glutamine synthetase cDNA. Northern hybridization analysis indicates that 1) GS-specific RNA increases many-fold during adipocyte differentiation and 2) dexamethasone increases and insulin decreases GS-specific RNA in 3T3-L1 adipocytes.