Vaddanahally T. Maddaiah

Growth hormone and liver mitochondria: Effects on cytochromes and some enzymes

Abstract The effects of hypophysectomy and subsequent growth hormone treatment on liver mitochondrial levels of cytochromes, flavoprotein, cytochrome oxidase and NADH oxidase were studied in rats. Hypophysectomy lowered cytochrome a , a 3 , b , c , and c 1 , concentrations and cytochrome oxidase activity. On the other hand, flavoprotein concentration and NADH oxidase activity were increased. Administration of either human or bovine growth hormone restored the cytochrome levels. Cytochrome oxidase activity was also increased but did not reach its original level. Only the bovine hormone exerted a lowering effect on flavoprotein and NADH oxidase activities. These and other results suggest that growth hormone influences mitochondrial electron transport.

Growth hormone and liver mitochondria: effects on phospholipid composition and fatty acyl distribution.

Effects of growth hormone on phospholipid composition and fatty acyl distribution were studied in liver mitochondria of hypophysectomized rats. After hypophysectomy, only cardiolipin showed a 25% decrease. Its fatty acyl distribution, which consisted mainly of linoleic acid (55–60%) and oleic acid (20%), was unchanged. In phosphatidylcholine and phosphatidylethanolamine fractions the contents of docosahexaenoic and arachidonic acids were decreased with a concomitant increase in linoleic acid content. These changes could be accounted for by small but significant decreases in the activities of Δ9-desaturase (sucrose-induced), Δ5-desaturase and mitochondrial elongation enzymes. The activities of Δ6-desaturase NADH cytochrome b5 ferri-reductase, cytochrome b5, NADH cytochrome c reductase and microsomal elongation enzymes remained virtually unchanged. Injection of bovine growth hormone daily for seven days restored cardiolipin and fatty acyl distribution and the enzyme activities. From these and other results, we conclude that growth hormone-dependent increase of respiratory activity of liver mitochondria may be partly mediated by the hormonal effects on membrane lipid distribution.

Hormones and liver mitochondria: Effects of growth hormone and thyroxine on respiration, fluorescence of 1-anilino-8-naphthalene sulfonate and enzyme activities of complex I and II of submitochondrial particles

Abstract The effects of hypophysectomy and subsequent administration of bovine growth hormone (0.1 IU/100 g body wt) and l -thyroxine (5 μg/100 g body wt) on respiration, energization-dependent fluorescence of 1-anilino-8-naphthalene sulfonate, NADH dehydrogenase, energy-independent nicotinamide nucleotide transhydrogenase, and succinate dehydrogenase activities were investigated in submitochondrial particles of rat liver. Hormones were injected daily for 7 days. Hypophysectomy decreased the respiratory rate with NADH or succinate and the activities of the three enzymes. Administration of growth hormone increased the respiration but showed selectivity toward NADH. Thyroxine increased the respiration more than growth hormone did with both substrates. Growth hormone increased the activities of NADH dehydrogenase and transhydrogenase whereas thyroxine increased the activity of only succinate dehydrogenase. After growth hormone treatment transhydrogenase activity was increased to about three times that of controls which may have significance in some processes mediated either directly or permissively by growth hormone. When both hormones were injected together, there was a significant decrease in the thyroxine-dependent rise in respiration on succinate as well as the growth hormone-dependent rise in enzyme activities. Fluorescence yield of 1-anilino-8-naphthalene sulfonate in unenergized submitochondrial particles remained unchanged independent of the hormonal status. Energization with succinate or NADH increased the fluorescence yield by about 2–20 times. Several parameters of energizationdependent fluorescence were decreased after hypophysectomy. In restoring these parameters, growth hormone and thyroxine showed specificity toward the energization substrate NADH and succinate, respectively. From the present results we conclude that (a) growth hormone and thyroxine regulate mitochondrial activity by affecting different segments of the respiratory chain, namely Complex I and Complex II, respectively, and (b) growth hormone and thyroxine exert moderating effects on one another.

Distribution of 3H-acetyl human growth hormone in subcellular fractions of rat liver

Abstract 3 H-Acetyl human growth hormone ( 3 H-HGH) (∼60 μg) was injected intravenously into hypophysectomized rats. Radioactivity appeared in all the subcellular fractions of liver as early as 2 minutes after the injection. Until 10 minutes postinjection virtually all of the tissue radioactivity was precipitable with trichloroacetic acid, whereas at 20 and 40 minutes after injection all of the radioactivity was soluble in trichloroacetic acid. Liver uptake (cpm/mg protein) peaked at about 20 minutes postinjection. The appearance of radioactivity in the crude nuclear fraction closely followed that of the whole homogenate. Radioactivity in the microsomal and mitochlondrial fractions was highest at the early time periods (up to 10 minutes) and then declined progressively. The radioactivity in the final supernatant or cytoplasmic fraction increased slowly, peaked at 20 minutes, and remained the highest. Pretreatment with unlabeled or 14 C-labeled HGH significantly reduced the mitochondrial and microsomal uptake of 3 H-HGH. In contrast with these results, when 3 H-HGH was added in vitro just before homogenization and fractionation of liver, most of the radioactivity (77%) appeared in the cytoplasmic fraction. These results are interpreted to mean that the appearance of 3 H-HGH in the particulate fractions at these early time periods may have functional significance, and that important initial binding sites of growth hormone in the liver cell may be mitochondria and microsomes.

Effects of growth hormone on respiration and ATPase activity of rat liver and heart mitochondria.

Abstract The effects of hypophysectomy on respiration and ATPase activity of mitochondria of rat heart and liver were investigated. Hypophysectomy decreased significantly the rate of State 3 respiration and ATPase activity of liver but not of heart mitochondria. Heart cytochrome oxidase activity was also not affected. ADP:O and respiratory control ratios were not affected. Administration of bovine growth hormone (bGH) to hypophysectomized rats partially restored the rate of respiration and ATPase activity of liver mitochondria. However, hypophysectomy decreased heart mitochondrial protein synthesis as measured by in vivo and in vitro incorporation of radioactive leucine, but did not affect the in vitro uptake of the amino acid. Administration of bGH restored the incorporation capacity to the normal level. Similar effects on liver mitochondrial protein synthesis have already been reported. These data suggest that the effects of growth hormone on mitochondrial function may vary with the two tissues.

Effects of ammonium chloride, salicylate, and carnitine on palmitic acid oxidation in rat liver slices.

ABSTRACT: To explore the possible association of hyperlipidemia with hyperammonemia and aspirin ingestion, the effects of NH4 +, salicylate, and carnitine on the oxidation of [l-14C]palmitic acid to acid-soluble products (ASP) and to CO2 were investigated in rat liver slices, DL-carnitine (5 mM) increased total oxidation (ASP + CO2) more than oxidation to CO2. KCN (1.5 mM) inhibited more than 90% of the oxidation. NH4Cl inhibited the oxidation that reached a maximum at about 40 mM, but the inhibition of oxidation to CO2 (63%) was larger than that of total oxidation (30%). Carnitine did not influence NH4 + inhibition, which is consistent with the results reported for isolated mitochondria. Salicylate effects depended on salicylate concentration as well as on the presence of carnitine. In the absence of carnitine, inhibition of total oxidation reached 90% at 3 mM salicylate but that of oxidation to CO2 reached 50%. Velocity calculated at saturating palmitic acid concentration for total oxidation was slightly increased by 0.75 mM salicylate, but the increase for oxidation to CO2 was larger. At 3 mM salicylate, velocity at saturating palmitic acid concentration for the oxidation was decreased, but the decrease for oxidation to CO2 was smaller than for total oxidation. Carnitine partially relieved the inhibition of total oxidation and further increased the formation of CO2. The combination of 20 mM NH4Cl and 0.75 mM salicylate inhibited total oxidation, which was more than additive of the individual effects, and carnitine partially relieved the inhibition. It is concluded that NH4 + exerted a stronger inhibition of oxidation to CO2 than of oxidation to ASP, whereas salicylate strongly inhibited the oxidation to ASP but increased the oxidation to CO2 by uncoupling mitochondrial oxidative phosphorylation. Therefore, hyperammonemia and aspirin ingestion can inhibit fatty acid oxidation and mitochondrial metabolism that could lead to the pathophysiology seen in some childhood diseases such as Reyes syndrome. Carnitine therapy might offer some benefits.

Growth hormone and rat liver mitochondria: effects on urea cycle enzymes.

Abstract Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.

Growth hormone and liver mitochondria effect on morphology and protein turnover

Abstract Electron micrographs of hepatocytes from hypophysectomized rats when compared with those of normal rats showed increased number or crowding of mitochondria per unit cytoplasmic volume with decreased endoplasmic reticulum, decreased mitochondrial matrix density, and the presence of partitioned mitochondria. After hypophysectomy, the recovery of mitochondrial proteins per gram of wet liver, by centrifugation, was increased. The mean half-life value for the turnover of total mitochondrial proteins was doubled when measured by injection of either l -[4,5- 3 H]leucine or l -[guanidino- 14 C]arginine. These changes were reversed towards normal by subsequent treatment with growth hormone. These and other results suggest a role for GH in the maintenance of a normal population of properly functioning mitochondria in liver.

Inactivation of microsomal glucose-6-phosphatase by pyridoxal-5′-phosphate

Summary Glucose-6-phosphatase activity was inactivated when liver microsomes were incubated with pyridoxal-5′-phosphate at 30° in 0.05 M Tris-buffer, pH 7.0. Addition of glucose-6-phosphate completely protected the enzyme from inactivation. Disappearance of enzymatic activity was exponential with time and the inactivation was proportional to pyridoxal-5′-phosphate concentration below 25 mM.

Influence of growth hormone and thyroxine on thermotropic effects of respiration and 1-anilino-8-naphthalene sulfonate fluorescence and on lipid composition of cardiac membranes

The effects of hypophysectomy and subsequent administration of growth hormone and/or L-thyroxine on thermotropic properties of State 3 respiration (ADP-induced), cholesterol, phospholipid and fatty acid composition of phospholipid fraction were examined in myocardial mitochondria of rats. Temperature-dependence of 1-anilino-8-naphthalene sulfonate fluorescence was determined in vesicles prepared from lipids of heart mitochondria. Transition temperature obtained from the Arrhenius plots of respiration occurred at 21 and 24 degrees C for heart mitochondria of normal and hypophysectomized rats, respectively. Most notably, after hypophysectomy the rate of respiration was lower below 24 degrees C, but was progressively higher above that temperature when compared to normal rats. The energy of activation was 148 and 36% larger below and above the transition temperature, respectively. Growth hormone restored almost completely the energy of activation and respiratory rates to normal levels. Administration of L-thyroxine, with or without growth hormone, did not significantly change the rate of respiration but decreased the transition temperature to 17.7-17.9 degrees C. Lipid and phospholipid content, as well as percent distribution of phospholipids and their fatty acid composition were not statistically different among the different groups of rats. Only cholesterol content was increased after hypophysectomy. Administration of growth hormone and thyroxine did not significantly change the total unsaturation index of fatty acids, but growth hormone increased the content of arachidonic acid (20 : 4) by 70% but decreased the docosahexaenoic acid (22 : 6) three times which may have a beneficial effect on mitochondrial membranes. These and other results suggest that hormones exert different effects on subcellular organelles in different tissues, like heart and liver.