Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Effects of chloroquine on lysosomes and endocytosis by liver cells in vivo.

1. Chloroquine accumulation in rat liver after a single and repeated drug administration and lysosomal changes resembling some symptoms of lysosomal storage diseases were observed. 2. Repeated chloroquine treatment of rats resulted in increased activity of liver lysosomal enzymes acid phosphatase and beta-galactosidase and a significant enhancement of the activities of cathepsin D and cysteine proteinases were found. 3. No changes in the activity of liver macrophages (as assessed by the colloidal carbon clearance test) or in fluid-phase endocytosis of the marker 125I-polyvinyl-pyrrolidone by hepatocytes in vivo were found.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Effects of lidocaine on the lysosomes of cultured skin fibroblasts

Exposure of cultured human skin fibroblasts to lidocaine at concentrations over 3 mM for more than 6 hours caused dramatic vacuolation of the cells. The vacuoles were formed by expansion of lysosomes. The most likely explanation for this phenomenon is that lidocaine accumulated in the lysosomes by an “amine trapping” mechanism similar to that proposed for chloroquine.     

The influence of lysosomes on glycogen metabolism.

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6'-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.     

Effects of polycyclic aromatic hydrocarbons on molluscan lysosomes and endoplasmic reticulum

Summary Some effects of two isomeric polycyclic aromatic hydrocarbons, anthracene and phenanthrene, on the fine structure and cytochemistry of digestive cells in the marine musselMytilus edulis have been investigated. The cytochemical results show that increasing concentrations of anthracene and phenanthrene have different effects on the acid labilization time for laten -glucuronidase which is used to measure the stability of lysosomal membranes. At the ultrastructural level the limiting membranes of secondary lysosomes appear multilayered, with discontinuities and overlaps. Under the conditions of the experiment, only phenanthrene produces changes in this configuration. Both macroautophagic and microautophagic processes occur in the control and hydrocarbon treatments, and complementary data from other studies indicate that autophagic processes are enhanced by polycyclic aromatic hydrocarbons. Phenanthrene also causes proliferation of the smooth endoplasmic reticulum in the digestive cells, although cytochemical measurements of smooth endoplasmic reticulum-associated NADPH-ferrihemoprotein reductase show that anthracene stimulates activity over a greater range of concentrations than phenanthrene. The different effects of the two isomers is taken as evidence that the molecular configuration of the compound determines its reactivity with membranes and its subsequent effect on the physiology of the cells.     

Effects of ten steroids on acridine orange uptake and beta-N-acetylglucosaminidase levels in rat liver lysosomes.

Ten steroids have been compared for their ability to modify the rate of uptake of acridine orange by rat liver and by rat liver lysosomes in vivo. The short-term effects of the ten steroids on the specific activity of a lysosomal enzyme, beta-N-acetylglucosaminidase, were also compared. Five of the ten steroids were administered as tritium-labelled compounds and the concentration of steroids or metabolites was measured in rat liver and liver lysosomes at 2.5h and 3.75h after administration. Cortisone acetate, etiocholanolone (5-beta-androstan-3-alpha-01-17-one) and testosterone accelerate and increase the uptake of acridine orange by rat liver lysosomes. Deoxycorticosterone, corticosterone, triamcinolone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione), estradiol-17-beta and progesterone appear to inhibit the uptake of acridine orange by rat liver lysosomes at 2.5 hours. Cortisol and dexamethasone (9-alpha-fluoro-11-beta, 17, 21-trihydroxy-16-alpha-methyl-pregna-1, 4-diene-3, 20-dione) had little effect. All steroids with the exception of etiocholanolone and deoxycorticosterone increase with the specific activity of beta-N-acetylglucosaminidase in the lysosomal fraction at 2.5h. None of the effects at 2.5h are due to lowered protein levels. Lysosomal concentrations of radioactivity following the administration of tritiated steroids were greated for the glucocorticoids, corticosterone and cortisol. Estradiol-17-beta, progesterone and testosterone showed much lower concentrations of radioactivity in isolated lysosomes. Most of the lysosomal radioactivity (73-96%) was associated with the soluble fraction of the disrupted lysosomes.     

Studies on the involvement of lysosomes in estrogen action, I. Isolation and enzymatic properties of pig endometrial lysosomes.

Pig endometrium cells, collected by curettage and homogenized in an all-glass Potter Elvehjem homogenizer, gave a considerably higher yield of intact mitochondria and lysosomes than homogenates of whole uterus obtained with the Ultraturrax or the Parr bomb. After homogenization of the cells and subfractionation in the presence of Mg2, mitochondria and lysosomes equilibrated at the same modal density in isopycnic centrifugation. Homogenization and subfractionation in buffers devoid of divalent cations and containing EDTA resulted in a decrease in the buoyant density of mitochondria, allowing for a separation from lysosomes. The pH optima and the specific activities of two mitochondrial enzymes and eight hydrolyases used as marker enzymes were determined. The morphological characteristics of fractions were established by electron microscopy. Preliminary results indicate an involvement of lysosomes in steroid metabolism rather than in steroid and receptor translocation into the nucleus.     

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.