Calcitonin stimulates glycogenolysis in the liver of fasted rats

The effect of calcitonin (CT) on glycogenolysis in the liver was investigated in fasted rats. The fasting produced a marked decrease in the hepatic glycogen content. Thyroparathyroidectomy (TPTX) significantly prevented the decline in hepatic glycogen by fasting as compared with sham operation. This prevention by TPTX was clearly relieved by the subcutaneous administration of CT (80 MRC mU/100 g body weight). The appreciable effect of the hormone was also observed at the dose of 20 and 40 MRC mU CT/100 g body weight. The present results suggest that CT plays a physiological role in the stimulation of hepatic glycogenolysis after fasting in rats.     

Effects of glucagon-like peptide 1 on the kinetics of glycogen synthase a in hepatocytes from normal and diabetic rats

Glucagon-like peptide 1(7-36)amide (GLP-1) is currently under investigation as a possible tool in the treatment of non-insulin-dependent diabetes mellitus. In addition to enhancing nutrient-stimulated insulin release, the peptide also favors glycogen synthesis and glucose use in liver, muscle, and adipose tissue. GLP-1 also activates glycogen synthase a in hepatocytes from both normal and diabetic rats. In the present study, the kinetic aspects of such an activation were investigated in hepatocytes from normal rats and from animals rendered diabetic induced by injection of streptozotocin, either in the adult age (insulin-dependent diabetes mellitus model) or in days 1 or 5 after birth (non-insulin-dependent diabetes mellitus models). GLP-1 increased, in a dose-dependent manner, glycogen synthase a activity in the hepatocytes from all groups studied. The activation of the enzyme reached a steady state within 1 min exposure to GLP-1, which, at 10(-12) M, caused a half-maximal activation. When comparing fed vs. overnight-starved normal rats, a somewhat lower basal activity of glycogen synthase a in fasted animals (P < 0.05) coincided with a greater relative increment in reaction velocity in response to GLP-1. The basal activity of glycogen synthase a and the relative extent of its inhibition by glucagon or activation by insulin and GLP-1 were modulated by the extracellular concentration of D-glucose. The activation of glycogen synthase a by either insulin or GLP-1 resulted not solely in an increase in maximal velocity but also in a decrease in affinity of the enzyme for uridine diphosphate-glucose; in diabetic animals, the capacity of insulin or GLP-1 to increase the maximal velocity and Michaelis-Menten constant were less marked than in normal rats. In conclusion, this study indicates that the GLP-1-induced activation of glycogen synthase a displays attributes of rapidity, sensitivity, and nutritional dependency that are well suited for both participation in the physiological regulation of enzyme activity and therapeutic purpose.     

Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats

Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute s.c. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [3H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose-dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Histochemical, biochemical and ultrastructural studies on the liver of fasted rats

The subjection of rats with body weight 150 +/- 10 g to complete starvation for a period of four days leads to a diminution of total protein, total lipids, blood sugar, body weight and liver weight. Lipid dystrophy develops in the liver, as well as deposition of lipofuscin-like pigment and atrophy. Lipid dystrophy and desposition of pigment increase during the first three days and abruptly decrease during the fourth. Atrophy is a progressive process. The delineation of three phases in the atrophic - dystrophic process is possible with the application of histological, enzyme-histochemical, morphometric, biochemical and electron microscopic methods: Phase I (first 24 hours) - a common adaptive phase. It engages both the liver, which must utilize the increased nutrients from the organism depots and the homeostatic mechanisms of the organism as a whole. Phase II - (second and third 24 hours) - alterative-restorative, manifested markedly at the liver parenchimal level and especially by autophagic lysosome function. Phase III - (fourth 24 hours) - alterative. Exhaustion of adaptive-restorative liver process (and the hepatocyte in particular), and the organism as a whole as well.     

Mutations in the liver glycogen synthase gene in children with hypoglycemia due to glycogen storage disease type 0

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology. Recent studies have shown that genetic factors and both cellular and humoral immunological abnormalities are important in the pathogenesis of PSC. The most prominent autoantibodies in PSC are anti-neutrophil cytoplasmic antibodies (ANCA). The autoepitopes of ANCA in PSC are not well defined. The aim of this study was to identify corresponding ANCA autoantigens in patients with PSC. A biochemical approach with enrichment and partial purification of soluble neutrophil proteins, detection of autoantibodies by Western blot and partial amino acid sequencing were used. Two new autoantigen/autoantibody systems in patients with PSC were detected: catalase and alpha-enolase. The presence of catalase autoantibodies in 9/15 (60%) and alpha-enolase autoantibodies in 4/15 (27%) was confirmed by ELISA and Western blot. Furthermore, we showed immunoreactions of PSC sera with human biliary epithelial cells, showed the reduction of fluorescence in anti-catalase absorption experiments and observed partial co-localization of anti-catalase antibodies and PSC sera in double-staining experiments on biliary epithelial cells. The anti-catalase antibody-positive PSC patients had a more severe course of disease with a significantly higher alkaline phosphatase compared with the anti-catalase-negative PSC patients (P < 0.06). All ulcerative colitis control sera were anti-catalase antibody-negative. The identified antigens catalase and alpha-enolase can partly explain the ANCA fluorescence on ethanol-fixed and formaldehyde-fixed granulocytes in patients with PSC. Catalase is an important anti-oxidant enzyme and prevents cell damage from highly reactive oxygen-derived free radicals. Catalase autoantibodies might play a pathogenic role in patients with PSC. Our findings support the hypothesis that oxidative stress is one of the pathogenic mechanisms in patients with PSC.     

Effect of the sulfonylurea glyburide on glycogen synthase activity in alloxan-induced diabetic rat adipocytes

1. The effect of glyburide (glibenclamide) treatment in vivo on the adipose tissue glycogen synthase activity of type II diabetic rats has been studied. 2. Three week treatment of diabetic animals with glyburide (5 mg/kg orally, in saline) increased adipose glycogen synthase activity and decreased blood glucose levels. 3. These results demonstrate that the sulfonylurea glyburide is capable of exerting direct insulin-like effect on adipose glycogen-synthase activity of type II diabetic rats in vivo.     

Isolation and characterization of glycogen synthase in Dictyostelium discoideum

We have partially purified the protein and isolated the glcS gene for glycogen synthase in Dictyostelium. glcS mRNA is present throughout development and is the product of a single gene coding for 775 amino acids, with a predicted molecular mass of 87 kD. The sequence is highly similar to glycogen synthase from human muscle, yeast, and rat liver, diverging significantly only at the amino and carboxy termini. Phosphorylation and UDPG binding sites are conserved, with K(m) values for UDPG being comparable to those determined for other organisms, but in vitro phosphorylation failing to convert between the G6P-dependent (D) and -independent (I) forms. Enzyme activity is relatively constant throughout the life cycle: the I form of the enzyme isolates with the soluble fraction in amoebae, switches to the D form, becomes pellet-associated during early development, and finally reverts during late development to the I form, which again localizes to the soluble fraction. Deletion analysis of the promoter reveals a GC-rich element which, when deleted, abolishes expression of glcS.     

Spatial learning deficit and reduced hippocampal ChAT activity in rats after an ICV injection of streptozotocin

ICV injections of streptozotocin (STREP) lower the glucose utilization of the brain and affect the cholinergic system. The present study was designed to evaluate whether STREP-treated rats have an impaired spatial discrimination performance in the Morris spatial navigation task. Performance in this task is sensitive to treatment with cholinergic antagonists. In contrast to young rats, middle-aged STREP-treated rats tended to have an impaired spatial discrimination performance in the Morris task at the end of training. In middle-aged STREP-treated rats, but not in control rats, spatial discrimination performance was associated with hippocampal choline acetyltransferase (ChAT) activity. The correlation between spatial discrimination performance in the Morris task and the decrease in hippocampal ChAT activity resembles the relation between cognitive and biochemical changes observed in Alzheimer's disease. Our findings suggest that STREP treatment of middle-aged rats may provide a relevant model for dementia.     

Intracellular glucose metabolism after long term metabolic control with glyburide: improved glucose oxidation with unchanged glycogen synthase activity

To determine whether improved metabolic control with long term glyburide treatment alters intracellular glucose metabolism independent of effects on glucose uptake (GU), we studied eight obese patients with noninsulin-dependent diabetes mellitus before and 7 months after glyburide therapy. Indirect calorimetry and skeletal muscle biopsies were performed in the basal state and during 300 pmol/m2.min insulin infusions, with glucose turnover rates determined by [3-3H]glucose turnover. During the glucose clamps, rates of GU were matched before and after treatment using equivalent hyperinsulinemia and variable levels of hyperglycemia. After glyburide treatment, rates of GU were decreased in the basal state [4.16 +/- 0.57 vs. 3.29 +/- 0.37 mg/kg fat free mass (FFM)/min; P < 0.05], but similar during glucose clamps (11.53 +/- 1.42 vs. 11.93 +/- 1.32 mg/kg FFM.min; P = NS) according to study design. In both the basal state and during glucose clamps after glyburide therapy, rates of glucose oxidative metabolism (Gox) increased by 68-78% [1.21 +/- 0.16 vs. 2.03 +/- 0.31 mg/kg FFM.min (P < 0.05) and 3.13 +/- 0.51 vs. 5.58 +/- 0.55 mg/kg FFM.min (P < 0.05), respectively], and rates of nonoxidative glucose metabolism decreased [2.96 +/- 0.68 vs. 1.25 +/- 0.21 mg/kg FFM.min (P < 0.05) and 8.40 +/- 1.50 to 6.30 +/- 1.40 mg/kg FFM.min (P < 0.01), respectively]. Circulating plasma FFA levels and rates of fat oxidation (Fox) remained unchanged in both the basal state and during clamp studies. Skeletal muscle glycogen synthase (GS) activity, expressed as fractional velocity, was unchanged by glyburide therapy (2.2 +/- 0.8 vs. 2.7 +/- 0.3% in the basal state and 7.3 +/- 1.8 vs. 6.1 +/- 0.9% during clamps; both P = NS). In summary, at both matched (during clamp studies) and unmatched (during basal studies) rates of GU, improved metabolic control with glyburide therapy resulted in marked improvement of Gox independent of the effects on GU. The improvement in Gox was not associated with changes in Fox, circulating FFA, or muscle GS activity. These data indicate that long term metabolic control achieved by glyburide therapy markedly improves Gox, but not skeletal muscle GS activity, in noninsulin-dependent diabetes mellitus independent of GU and Fox.     

Plasma cocaine levels and locomotor activity after systemic injection in virgin and in lactating maternal female rats

We have determined the temporal pattern of plasma cocaine levels and increased activity that result from acute systemic injections of cocaine to female rats in two different endocrine and behavioral states, in nonmaternal virgins and in lactating maternal dams. Plasma levels of cocaine as well as ambulatory and rearing activity were determined every 30 min for a total of 300 min after subcutaneous injections of either 10, 20, or 40 mg/kg of cocaine. Virgin females had no prior drug history, whereas lactating, maternal dams had received two cocaine injections before activity testing. Within 30 min after an injection, cocaine in the plasma and activity were substantially elevated, and generally remained so for 270-300 min. Overall, plasma cocaine levels and activity were well correlated and followed a predictable dose-response pattern. The onset, peak, duration, and decline of activity corresponded generally to the onset, peak, duration, and decline of plasma cocaine. For virgins, mean ambulatory activity increased 2.5-4.0-fold over baseline, whereas in lactating females activity increased 5-11-fold over baseline. Stereotypy did not occur. Although the general responsivity of these females to cocaine was very similar to that reported for males, there are differences in the timing of peak activity and the return of activity to baseline when the virgins and the lactating dams are compared to each other and to reports by others on male rats. These data support the hypothesis that endocrine or behavioral state may influence the responsiveness of animals to cocaine.     

Electrophoretic investigations of blood serum in fasted rats

An affinity column has been synthesized consisting of p-aminophenyl 1-thio-beta-L-fucopyranoside residues attached to Sepharose 4B through succinylated diaminodipropylamine bridges. Surprisingly, it has been found to bind beta-N-acetylglucosaminidase in the serum of Limulus polyphemus (horseshoe crab). The enzyme is eluted with N-acetyl-D-glucosamine at a concentration of 2 mg/ml and with other sugars at higher concentrations. A highly purified enzyme free from other glycosidases is obtained. The enzyme is not eluted by solutions of salt.     

Enzymatic activity in the liver and lipogenesis processes in the fatty tissue of rats after a space flight

The rats flown aboard Cosmos-782 showed a significant increase in the activity of tyrosine aminotransferase and tryptophan pyrolase, i. e. the enzymes whose activity depends on the corticosterone level. The synchronous rats displayed a small increase in the enzyme activity. The flight and synchronous animals exhibited a slight increase in the activity of gluconeogenetic enzymes and a decrease in the activity of glucose-6-phosphatase. Immediately after flight and, to a lesser extent, after the synchronous experiment the activity of lipogenetic enzymes decreased. On the R+25 day the enzyme activity remained unchanged. The study of lipogenesis in the epididymal fat, using C14-glucose incorporation into lipids, did not reveal any differences in the flight and synchronous rats. The findings demonstrated that changes in the enzyme activity induced by the flight and synchronous experiments returned to the normal during readaptation.     

Influence of tryptophan on the level of hepatic microsomal cytochrome P-450 in well-fed normal, adrenalectomized, and phenobarbital-treated rats

In well-fed normal male rats, either force-feeding of tryptophan or a single injection of phenobarbital produced significant increments in hepatic microsomal cytochrome P-450 and the associated aniline hydroxylase activity. Administration of both tryptophan and phenobarbital together resulted in even greater stimulation than when the compounds were given alone. Adrenalectomy lowered instead the cytochrome P-450 concentration in comparison with that of normal rats, and administration of tryptophan and phenobarbital in this condition produced no significant increment in cytochrome P-450 concentration. In addition, phenobarbital administered either singly or in combination with tryptophan resulted in 80% mortality, which was reduced to zero by pretreatment with cortisol. While in cortisol-treated adrenalectomized rats administration of phenobarbital caused a 56% increment in cytochrome P-450 as compared to controls, tryptophan produced only a minor (9%) increase. In normal, as well as in adrenalectomized rats, tryptophan and phenobarbital administered either idividually or together increased microsomal protein concentration. In normal rats actinomycin-D treatment reduced both cytochrome P-450 and microsomal protein concentrations below that of the non-treated control levels. Further administration of either tryptophan or phenobarbital slightly increased the level of cytochrome P-450, and the two compounds together caused 40 and 21% increments of the same compared to actinomycin-treated and non-treated controls, respectively.     

Purification and characterization of bovine heart glycogen synthase kinase-3

Glycogen Synthase Kinase-3 (GSK-3) was isolated from bovine heart tissue extracts by a procedure involving ammonium sulfate fractionation, followed by chromatography on phosphocellulose, Cibacron blue 3GA-agarose, DEAE-Sephacel, CM-Sepharose, heparin-agarose, myelin basic protein-Sepharose, and LiChrospher 1000 C00-. GSK-3 was identified by its activation of protein phosphatase-1i (PP-1i). The purified enzyme had a specific activity of 25,500 units of protein phosphatase-1i activated/mg protein. The enzyme is an asymmetric monomeric protein of 53 kDa. The molecular size and retention of activity after autophosphorylation indicated that the isolated enzyme was the GSK-3 alpha-isoform.     

Glucose-6-P control of glycogen synthase phosphorylation in yeast

The SNF1 gene encodes a protein kinase necessary for expression of glucose-repressible genes and for the synthesis of the storage polysaccharide glycogen. From a genetic screen, we have found that mutation of the PFK2 gene, which encodes the beta-subunit of 6-phosphofructo-1-kinase, restores glycogen accumulation in snf1 cells. Loss of PFK2 causes elevated levels of metabolites such as glucose-6-P, hyperaccumulation of glycogen, and activation of glycogen synthase, whereas glucose-6-P is reduced in snf1 cells. Other mutations that increase glucose-6-P, deletion of PFK1, which codes for the alpha-subunit of 6-phosphofructo-1-kinase, or of PGI1, the phosphoglucoisomerase gene, had similar effects on glycogen metabolism as did pfk2 mutants. We propose that elevated glucose-6-P mediates the effects of these mutations on glycogen storage. Glycogen synthase kinase activity was reduced in extracts from pfk2 cells but was restored to that of wild type if the extract was gel-filtered to remove small molecules. Also, added glucose-6-P inhibited the glycogen synthase kinase activity in extracts from wild-type cells, half-maximally at approximately 2 mM. We suggest that glucose-6-P controls glycogen synthase activity by two separate mechanisms. First, glucose-6-P is a direct activator of glycogen synthase, and second, it controls the phosphorylation state of glycogen synthase by inhibiting a glycogen synthase kinase.     

Sequential changes of sonographic and computed tomography findings in the normal rabbit liver after percutaneous ethanol injection. Correlation with pathologic findings

BACKGROUND: A neural network computer model described in a companion paper predicted the effects of increased dopamine transmission on selective attention under two different hypotheses. METHODS: To evaluate these predictions we conducted an empirical study in human subjects of D-amphetamine effects on performance of the Eriksen response competition task. Ten healthy volunteers were tested before and after placebo or D-amphetamine in a double-blind cross-over design. RESULTS: D-amphetamine induced a speeding of reaction time overall and an improvement of accuracy at fast reaction times but only in the task condition requiring selective attention. CONCLUSIONS: This pattern of results conforms to the prediction of the model under the hypothesis that D-amphetamine primarily affects dopamine transmission in cognitive rather than motor networks. This suggests that the principles embodied in parallel distributed processing models of task performance may be sufficient to predict and explain specific behavioral effects of some drug actions in the central nervous system.     

Changes in the normal liver fibrinolytic activity after portacaval anastomosis in experimental animals (1st communication)

One hundred twenty-one patients with disseminated malignant melanoma were treated with BCNU, vincristine, DTIC, and chlorpromazine (BVD). A response rate of 22% was observed; 28% of the patients had stable disease and 50% had increasing disease. Similar response rates were obtained with both the high dose and low dose treatment schedules. Patients who exhibited some degree of improvement during their initial course of treatment had the highest overall response rate (72%) to BVD chemotherapy. The median survival from onset of therapy was six months for all patients and 18 months for patients who responded to chemotherapy. The median duration of response was 9.9 months. Thus, the addition of chlorpromazine to BVD chemotherapy did not increase tumor response, and the overall results obtained were comparable to DTIC alone. Patients were found to be lymphopenic prior to the onset of therapy. Their median absolute lymphocyte count was 1800/mm3. Those patients with absolute lymphocyte counts above the 2710/mm3 normal mean had significantly higher response rates (35% vs. 19%, P less than .05) and longer survivals (9.8 months vs. 4.3 months, P less than .05) than patients with lower initial lymphocyte levels. Pretreatment eosinophil and monocyte counts were not closely correlated with patient response or survival.