Inhibition of glycogenolysis in primary rat hepatocytes by 1, 4-dideoxy-1,4-imino-D-arabinitol.
(1/23)
1,4-Dideoxy-1,4-imino-d-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DAB(medium-concentration). In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC(50) values of 1.0+/-0.3 and 1.1+/-0.2 microM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation. (+info)
Lack of hepatic "interregulation" during inhibition of glycogenolysis in a canine model.
(2/23)
It has been proposed that the glycogenolytic and gluconeogenic pathways contributing to endogenous glucose production are interrelated. Thus a change in one source of glucose 6-phosphate might be compensated for by an inverse change in the other pathway. We therefore investigated the effects of 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), a potent glycogen phosphorylase inhibitor, on glucose production in fasted conscious dogs. When dogs were treated acutely with high glucagon, glucose production rose from 1.93 +/- 0.14 to 3.07 +/- 0.37 mg x kg(-1) x min(-1) (P < 0.01). When dogs were treated acutely with DAB in addition to high glucagon infusion, the stimulation of the glycogenolytic rate was completely suppressed. Glucose production rose from 1.85 +/- 0.20 to 2.41 +/- 0.17 mg x kg(-1) x min(-1) (P < 0.05), which was due to the increase in gluconeogenesis from 0.93 +/- 0.09 to 1.54 +/- 0.08 mg x kg(-1) x min(-1) (P < 0.001). In conclusion, infusion of DAB inhibited glycogenolysis; however, the absolute contribution of gluconeogenesis to glucose production was not affected. These results suggest that inhibition of glycogenolysis could be an effective antidiabetic treatment. (+info)
Evidence against glycogen cycling of gluconeogenic substrates in various liver preparations.
(3/23)
The effect of inhibition of glycogen phosphorylase by 1,4-dideoxy-1,4-imino-d-arabinitol on rates of gluconeogenesis, gluconeogenic deposition into glycogen, and glycogen recycling was investigated in primary cultured hepatocytes, in perfused rat liver, and in fed or fasted rats in vivo clamped at high physiological levels of plasma lactate. 1,4-Dideoxy-1,4-imino-d-arabinitol did not alter the synthesis of glycerol-derived glucose in hepatocytes or lactate-derived glucose in perfused liver or fed or fasted rats in vivo. Thus, 1,4-dideoxy-1,4-imino-d-arabinitol inhibited hepatic glucose output in the perfused rat liver (0.77 +/- 0.19 versus 0.33 +/- 0.09, p < 0.05), whereas the rate of lactate-derived gluconeogenesis was unaltered (0.22 +/- 0.09 versus 0.18 +/- 0.08, p = not significant) (1,4-dideoxy-1,4-imino-d-arabinitol versus vehicle, micromol/min * g). Overall, the data suggest that 1,4-dideoxy-1,4-imino-d-arabinitol inhibited glycogen breakdown with no direct or indirect effects on the rates of gluconeogenesis. Total end point glycogen content (micromol of glycosyl units/g of wet liver) were similar in fed (235 +/- 19 versus 217 +/- 22, p = not significant) or fasted rats (10 +/- 2 versus 7 +/- 2, p = not significant) with or without 1,4-dideoxy-1,4-imino-d-arabinitol, respectively. The data demonstrate no glycogen cycling under the investigated conditions and no effect of 1,4-dideoxy-1,4-imino-d-arabinitol on gluconeogenic deposition into glycogen. Taken together, these data also suggest that inhibition of glycogen phosphorylase may prove beneficial in the treatment of type 2 diabetes. (+info)
The effect of glucose on the potency of two distinct glycogen phosphorylase inhibitors.
(4/23)
Two distinct glycogen phosphorylase inhibitors, 5-chloro-1H-indole-2-carboxylic acid [1-(4-fluorobenzyl)-2-(4-hydroxy-piperidin-1-yl)-2-oxoethyl]amide (CP-320,626) and 1,4-dideoxy-1,4-D-arabinitol (DAB), were characterized in vitro with respect to the influence of glucose on their potencies. CP-320,626 has previously been shown to bind to a newly characterized indole site, whereas DAB seems to act as a glucose analogue, but with slightly different properties from those of glucose. When analysed in pig liver glycogen phosphorylase a (GPa) activity assays, the two inhibitors showed very different properties. When GPa activity was measured in the physiological direction (glycogenolysis), DAB was the most potent inhibitor with an IC(50) value of 740+/-9 nM compared with the IC(50) value for CP-320-626 of 2.39+/-0.37 microM. There was no effect of glucose on the inhibitory properties of DAB, whereas a glucose analogue N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc) antagonized the effect of DAB. Likewise, there was no synergistic effect of CP-320,626 and glucose, whereas CP-320,626 and 1-GlcNAc inhibited GPa in synergy. Moreover, the synergistic effect of glucose and CP-320,626 was GPa-isoform-specific, since CP-320,626 and glucose inhibited rabbit muscle GPa in synergy when the GPa activity was measured towards glycogenolysis. When GPa activity was measured towards glycogen synthesis, CP-320,626 showed a synergistic effect with glucose, whereas the effect of DAB was slightly antagonized by glucose in this assay direction. Caffeine was included in the investigation as a control GP inhibitor, and both glucose and 1-GlcNAc potentiated the effect of caffeine independent of the assay direction. In primary cultured rat hepatocytes 1-GlcNAc and CP-320,626 inhibited basal and glucagon-induced glycogenolysis in synergy, whereas the ability of DAB to inhibit basal or glucagon-induced glycogenolysis was unaltered by 1-GlcNAc. Glucose had no effect on either CP-320,626 or DAB inhibition of glycogenolysis in cultured rat hepatocytes. In conclusion, the present study shows that the two GP inhibitors are kinetically very distinct and neither of the inhibitors demonstrates a physiologically relevant glucose dependence in vitro. (+info)
Diverse effects of two allosteric inhibitors on the phosphorylation state of glycogen phosphorylase in hepatocytes.
(5/23)
Two distinct allosteric inhibitors of glycogen phosphorylase, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) and CP-91149 (an indole-2-carboxamide), were investigated for their effects on the phosphorylation state of the enzyme in hepatocytes in vitro. CP-91149 induced inactivation (dephosphorylation) of phosphorylase in the absence of hormones and partially counteracted the phosphorylation caused by glucagon. Inhibition of glycogenolysis by CP-91149 can be explained by dephosphorylation of phosphorylase a. This was associated with activation of glycogen synthase and stimulation of glycogen synthesis. DAB, in contrast, induced a small degree of phosphorylation of phosphorylase. This was associated with inactivation of glycogen synthase and inhibition of glycogen synthesis. Despite causing phosphorylation (activation) of phosphorylase, DAB is a very potent inhibitor of glycogenolysis in both the absence and presence of glucagon. This is explained by allosteric inhibition of phosphorylase a, which overrides the increase in activation state. In conclusion, two potent phosphorylase inhibitors exert different effects on glycogen metabolism in intact hepatocytes as a result of opposite effects on the phosphorylation state of both phosphorylase and glycogen synthase. (+info)
Hepatic glycogen breakdown is implicated in the maintenance of plasma mannose concentration.
(6/23)
D-mannose is an essential monosaccharide constituent of glycoproteins and glycolipids. However, it is unknown how plasma mannose is supplied. The aim of this study was to explore the source of plasma mannose. Oral administration of glucose resulted in a significant decrease of plasma mannose concentration after 20 min in fasted normal rats. However, in fasted type 2 diabetes model rats, plasma mannose concentrations that were higher compared with normal rats did not change after the administration of glucose. When insulin was administered intravenously to fed rats, it took longer for plasma mannose concentrations to decrease significantly in diabetic rats than in normal rats (20 and 5 min, respectively). Intravenous administration of epinephrine to fed normal rats increased the plasma mannose concentration, but this effect was negated by fasting or by administration of a glycogen phosphorylase inhibitor. Epinephrine increased mannose output from the perfused liver of fed rats, but this effect was negated in the presence of a glucose-6-phosphatase inhibitor. Epinephrine also increased the hepatic levels of hexose 6-phosphates, including mannose 6-phosphate. When either lactate alone or lactate plus alanine were administered as gluconeogenic substrates to fasted rats, the concentration of plasma mannose did not increase. When lactate was used to perfuse the liver of fasted rats, a decrease, rather than an increase, in mannose output was observed. These findings indicate that hepatic glycogen is a source of plasma mannose. (+info)
Characterization of a human core-specific lysosomal {alpha}1,6-mannosidase involved in N-glycan catabolism.
(7/23)
In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism. (+info)
Mulberry latex rich in antidiabetic sugar-mimic alkaloids forces dieting on caterpillars.
(8/23)
Since ancient times, mulberry leaves (Morus spp.) have been used to rear the silkworm Bombyx mori. Because the silkworm grows well on mulberry leaves, the toxicities and defensive activities of these leaves against herbivorous insects have been overlooked. Here we show that mulberry leaves are highly toxic to caterpillars other than the silkworm B. mori, because of the ingredients of the latex, a milky sap exuded from mulberry leaf veins. The toxicity of mulberry leaves was lost when the latex was eliminated from the leaves, and artificial diets containing latex showed toxicity. Mulberry latex contained very high concentrations of alkaloidal sugar-mimic glycosidase inhibitors reported to have antidiabetic activities, such as 1,4-dideoxy-1,4-imino-D-arabinitol, 1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-D-ribitol. The overall concentrations of these inhibitors in latex reached 1.5-2.5% (8-18% dry weight) in several mulberry varieties, which were approximately 100 times the concentrations previously reported from whole mulberry leaves. These sugar-mimic alkaloids were toxic to caterpillars but not to the silkworm B. mori, indicating that the silkworm can circumvent the mulberry tree's defense. Our results suggest that latex ingredients play key roles in defense of this tree and of other plants against insect herbivory, and they imply that plant latexes are treasuries of bioactive substances useful as medicines and pesticides. (+info)