Endotoxin stimulates glycogenolysis in the liver by means of intercellular communication

Escherichia coli endotoxin (lipopolysaccharide) was shown to increase glycogenolysis in the perfused liver 2-3-fold. In isolated parenchymal liver cells, however, endotoxin did not influence glycogenolysis, whereas stimulation by endotoxin of glycogenolysis in the perfused liver could be blocked by aspirin. This suggests that the effect of endotoxin on liver glycogenolysis is mediated by eicosanoids. The amount of prostaglandin D2 (which is the major prostanoid formed by Kupffer cells) in the liver perfusates was increased 5-fold upon endotoxin addition, with a time course which preceded the increase in glucose output. It is concluded that endotoxin stimulates glycogenolysis in the liver by stimulating prostaglandin D2 release from Kupffer cells, with a subsequent activation of glycogenolysis in parenchymal liver cells. This mechanism of intercellular communication may be designed to provide the carbohydrate source of energy necessary for the effective destruction of invaded microorganisms, by phagocytic cells, including the Kupffer cells.     

Prostaglandin D2 mediates the stimulation of glycogenolysis in the liver by phorbol ester.

The tumour-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), when added to the perfused liver, stimulates glycogenolysis 2-fold. This stimulation is not seen when aspirin is present in the perfusion medium. In isolated parenchymal liver cells. PMA is not able to stimulate glycogenolysis, suggesting that its effect on glycogenolysis might be indirect and depends on the presence of the non-parenchymal liver cell types. To test the possible operation of an indirect mechanism, we measured the amount of prostaglandin (PG) D2 in liver perfusates. After addition of PMA, the amount of PGD2 is doubled, in parallel with the increase in glycogenolysis. Glycogenolysis in both isolated parenchymal liver cells and perfused liver could be stimulated by the addition of PGD2. Our data indicate that stimulation of glycogenolysis in the liver by PMA may be mediated by non-parenchymal liver cells, which produce PGD2 in response to PMA. Subsequently PGD2 activates glycogenolysis in the parenchymal liver cells. The intercellular communication inside the liver in response to PMA adds a new mechanism to the complex regulation of glucose homoeostasis by the liver.     

The anomeric malaise: a manifestation of B-cell glucotoxicity.

In non-insulin-dependent diabetic subjects and in various animal models of spontaneous or experimental chronic hyperglycaemia, the secretory response of the pancreatic B-cell to a rapid rise in extracellular D-glucose concentration is characterized by a paradoxical, early and transient fall in insulin output and/or an altered anomeric specificity. These two features of B-cell glucotoxicity may be accounted for by the accumulation of glycogen in the B-cell and the interference of changes in glycogenolysis with the hexose-induced increase in glycolytic flux. The inhibitory action of D-glucose upon glycogenolysis displaying alpha-stereospecificity, the metabolic and secretory response to alpha-D-glucose is expected to be more severely affected than that evoked by the beta-anomer. Such a preferential alteration of the response to alpha-D-glucose was indeed documented in diabetic subjects, BB rats, duct-ligated rabbits, and adult rats either injected with streptozotocin during the neonatal period or rendered hyperglycaemic by the repeated administration of diazoxide. In these experimental models, the attenuation, suppression or even reversal of the anomeric preference in insulin release appeared related to the severity and duration of the hyperglycaemic state. A clear distinction ought to be made between these features of B-cell glucotoxicity and other etiopathogenic factors of B-cell dysfunction, such as the long term deleterious effect of streptozotocin upon the activity of key mitochondrial dehydrogenases.     

Regulation of glycogenolysis in the liver of the newborn rat in vivo inhibitory effect of glucose

Newborn rats were injected immediately after delivery with glucose or glucose plus mannoheptulose, and the time-courses of liver glycogen, plasma glucose, insulin and glucagon concentration were studied. The administration of glucose prevented both liver glycogenolysis and the increase in plasma glucagon concentration which normally occurs immediately after delivery. In addition, the administration of glucose prevented the decrease of plasma glucose and insulin concentration which normally occurs during the first hour of extrauterine life. Supplementation of glucose with mannoheptulose prevented the increase of plasma insulin concentrations caused by the administration of glucose; liver glycogenolysis, however, was not stimulated in these circumstances. The increase in the rate of glycogenolysis caused by the administration of glucagon was prevented in newborn rats previously treated with glucose. These results suggest that glucose exerts an inhibitory effect on the stimulation of neonatal liver glycogenolysis by glucagon.     

Influence of extracellular phosphate concentrations on the regulation of hepatic glucose output

Experiments were carried out to investigate the role of extracellular phosphate in the hormonal regulation of glycogenolysis in perfused fed-rat liver. Omission of phosphate from the perfusate did not affect the ATP, ADP and AMP contents of the tissue and the basal glucose output from the perfused liver. However, it inhibited significantly the glycogenolysis induced by glucagon, cyclic AMP, phenylephrine and vasopressin but not that induced by 2,4-dinitrophenol. In the absence of perfusate phosphate, the increase in phosphorylase a activity caused by the addition of glucagon, phenylephrine and vasopressin was significantly less than that observed in the presence of perfusate phosphate. Insulin inhibition of the glucagon- or cyclic AMP-induced glycogenolysis was abolished when the perfusion was carried out with the phosphate-free buffer. However, the inhibitory effect of insulin on phenylephrine-induced glycogenolysis was clearly demonstrated even when the perfusate contained no phosphate. These data indicate that in the phosphate-depleted liver, the hormonal control of phosphorylation and dephosphorylation of phosphorylase is impaired. The difference in the phosphate dependency of insulin action on glucagon-and alpha-adrenergic agonist-induced glycogenolysis suggests that the mechanism or site of insulin action on glucagon and phenylephrine is different.     

Influence of cAMP and calcium on epinephrine-mediated hepatic glycogenolysis in rainbow trout,Oncorhynchus mykiss

1. The role of cAMP and of calcium in mediating epinephrine-stimulated glycogenolysis was studied by incubating rainbow trout liver in vitro.2. Epinephrine significantly stimulates glucose release from liver pieces incubated in either calciumcontaining or calcium-free medium. However, the development of the glycogenolytic profile occurred more rapidly in the presence of calcium.3. The β-antagonist, propranolol, inhibited epinephrine-stimulated glucose release from liver pieces incubated in either calcium-containing or calcium-free medium.4. Calcium ionophore, A3187, stimulated glucose release from liver pieces incubated in calciumcontaining medium. Verapamil, a putative calcium channel blocker, had no effect on A23187-stimulated glycogenolysis. However, verapamil completely inhibited epinephrine-stimulated glycogenolysis.5. Dibutyryl cAMP and IBMX, singly or together, stimulated glucose release from liver pieces. cAMP-mediated glycogenolysis was more pronounced in liver pieces incubated in calcium-containing medium.6. These results indicate that epinephrine-stimulated hepatic glycogenolysis in rainbow trout proceeds through the activation of β-adrenergic receptors and that both cAMP and calcium are involved in the post-receptor signal transduction process.     

Effects of vasoactive intestinal peptide on glycogenolysis in cultured liver cells.

When isolated rat liver cells were incubated in the presence of vasoactive intestinal peptide at the concentrations ranging from 0.2 microgram to 2 micrograms per ml, glycogenolysis was maximally stimulated within 15 min. However, somatostatin inhibited the liver glycogenolysis. The combined addition to the incubation medium showed that insulin and somatostatin inhibited the stimulated glycogenolysis induced by vasoactive intestinal peptide, while vasoactive intestinal peptide plus secretin showed no additive effect on glycogenolysis, as compared with single the addition of vasoactive intestinal peptide. On the other hand, the additon of glucagon to vasoactive intestinal peptide showed additive effects on glycogenolysis. These results suggest that the receptor site for vasoactive intestinal peptide may be distinguishable from that for glucagon. Extracellular calcium ions were demonstrated to play an important role in the modulation of vasoactive intestinal peptide-induced glycogenolysis. The evidence presented in this paper indicates that glucose metabolism may be partly regulated by the direct action of vasoactive intestinal peptide on hepatocytes, which is referred to as an enterohepatic axis and that the axis is inhibited by insulin and somatostatin.     

Hexose metabolism in pancreatic islet cells: the coupling between hexose phosphorylation and mitochondrial respiration

The possible relevance of D-glucose phosphorylation by mitochondria-bound hexokinase to the control of respiration was examined in mitochondria prepared from either tumoral pancreatic islet cells (RINm5F line) or normal rat liver. In both systems, ATP generated by mitochondria exposed to ADP and succinate could serve as a substrate for the phosphorylation of D-glucose. However, after exposure to exogenous ADP in the presence of succinate, only mitochondria isolated from RINm5F cells displayed a sizeable increase in O2 consumption in response to a subsequent administration of D-glucose. In this respect, the discrepancy between mitochondria from islet cells and liver, respectively, was found to be attributable to the much lower hexokinase activity, relative to respiratory rate, in liver than in RINm5F cell mitochondria. It is speculated that the coupling between hexose phosphorylation and respiration in islet cells may prime the mitochondria to generate ATP during the early metabolic and secretory response to a rise in extracellular D-glucose concentration.     

Stimulation of hepatic glycogenolysis by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) via cyclooxygenase products

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) stimulates glycogenolysis in perfused rat liver. The effect of TPA was blocked by indomethacin and bromophenacyl bromide. The effect of TPA on glucose output was transient in spite of the continuous presence of the phorbol ester in the perfusion medium. Addition of platelet activating factor (PAF) after the effect of TPA did not stimulate glycogenolysis. In contrast, vasopressin was able to stimulate glucose output under these conditions. Interestingly, as previously reported, PAF produced also transient stimulation of glycogenolysis; the addition of TPA after the effect of PAF had declined, was also unable to increase glucose output by the liver. It is suggested that both PAF and TPA stimulate hepatic metabolism through the generation of cyclooxygenase products.     

Possible involvement of cyclooxygenase products in the actions of platelet-activating factor and of lipoxygenase products in the vascular effects of epinephrine in perfused rat liver

Platelet-activating factor (PAF) stimulates glycogenolysis and induces vasoconstriction in perfused rat liver. The effect of PAF was rapid but transient and it was blocked by indomethacin and bromophenacyl bromide which suggests a role of cyclooxygenase metabolites in its action. The homologous desensitization of glycogenolysis produced by PAF and the sensitivity of its actions to inhibitors of cyclooxygenase and phospholipase A2 markedly differentiate the mechanism of action of this agent with that of alpha 1-adrenergic agents, vasopressin or angiotensin II. No effect of PAF in isolated hepatocytes was observed which suggest that cells other than hepatocytes could be involved in its action in perfused liver. In addition nordihydroguaiaretic acid and bromophenacyl bromide abolished the vascular effect (but not the glycogenolysis) produced by epinephrine which suggest a role for lipoxygenase products in this effect.     

Hexose metabolism in pancreatic islets: preferential utilization of mitochondrial ATP for glucose phosphorylation

The respective contribution of exogenous and intramitochondrially formed ATP to D-glucose phosphorylation by mitochondria-bound hexokinase was examined in both rat liver and pancreatic islet mitochondria by comparing the generation of D-glucose 6-[32P]phosphate from exogenous [gamma-32P]ATP to the total rate of D-[U-14C]glucose phosphorylation. In liver mitochondria, the fractional contribution of exogenous ATP to D-glucose phosphorylation ranged from 4 to 74%, depending on the availability of endogenous ATP formed by either oxidative phosphorylation or in the reaction catalyzed by adenylate kinase. Likewise, in islet mitochondria exposed to exogenous ATP but deprived of exogenous nutrient, about 60% of D-glucose phosphorylation was supported by mitochondrial ATP. Such a fractional contribution was further increased in the presence of ADP and succinate, and suppressed by mitochondrial poisons. It is concluded that, in islet like in liver mitochondria, mitochondrial ATP is used preferentially to exogenous ATP as a substrate for D-glucose phosphorylation by mitochondria-bound hexokinase. This may favour the maintenance of a high cytosolic ATP concentration in glucose-stimulated islet cells.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene, in the glycogenolytic action of alpha-adrenergic stimulation in perfused rat liver

To identify the role of Ca2+ mobilization from intracellular pool(s) in the action of alpha-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5 X 10(-5) M) inhibited both glycogenolysis and 45Ca efflux induced by 5 X 10(-7) M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca2+ concentration induced by phenylephrine both in the presence and absence of extracellular Ca2+. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of alpha-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

Studies on the role of cyclic guanosine 3':5'-monophosphate and extracellular Ca2+ in the regulation of glycogenolysis in rat liver cells.

Catecholamines increased guanosine 3':5'-monophosphate (cyclic GMP) accumulation by isolated rat liver cells. The increases in cyclic GMP due to 1.5 muM epinephrine, isoproterenol, or phenylephrine were blocked by phenoxybenzamine but not by propranolol. The possibility that cyclic GMP is involved in the glycogenolytic action of catecholamines seems unlikely since cyclic GMP accumulation is also elevated by carbachol, insulin, A23187, and to a lesser extent by glucagon. Furthermore, carbachol had little effect on glycogenolysis while insulin actually inhibited hepatic glycogenolysis. The rise in cyclic GMP due to carbachol was abolished by atropine and that due to all agents was markedly reduced by the omission of extracellular calcium. However, the glycogenolytic action of glucagon and catecholamines was only slightly inhibited by the omission of calcium. The only agent which was unable to stimulate glycogenolysis in calcium-free buffer was the divalent cation ionophore A23187. There was a drop in ATP content of liver cells during incubation in calcium-free buffer which was accompanied by an inhibition of glucagon-activated adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The presence of calcium inhibited the rise in adenylate cyclase activity of lysed rat liver cells due to glucagon or isoproterenol but not that due to fluoride. These results suggest that the stimulation by catecholamines and glucagon of glycogenolysis is not mediated through cyclic GMP nor does it depend on the presence of extracellular calcium. Cyclic GMP accumulation was increased in liver cells by agents which either inhibit, have little affect, or accelerate glycogenolysis. The significance of elevations of cyclic GMP in rat liver cells remains to be established.     

Assessment of the role of Ca2+ mobilization from intracellular pool(s), using dantrolene,in the glycogenolytic action of α-adrenergic stimulation in perfused rat liver

To identify the role of Ca²⁺ mobilization from intracellular pool(s) in the action of α-adrenergic agonist, the effects of dantrolene on phenylephrine-induced glycogenolysis were investigated in perfused rat liver. Dantrolene (5·10⁻⁵ M) inhibited both glycogenolysis and ⁴⁵Ca efflux induced by 5·10⁻⁷ M phenylephrine. The inhibition by dantrolene was observed in the presence and absence of perfusate calcium. In contrast, dantrolene did not inhibit glycogenolysis induced by glucagon. To confirm the specificity of dantrolene action on calcium release in liver, experiments were also carried out using isolated hepatocytes. Dantrolene did not affect phenylephrine-induced production of inositol 1,4,5-trisphosphate. The compound did inhibit a rise in cytoplasmic Ca²⁺ concentration induced by phenylephrine both in the presence and absence of extracellular Ca²⁺. Thus, these results suggest that calcium release from an intracellular pool is essential for the initiation of α-adrenergic stimulation of glycogenolysis in the perfused rat liver.     

Structural specificity for prostaglandin effects on hepatocyte glycogenolysis.

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.     

Interference of D-mannoheptulose with D-glucose phosphorylation,metabolism and functional effects: Comparison between liver,parotid cells and pancreatic islets

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.     

Comparative activities of glycogen phosphorylase and gamma-amylase in livers of carp (Cyprinus carpio) and goldfish (Carassius auratus).

1. The activity of glycogen phosphorylase in goldfish liver is fivefold greater than that in carp liver, suggesting that the enzyme may not be as important in regulating glycogenolysis in the latter species. 2. The activity of gamma-amylase is comparable in carp and goldfish liver. 3. The activity of hepatic gamma-amylase is approximately one-half that of glycogen phosphorylase in carp whereas in goldfish, the activity of gamma-amylase is less than one-sixth that of phosphorylase. Hepatic gamma-amylase may be an important glycogenolytic enzyme in carp but makes an insignificant contribution to glycogenolysis in goldfish.     

Relative contribution of glycogenolysis and gluconeogenesis to basal, glucagon- and nerve stimulation-dependent glucose output in the perfused liver from fed and fasted rats

The relative contribution to basal, glucagon- and nerve stimulation-enhanced glucose output of glycogenolysis (glucose output in the presence of the gluconeogenic inhibitor mercaptopicolinate) and gluconeogenesis (difference in glucose output in the absence and presence of the inhibitor) was investigated in perfused livers from fed rats with high and from fasted animals with low levels of glycogen. 1) Basal glucose output in both states was due only to gluconeogenesis. 2) Glucagon-enhanced glucose output was due about equally to glycogenolysis and gluconeogenesis in the fed state, but predominantly to gluconeogenesis (80%) in the fasted state. 3) Nerve stimulation-increased glucose output was due mainly to glycogenolysis (65%) in the fed state and about equally to both processes in the fasted state. The results suggest that under basal conditions of normal demands the liver supplies glucose only via gluconeogenesis and thus spares its glycogen stores, and that in situations of enhanced demands signalled by an increase in glucagon or sympathetic tone the liver liberates glucose mainly via glycogenolysis.     

Studies on the inhibition of glycogenolysis by D-galactosamine in rat liver hepatocytes.

Effect of galactosamine on glycogenolysis was studied in isolated hepatocytes. It was found that addition of galactosamine strongly inhibited glycogenolysis in normal hepatocytes. Galactosamine-inhibited glycogenolysis was not stimulated by epinephrine or glucagon. This inhibition was specific as no such inhibition was observed with galactose, 2-deoxy-glucose or glucosamine. The glucagon-stimulated cyclic AMP formation in galactosamine-treated hepatocytes was the same as in normal cells; Glc-1-P and Glc-6-P did not accumulate nor was lactate formation enhanced. The glucose production by hepatocytes from regenerating liver was only slightly inhibited by galactosamine and glucagon addition stimulated glycogenolysis in the presence of the amino sugar.     

Interrelationship of insulin and glucagon ratios on carbohydrate metabolism in isolated hepatocytes containing high glycogen.

The effect of physiological concentrations of glucagon and insulin on glycogenolysis was studied in the presence and absence of substrates in isolated hepatocytes containing high glycogen. In the absence of substrates glucagon stimulated glycogenolysis at 10^?14M concentration, and addition of 100 μunits of insulin partially inhibited glucagon stimulated glycogenolysis (10^?14M to 10^?11M). However, in the presence of substrates, insulin completely inhibited glucagon stimulated glycogenolysis (10^?14M to 10^?11M), indicating that molar glucagon and insulin ratios control carbohydrate metabolism in liver. Additional studies showed incorporation of amino acid into protein was linear for only 3 to 4 hr in cells containing low glycogen, whereas in cells containing high glycogen, incorporation was linear for 8 to 10 hr.