Content uploaded by Louis Hue
Author content
All content in this area was uploaded by Louis Hue
Content may be subject to copyright.
A preview of the PDF is not available
The effect of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations
Abstract
In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.
... Glycogen synthase activity was determined by measuring incorporation of the 14 C-glucosyl moiety of UDP-[ 14 C]glucose into glycogen (Thomas et al. 1968;Hue et al. 1975;Nuttall and Gannon 1989). Hepatocytes (5.0 × 10 5 ) were homogenized in 0.5 ml of a buffer containing 100 mM NaF, 20 mM EDTA, 0.5% glycogen, 1% protease inhibitor, 1% phosphatase inhibitor cocktail, and 50 mM glycylglycine (pH 7.4). ...
... Glycogen phosphorylase activity was measured by determining the level of free phosphate that is released during the reversed reaction, i.e., synthesis of glycogen from glucose-1-phosphate (Hue et al. 1975;Saheki et al. 1985;Stalmans and Hers 1975). Hepatocytes were lysed in a buffer containing 100 mM NaF, 20 mM EDTA, 0.5% glycogen, 1% protease inhibitor, 1% phosphatase inhibitor cocktail, and 50 mM glycylglycine (pH 7.4); supernate obtained after 9000×g centrifugation was used for the assay. ...
Environmental exposure to inorganic arsenic (iAs) has been shown to disturb glucose homeostasis, leading to diabetes. Previous laboratory studies have suggested several mechanisms that may underlie the diabetogenic effects of iAs exposure, including (i) inhibition of insulin signaling (leading to insulin resistance) in glucose metabolizing peripheral tissues, (ii) inhibition of insulin secretion by pancreatic β cells, and (iii) dysregulation of the methylation or expression of genes involved in maintenance of glucose or insulin metabolism and function. Published studies have also shown that acute or chronic iAs exposures may result in depletion of hepatic glycogen stores. However, effects of iAs on pathways and mechanisms that regulate glycogen metabolism in the liver have never been studied. The present study examined glycogen metabolism in primary murine hepatocytes exposed in vitro to arsenite (iAs³⁺) or its methylated metabolite, methylarsonite (MAs³⁺). The results show that 4-h exposures to iAs³⁺ and MAs³⁺ at concentrations as low as 0.5 and 0.2 µM, respectively, decreased glycogen content in insulin-stimulated hepatocytes by inhibiting insulin-dependent activation of glycogen synthase (GS) and by inducing activity of glycogen phosphorylase (GP). Further investigation revealed that both iAs³⁺ and MAs³⁺ inhibit insulin-dependent phosphorylation of protein kinase B/Akt, one of the mechanisms involved in the regulation of GS and GP by insulin. Thus, inhibition of insulin signaling (i.e., insulin resistance) is likely responsible for the dysregulation of glycogen metabolism in hepatocytes exposed to iAs³⁺ and MAs³⁺. This study provides novel information about the mechanisms by which iAs exposure impairs glucose homeostasis, pointing to hepatic metabolism of glycogen as one of the targets.
... K is an important cofactor for glycogen www.nature.com/scientificreports/ phosphorylase, an enzyme participating in the glycogenolysis process 22 . Its reduced level may be, therefore, connected with the limited requirement for the enzyme when low levels of glycogen are observed 23 . . ...
The ketogenic diet (KD) is a low-carbohydrate and high-fat diet that gains increasing popularity in the treatment of numerous diseases, including epilepsy, brain cancers, type 2 diabetes and various metabolic syndromes. Although KD is effective in the treatment of mentioned medical conditions, it is unfortunately not without side effects. The most frequently occurring undesired outcomes of this diet are nutrient deficiencies, the formation of kidney stones, loss of bone mineral density, increased LDL (low-density lipoprotein) cholesterol levels and hormonal disturbances. Both the diet itself and the mentioned adverse effects can influence the elemental composition and homeostasis of internal organs. Therefore, the objective of this study was to determine the elemental abnormalities that appear in the liver, kidney, and spleen of rats subjected to long-term KD treatment. The investigation was conducted separately on males and females to determine if observed changes in the elemental composition of organs are gender-dependent. To measure the concentration of P, S, K, Ca, Fe, Cu, Zn and Se in the tissues the method of the total reflection X-ray fluorescence (TXRF) was utilized. The obtained results revealed numerous elemental abnormalities in the organs of animals fed a high-fat diet. Only some of them can be explained by the differences in the composition and intake of the ketogenic and standard diets. Furthermore, in many cases, the observed anomalies differed between male and female rats.
... Previously has been observed that potassium depletion leads to changes in glucose homeostasis and appears to be associated with abdominal obesity in patients with diuretic therapy. [32] Because of the fundamental role of K in insulin secretion and carbohydrate metabolism, [33,34] higher K intake may play a effective role in improving insulin secretion and impaired fasting glucose [35] also proposed that K intake may effects on body weight by reducing inflammation, improvement in insulin sensitivity, [36] and beneficial effects on fat deposition/mobilization or energy balance. [37] ...
Backgrounds:
Dietary sodium (Na) and potassium (K) relationship with chronic disease has drawn more attention recently. Epidemiological studies reported controversial findings about high salt and Na diets with the risk of nonalcoholic fatty liver disease (NAFLD) and studies about the association between K and NAFLD are scare. Present study aimed to examine the associations between dietary intake of Na, K, and Na:K ratio with the risk of NAFLD.
Methods:
We analyzed data from a case-control study of 225 patients with NAFLD cases and 450 controls. Dietary intake of Na and K measured using a validated 168 item food frequency questionnaire. Adjusted logistic regression models were used to report odds ratio (OR) 95% confidence interval (CI) of NAFLD across tertiles of Na, K, and Na:K ratio.
Results:
The mean ± standard deviation of age and body mass index of participants (47% female) were 38.1 ± 8.8 years and 26.8 ± 4.3 Kg/m2. In the age- and sex-adjusted model, there was any significant association between Na, K, and Na: K ratio with the risk of NAFLD. In the final adjusted model, the OR (95%CI) of the highest vs the lowest tertiles of K, Na, and Na:K was 0.39 (0.19-0.80), 0.71 (0.40-1.25), and 1.10 (0.61-1.97), respectively.
Conclusion:
The present study indicates that higher dietary K was related to lower odds of NAFLD; however, there was no association between dietary Na and Na: K ratio with odds of NAFLD.
... Glycogen reserves, however, provide only a short-term supply of glucose and become especially critical beyond 18-24 h fasting (Klover et al., 2004). Over the years, evidence has mounted that under physiological conditions glucose utilisation by liver is rather limited and that glucose is in fact a poor precursor for glycogen (Boyd et al., 1981;Hue et al., 1975). This finding has been defined as the 'glucose paradox'. ...
... G1P is the substrate of the phosphoglucomutase, which transform it into glucose-6-P (G6P) and hence direct the metabolic machinery towards the production of ATP to regulate cellular functions such as muscle contraction [36]. However, the liver isoform hydrolyzes glycogen from internal liver stores to release G1P to maintain the physiological glucose levels in the bloodstream [37]. Further, the activation mechanisms are different; the liver isozyme is only activated by reversible phosphorylation of S15, while the other two isoforms can also be regulated by allosteric mechanisms (Figure 3). ...
McArdle disease, also known as glycogen storage disease type V (GSDV), is characterized by exercise intolerance, the second wind phenomenon, and high serum creatine kinase activity. Here, we recapitulate PYGM mutations in the population responsible for this disease. Traditionally, McArdle disease has been considered a metabolic myopathy caused by the lack of expression of the muscle isoform of the glycogen phosphorylase (PYGM). However, recent findings challenge this view, since it has been shown that PYGM is present in other tissues than the skeletal muscle. We review the latest studies about the molecular mechanism involved in glycogen phosphorylase activity regulation. Further, we summarize the expression and functional significance of PYGM in other tissues than skeletal muscle both in health and McArdle disease. Furthermore, we examine the different animal models that have served as the knowledge base for better understanding of McArdle disease. Finally, we give an overview of the latest state-of-the-art clinical trials currently being carried out and present an updated view of the current therapies.
... Glucose concentrations of 5.5 mM as a control solution and 20-30 mM as a relatively high solution are used to determine the effect of high glucose on cells [22,23]. Conversely, various glucose concentrations (e.g., 11 mM as a control solution and 50 mM as a relatively high solution) have been used depending on the cell species and purpose of the study [24][25][26][27]. In the experiments using human dermal fibroblast, cell proliferation and collagen synthesis were significantly increased in 15 mM than 5mM [28,29]. ...
The aim of this study is to investigate the mechanisms linking high glucose to gingival wound healing. Bilateral wounds were created in the palatal gingiva adjacent to maxillary molars of control rats and rats with streptozotocin-induced diabetes. After evaluating postsurgical wound closure by digital imaging, the maxillae including wounds were resected for histological examinations. mRNA expressions of angiogenesis, inflammation, and oxidative stress markers in the surgical sites were quantified by real-time polymerase chain reaction. Primary fibroblast culture from the gingiva of both rats was performed in high glucose and normal medium. In vitro wound healing and cell proliferation assays were performed. Oxidative stress marker mRNA expressions and reactive oxygen species production were measured. Insulin resistance was evaluated via PI3K/Akt and MAPK/Erk signaling following insulin stimulation using Western blotting. To clarify oxidative stress involvement in high glucose culture and cells of diabetic rats, cells underwent N-acetyl-L-cysteine treatment; subsequent Akt activity was measured. Wound healing in diabetic rats was significantly delayed compared with that in control rats. Nox1, Nox2, Nox4, p-47, and tumor necrosis factor-α mRNA levels were significantly higher at baseline in diabetic rats than in control rats. In vitro study showed that cell proliferation and migration significantly decreased in diabetic and high glucose culture groups compared with control groups. Nox1, Nox2, Nox4, and p47 expressions and reactive oxygen species production were significantly higher in diabetic and high glucose culture groups than in control groups. Akt phosphorylation decreased in the high glucose groups compared with the control groups. Erk1/2 phosphorylation increased in the high glucose groups, with or without insulin treatment, compared with the control groups. Impaired Akt phosphorylation partially normalized after antioxidant N-acetyl-L-cysteine treatment. Thus, delayed gingival wound healing in diabetic rats occurred because of impaired fibroblast proliferation and migration. Fibroblast dysfunction may occur owing to high glucose-induced insulin resistance via oxidative stress.
... As expected, each successive doubling in homogenate volume resulted in an approximate doubling in NADPH produced. Because glucose exerts feedback inhibition on Pyg activity [27], we added 5.0 mM glucose to duplicate reaction mixtures, which reduced total enzyme activity 65%. ...
Glycogen is a component of the nutrient rich, uterine glandular secretions (histrotroph) that support embryonic development prior to formation of the placenta. As a species exhibiting obligatory embryonic diapause, mink may produce 15 or more blastocysts, which do not implant until 60 days post coitum. Therefore, uterine glycogen reserves may play an essential role in reproductive success of mink. And yet, no systematic studies have been reported on uterine glycogen metabolism during the reproductive cycle. Therefore, our objectives were to (1): determine the glycogen content of the uterine endometrium, myometrium, glandular and luminal epithelia, during estrus, the peri-implantation period and post-implantation (pregnancy), and (2): correlate uterine glycogen content with expression of glycogen synthase (Gys1), glycogen phosphorylase (Pygm), glycogen synthase kinase-3beta (Gsk3b), hexokinase (HkI) and glucose-6-phosphatase (G6pc3). Mink uteri (n=3) were collected during estrus (day 0), the peri-implantation period (day 20) and pregnancy (day 31/32) from a local mink ranch. Total uterine glycogen concentrations decreased 55% between estrus and the peri-implantation period, and 85%, after implantation (P<0.01). The percent glycogen content of the endometrium (36.1± 2.01) and myometrium (31.6±1.20) at estrus was significantly reduced during the peri-implantation period (endometrium = 7.79± 1.51, myometrium = 17.49± 0.60; P<0.05), and was non-detectable during pregnancy at inter-implantation sites. The percent glycogen content of the glandular epithelia at estrus (31.92±6.9) decreased during the peri-implantation period to 0.96±0.08 and was undetectable during pregnancy (P<0.01). Similarly, the glycogen content of the luminal epithelia at estrus (34.9±1.6) decreased during the peri-implantation period (1.60±0.18) and was undetectable during pregnancy (P<0.01). Expression of Gys1 mRNA increased 40% during the peri-implantation period and 200% after implantation (P<0.05). Expression of the Pygm gene increased approximately 25% during the peri-implantation period (P<0.05) but did not differ between estrus and pregnancy. Uterine HkI mRNA expression was increased 75% during the peri-implantation period and 60% after implantation (P<0.05). The expression of G6pc3 mRNA increased 5-fold during the peri-implantation period and 3.5-fold during pregnancy (P<0.05). Our findings show that uterine glycogen reserves of the glandular and luminal epithelia were almost completely mobilized between estrus and the expected time of blastocyst implantation. It is therefore likely that optimal uterine glycogen accumulation, prior to mating, may be requisite to subsequent blastocyst survival and development, which could result in larger litter sizes. The simultaneous increase in Hk and Gys mRNA expression (which could promote glycogen storage) with increased Pyg and G6pc mRNA expression (contributing to glycogen catabolism and mobilization) may appear paradoxical. However, these enzyme expression patterns may be explained by the observation that both glucose and glycogen are secreted concomitantly into the uterine lumen. (Funded by Fur Commission USA & NIH INBRE P20RR016454)
(poster)
Glycogen phosphorylase (PG) is a key enzyme taking part in the first step of gly-cogenolysis. Muscle glycogen phosphorylase (PYGM) differs from other PG isoforms in expression pattern and biochemical properties. The main role of PYGM is providing sufficient energy for muscle contraction. However, it is expressed in tissues other than muscle, such as the brain, lym-phoid tissues, and blood. PYGM is important not only in glycogen metabolism, but also in such diverse processes as the insulin and glucagon signaling pathway, insulin resistance, necroptosis, immune response, and phototransduction. PYGM is implicated in several pathological states, such as muscle glycogen phosphorylase deficiency (McArdle disease), schizophrenia, and cancer. Here we attempt to analyze the available data regarding the protein partners of PYGM to shed light on its possible interactions and functions. We also underline the potential for zebrafish to become a convenient and applicable model to study PYGM functions, especially because of its unique features that can complement data obtained from other approaches.
The effect of insulin on hepatic glucose production has been studied in anesthetized rats in the postabsorptive state. Insulin decreases significantly hepatic glucose production within 5-10 min. It also increases the level of fructose 2,6-bisphosphate, via an increase in the Vmax of 6-phosphofructo-2-kinase and concomitantly decreased the activity of fructose-2,6-bisphosphatase, resulting in a 5-fold increase in the ratio of kinase/phosphatase. Insulin also increased the apparent Kd of pyruvate kinase for phosphoenolpyruvate. The changes in the activity of 6-phosphofructo-2-kinase and pyruvate kinase were measured after separation from possible modulators, and suggest a decrease in their phosphorylation state which cannot be attributed to a decrease in the level of cAMP or in the activity of cAMP-dependent protein kinase since these two parameters were not modified by insulin. In addition, neither the activity of phosphorylase a nor that of glycogen synthase were modified. The data strongly suggest that the increase in the glycolytic rate plays a role in the effect of insulin on hepatic glucose production and that insulin mediates its effect on the activity of these enzymes via one or more phosphatases.
The addition of glucose to a liver Sephadex filtrate causes a several fold increase in the rate of inactivation of phophorylase by phosphorylase phosphatase; this enzyme is also several times more active in liver preparations obtained from mice treated with glucocorticoids several hours before sacrifice. The glucose effect is less apparent in an unfiltered liver extract because of the high concentration of glucose and the inhibitory effect of AMP.
The effects of intravenous glucose, insulin and glucagon admininistration on the hepatic glycogen synthase and glycogen phosphorylase systems were assessed in the anesthetized rhesus monkey. Results were correlated with measurements of hepatic cyclic AMP (cAMP) concentrations and plasma glucose, insulin, and glucagon concentrations. Both glucose and insulin administration promoted significant inactivation of phosphorylase by 1 min, which was followed by more gradual activation of synthase. Neither glucose nor insulin caused significant changes in hepatic cAMP. Marked hyperglucagonemia resulting from insulin-induced hypoglycemia did not cause increases IN in hepatic cAMP, suggesting that the elevated insulin levels possibly inhibited glucagon action on the hepatic adenylate cyclase-cAMP system. Glucagon administration caused large increases in hepatic cAMP and activation of phosphorylase within 1 min, followed by more gradual inactivation of synthase when it had been previously activated by glucose. Concomitant glucose infusion, with resulting increased plasma insulin concentrations, markedly diminished the duration of hepatic cAMP elevations following glucagon adminstration, again suggesting an insulin inhibition of glucagon action on the hepatic adenylate-cAMP system.
The activity of liver phosphorylase b from several mammalian species has been studied. The enzyme from rat or mouse has a higher activity than the rabbit enzyme, which is itself more active than pig liver phosphorylase b .
The activity of liver phosphorylase b is influenced by anions and by AMP, and these effects are influenced by pH. Fluoride, which is currently added to the assay mixture of phosphorylase a in crude preparations, is about as active as sulfate as a stimulator of phosphorylase b .
When assayed at pH 6.1 and in the presence of 0.15 M NaF, the activity of rat liver phosphorylase b reaches 25% of that of the a enzyme; if 1 mM AMP is also present, this value rises to 50%.
Methods are described that allow the determination of liver phosphorylase a without interference of b , and the determination of total phosphorylase ( a + b ) in rat liver.
Summary Isolated hepatocytes and perfused rat livers respond to high external potassium concentrations with a remarkable potassium loss which is completely inhibited by ouabain. In contrast the liver swelling induced by high K+-concentrations in perfused livers is not influenced by ouabain.
The conversion of liver glycogen synthetase b into a in vivo, induced by glucose or by glucocorticoids, is further enhanced when both treatments are combined. Once activated, the enzyme is rapidly inactivated by an injection of glucagon, epinephrine or cyclic AMP. When given together with glucose, these substances prevent the usual activation of the enzyme.
The sensitivity of the mouse to glucagon was studied by the inactivation of liver glycogen synthetase and by the activation of liver phosphorylase; the first effect was obtained with lower doses than the second. On the other hand, about 100 times more glucagon was required to inactivate the synthetase previously activated by prednisolone than to prevent its activation by glucose. This difference in sensitivity can presumably not be explained by a different rate of destruction of cyclic AMP, as neither the total activity of the diesterase nor its affinity for the cyclic nucleotide is altered by the treatments. No evidence has been obtained suggesting that prednisolone lowers the sensitivity of the synthetase kinase towards cyclic AMP.
An increase in the perfusate glucose concentration from near zero to about 11 mM increased glycogen synthesis in the perfused, isolated rat liver from zero to a value about half the maximum seen in the intact animal. Increased synthesis appeard to due not only to provision of substrate but also to conversion of glycogen synthetase to the active form and of glycogen phosphorylase to the inactive form. These glucose effects, which are apparently independent of changes in levels of hormones or adenosine 3':5'-cyclic phosphate, may be physiologically significant for control of the blood glucose level.
In the isolated perfused rat liver administration of glucagon, or cyclic AMP, is followed by a biphasic response of K+ and Na+ movement. There is an initial uptake of these cations; during this period perfusate pH drops slightly. Subsequently an efflux of K+ and Na+ and a rise in pH of the perfusate occur. This alkalinization is associated with a decrease of intracellular pH. On the other hand, administration of tetracaine, a local anesthetic, is followed by a sharp drop of perfusate pH and intracellular alkalinization. This agent also interferes with the hormone of cyclic nucleotide-induced ion movements and metabolic effects. If the Na+ in the perfusate is replaced with an equimolar concentration of choline, glucagon or cyclic AMP-induced glycogenolysis and gluconeogenesis are inhibited. On the other hand, substitution of perfusate K+ by Na+ does not interfere with the glucagon or cyclic AMP-induced increase in glycogenolysis. Omission of either Na+ or K+ from the perfusate does not interfere with the glucagon-induced increase in cyclic AMP levels.
Rat liver hepatocytes were isolated by collagenase perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.
Synthetase D phosphatase activity in a liver glycogen pellet preparation is inhibited by ATP (physiological concentration). The inhibition can be reversed by glucose concentrations within the usual physiological range. Phosphophosphorylase activity decreases concomitantly with increasing synthetase I activity during the phosphatase incubation but the decrease is modest even in the presence of glucose. The glucose reversal of ATP inhibition is not the result of ATPase or glucokinase activities, which would reduce the ATP concentration. ATP, glucose and glucose 6-phosphate concentrations remain stable during the phosphatase assay.