1H NMR study of metabolic alterations in the small intestine of rats infected with Hymenolepis diminuta

• 1.1. 1H NMR spectra of the duodenum, jejunum and ileum tissues of the small intestine of a rat showed metabolic gradients.
• 2.2. The concentrations of metabolites in these gut regions were altered by the presence of the tapeworm Hymenolepis diminuta.
• 3.3. In the infected duodenum there was significantly less glycogen, glucose and phosphocreatine/creatine, but significantly more lactate than in the corresponding controls.
• 4.4. Infected jejunum contained significantly less betaine but significantly more succinate, alanine and lactate.
• 5.5. Infected ileum had significantly less glycogen and taurine but significantly more alanine and lactate.

     

The effect of time on physiological changes in eel Anguilla anguilla,induced by lindane

• 1.1. Eel were exposed to a sublethal concentration of lindane (0.335 ppm) for 6, 12, 24, 48, 72 and 96 hr.
• 2.2. Concentrations of glycogen, glucose, lactate, pyruvate and lipids were determined in gill tissue after lindane exposure.
• 3.3. Gill glycogen descreased and glucose levels increased at 6 hr of treatment, lactate and pyruvate concentration increased between 6 and 48 hr. Total lipid values decreased between 6 and 24 hr; thereafter, the levels increased up to 72 hr of exposure.
• 4.4. Clear changes were found in all parameters tested in gill tissues. The observed effects of lindane on metabolism in fish are discussed in relation to acute stress syndrome.

     

Liver glycogen in type 2 diabetic mice is randomly branched as enlarged aggregates with blunted glucose release

Glycogen is a vital highly branched polymer of glucose that is essential for blood glucose homeostasis. In this article, the structure of liver glycogen from mice is investigated with respect to size distributions, degradation kinetics, and branching structure, complemented by a comparison of normal and diabetic liver glycogen. This is done to screen for differences that may result from disease. Glycogen α-particle (diameter ～ 150 nm) and β-particle (diameter ～ 25 nm) size distributions are reported, along with in vitro γ-amylase degradation experiments, and a small angle X-ray scattering analysis of mouse β-particles. Type 2 diabetic liver glycogen upon extraction was found to be present as large loosely bound, aggregates, not present in normal livers. Liver glycogen was found to aggregate in vitro over a period of 20 h, and particle size is shown to be related to rate of glucose release, allowing a structure-function relationship to be inferred for the tissue specific distribution of particle types. Application of branching theories to small angle X-ray scattering data for mouse β-particles revealed these particles to be randomly branched polymers, not fractal polymers. Together, this article shows that type 2 diabetic liver glycogen is present as large aggregates in mice, which may contribute to the inflexibility of interconversion between glucose and glycogen in type 2 diabetes, and further that glycogen particles are randomly branched with a size that is related to the rate of glucose release.     

The properties and potential metabolic role of glucokinase in halotolerant obligate methanotroph <Emphasis Type="Italic">Methylomicrobium alcaliphilum</Emphasis> 20Z

Aerobic bacteria utilizing methane as the carbon and energy source do not use sugars as growth substrates but possess the gene coding for glucokinase (Glk), an enzyme converting glucose into glucose 6-phosphate. Here we demonstrate the functionality and properties of Glk from an obligate methanotroph Methylomicrobium alcaliphilum 20Z. The recombinant Glk obtained by heterologous expression in Escherichia coli was found to be close in biochemical properties to other prokaryotic Glks. The homodimeric enzyme (2 × 35 kDa) catalyzed ATP-dependent phosphorylation of glucose and glucosamine with nearly equal activity, being inhibited by ADP (K _i = 2.34 mM) but not affected by glucose 6-phosphate. Chromosomal deletion of the glk gene resulted in a loss of Glk activity and retardation of growth as well as in a decrease of intracellular glycogen content. Inactivation of the genes encoding sucrose phosphate synthase or amylosucrase, the enzymes involved in glycogen biosynthesis via sucrose as intermediate, did not prevent glycogen accumulation. In silico analysis revealed glk orthologs predominantly in methanotrophs harboring glycogen synthase genes. The data obtained suggested that Glk is implicated in the regulation of glycogen biosynthesis/degradation in an obligate methanotroph.     

The effect of the desert cobra (Walterinnesia aegyptia) crude venom and its protein fractions on the metabolic activity of cultured human fibroblasts

Fibroblast cultures were used to study the effect of crude venom and six venom protein fractions (F₂–F₇) fromWalterinnesia aegyptia) on their metabolic activity. This was done by incubation of six fibroblast cultures with 10 g of crude venom for 3 h at 37°C. The activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase were significantly lowered upon incubation with all fractions except F₂. Glycogen phosphorylase activity was significantly increased, leading to a significant concurrent drop of glycogen content. This effect was only seen for fractions F₃ and F₅. Creatine kinase activity and cellular ATP levels rose significantly upon incubation with all venom proteins except fractions F₂ and F₇. Increases were seen for aspartate and alanine amino-transferases by all venom proteins except fractions F₂ and F₄. Incubation of cell sonicates with all the venom proteins did not significantly alter activities of any of the parameters. Thus, fibroblasts in culture under such conditions appear to mobilize glycogen, phosphocreatine, and protein for ATP production to compensate for decreased glucose.Abbreviations ALT alanine aminotransferase - AST aspartate aminotransferase - ATP adenosine 5-triphosphate - CS citrate synthhase - GP glycogen phosphorylase - LDH lactate dehydrogenase - PFK phosphofructokinase     

Seasonal changes in the utilisation of 14C- and 3H-labelled glucose in a mantle tissue slice preparation of Mytilus edulis L.

• 1.1. Seasonal changes in ¹⁴C- and ³H-labelled glucose metabolism were studied in an in vitro preparation of the mantle tissue from Mytilus edulis L. throughout 1978–1979.
• 2.2. Incorporation of [1-¹⁴C] and [6-¹⁴C]glucose into glycogen and amino acids peaked in the summer, resulting in an increased rate of glucose utilisation. [2-³H]glucose utilisation data agreed with this finding.
• 3.3. Pentose phosphate pathway activity reached a maximum in the spring of 1979, but represented only a very small fraction of the total glucose utilisation.
• 4.4. In the winter, and during starvation experiments, the capacity for exogenous glucose utilisation fell, with a compensatory increase in tissue glycogen degradation. The contribution of the Embden-Meyerhof pathway to total carbohydrate metabolism appeared to remain stable throughout the year.

     

The glycogen content in stressed marine bivalves: The initial absence of a decrease

• 1.1. Changes in the glycogen content, condition, stomach content and acetic acid concentration of mussels Mytilus edulis and cockles Cerastoderma edule were followed during periods of up to 14 days of exposure (to air) at temperatures of 5 and 20°C.
• 2.2. In animals with a high glycogen content the glycogen is not used during the first 3 to 7 days, at high and low temperature respectively.
• 3.3. After this latent period the glycogen concentration often decreased, coinciding with a high mortality and an increase of the concentration of acetic acid.
• 4.4. In cockles with a low glycogen content, and kept at a high temperature, glycogen can be used from the beginning of the stress period.
• 5.5. Between species no clear differences were found.
• 6.6. The stomach content decreased during exposure; however, the stomach content amounted to only 0.5 to 0.7% of the body weight, and is thought to be of minor importance as an energy source during the stress period.
• 7.7. Especially at the higher temperatures glycogen finally is transformed into acetic acid.
• 8.8. It is concluded that during exposure, the animals do not die because of a lack of energy reserves, but because of a high accumulation of acids.

     

Effect of exposure to atmospheric air on blood glucose and lactate concentrations in two crustacean species: A role of the Crustacean hyperglycemic hormone (CHH)

• 1.1. In intact (control) crabs (Carcinus maenas) and crayfish (Orconectes limosus) a significant (P < 0.01) increase in both glucose and lactate concentrations in the blood was observed after exposure to air. Such changes were not observed in either eyestalk-less or eyestalk-less and saline injected animals (P > 0.05).
• 2.2. Injections of Crustacean hyperglycemie hormone (CHH) into eyestalk-less animals before exposure to air were able to reverse the effects of eyestalk ablation, i.e., significant increases (P < 0.01) in blood glucose and lactate could again be observed.
• 3.3. Significant hyperglycemia (P < 0.01), but no changes in lactate concentration (P > 0.05), was observed after injection of CHH in eyestalk-less submerged animals.
• 4.4. These results suggest that the increase in glycolysis after air exposure is facilitated by CHH, possibly by increased substrate availability due to glycogen degradation.

     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—I. Utilization of glycogen and accumulation of glycolytic end products in various tissues

• 1.1. Resting oxygen consumption at 10°C did not change from normoxia (150 mm Hg) down to an oxygen tension of 55 mm Hg for the flounder, Platichtys flesus.
• 2.2. Flounders exposed to hypoxia showed increased levels of blood glucose and lactate, dependent on the degree of hypoxia.
• 3.3. Due to hypoxia glycogen was depleted in the liver and swimming muscle but in the heart there was no significant change.
• 4.4. Liver glucose increased after 7 hr of hypoxia. Heart and muscle glucose did not change but the absolute glucose concentration in the heart was five times higher than in the muscle.
• 5.5. There is a transient accumulation of lactate in heart, liver and kidney after 7 hr of hypoxia while lactate accumulation in the swimming muscle is significant only after 21 hr of hypoxia.
• 6.6. Succinate only accumulated in the liver while alanine accumulated in muscle, heart and liver.

     

Fetal lung metabolism: Response to maternal fasting

• 1.1. Fetal lung metabolic response to maternal fasting late in gestation was investigated.
• 2.2. Maternal fasting 4 days before term was associated with low fetal plasma glucose and insulin levels but increased levels of fetal plasma glucagon, glycerol, lactate and fatty acids.
• 3.3. Fetuses from fasted mothers showed a significant decrease in body weight (30%), lung weight (30%) and lung glycogen (46%), but no change in lung protein, phospholipid or total lung DNA, suggesting that lung size is affected more than maturation.
• 4.4. Fetal lung slices incubated in vitro showed that lactate oxidation to CO₂ equalled that of glucose in control fetal lungs and was unaffected by maternal fasting, while glucose oxidation was depressed (23%).
• 5.5. Maternal fasting significantly decreased in vitro incorporation of [U-¹⁴C]-glucose, [U-¹⁴C]lactate and [1-¹⁴C]palmitate into lung phospholipids.
• 6.6. Fetal lungs from fasted mothers showed increased conversion of lactate to glucose, indicating gluconeogenic potential by fetal lung.
• 7.7. These studies show that plasma lactate serves as an important energy fuel and substrate for lipid synthesis for the fetal lung, and maternal fasting markedly alters fetal lung metabolism.

     

Glycogen synthetase in the sea mussel Mytilus edulis L.—II. Seasonal changes in glycogen content and glycogen synthetase activity in the mantle tissue

• 1.1. The glycogen content of the mantle tissue reached a maximum in the summer (May–July) with levels of 41.0–53.5% of the dry tissue weight.
• 2.2. Seasonal changes in glycogen synthetase activity showed that the I-activity (independent of G6P) increased up to 10-fold in June as compared with December. The measured I-activity of glycogen synthetase was sufficient to account for the accumulation of mantle glycogen in the summer.
• 3.3. The I-activity of glycogen synthetase declined rapidly in July of each year. A possible role for the inhibition of glycogen synthetase by high levels of tissue glycogen is suggested.
• 4.4. The I-activity in the mantle tissue of mussels on the shore was higher than that for animals starved in the laboratory for 2–3 days. The differences were minimal in early May but increased markedly in late May–July. Starved mussels returned to the shore showed an increase in I-activity of glycogen synthetase.
• 5.5. Injection of 30 μmol glucose into the adductor muscle increased the concentration of glucose in the mantle fluid to 2.0–2.5 mM. A similar injection of 60 μ mol glucose resulted in a time-dependent increase in the I-activity of glycogen synthetase.
• 6.6. Injection of mussels with mammalian insulin or anti-insulin serum had no effect on the activity of glycogen synthetase. Our results are at variance with those of other workers who have used the mammalian hormone in molluscan studies (see Discussion).

     

Is there a link between impaired glucose metabolism and protein kinase C activity in the diabetic heart?

The activity of the isoform of protein kinase C (PKC) is reduced in the diabetic heart. Since this isozyme has been implicated in insulin action, we tested the hypothesis that PKC contributes to the development of impaired glucose metabolism by the noninsulin-dependent diabetic heart. Exposure of the diabetic heart to buffer containing the protein kinase C activator, phorbol myristate acetate, increased PKC activity in the membrane. Associated with the improvement in PKC activity was a biphasic change in glucose metabolism. The initial phase was characterized by a breakdown in glycogen stores, a stimulation in glucose oxidation and a decrease in endogenous fatty acid oxidation. This was followed by a second phase in which the uptake of glucose was modestly stimulated. Nonetheless, since the phorbol ester did not overcome the diabetes-linked defect in pyruvate dehydrogenase, the increase in glycolytic flux was not associated with a rise in glucose oxidation. Consequently, nearly 50% of the triose units were diverted into lactate and pyruvate production and the generation of ATP from glucose was restricted. Since insulin promotes not only glucose uptake, but also glycogen synthesis and glucose oxidation, the phorbol ester and insulin effects are very different. Thus, the data do not support a role for PKC in the development of glucose metabolic defects in the hearts of noninsulin-dependent diabetic rats.     

Differential Regulation of Glycogenolysis by Mutant Protein Phosphatase-1 Glycogen-targeting Subunits

PTG and G_L are hepatic protein phosphatase-1 (PP1) glycogen-targeting subunits, which direct PP1 activity against glycogen synthase (GS) and/or phosphorylase (GP). The C-terminal 16 amino residues of G_L comprise a high affinity binding site for GP that regulates bound PP1 activity against GS. In this study, a truncated G_L construct lacking the GP-binding site (G_Ltr) and a chimeric PTG molecule containing the C-terminal site (PTG-G_L) were generated. As expected, GP binding to glutathione S-transferase (GST)-G_Ltr was reduced, whereas GP binding to GST-PTG-G_L was increased 2- to 3-fold versus GST-PTG. In contrast, PP1 binding to all proteins was equivalent. Primary mouse hepatocytes were infected with adenoviral constructs for each subunit, and their effects on glycogen metabolism were investigated. G_Ltr expression was more effective at promoting GP inactivation, GS activation, and glycogen accumulation than G_L. Removal of the regulatory GP-binding site from G_Ltr completely blocked the inactivation of GS seen in G_L-expressing cells following a drop in extracellular glucose. As a result, G_Ltr expression prevented glycogen mobilization under 5 mm glucose conditions. In contrast, equivalent overexpression of PTG or PTG-G_L caused a similar increase in glycogen-targeted PP1 levels and GS dephosphorylation. Surprisingly, GP dephosphorylation was significantly reduced in PTG-G_L-overexpressing cells. As a result, PTG-G_L expression permitted glycogenolysis under 5 mm glucose conditions that was prevented in PTG-expressing cells. Thus, expression of constructs that contained the high affinity GP-binding site (G_L and PTG-G_L) displayed reduced glycogen accumulation and enhanced glycogenolysis compared with their respective controls, albeit via different mechanisms.Hepatic glycogen metabolism plays a central role in the maintenance of circulating plasma glucose levels under various physiological conditions. The rate-controlling enzymes in glycogen metabolism, glycogen synthase (GS)² and glycogen phosphorylase (GP), are subject to multiple levels of regulation, including allosteric binding of activators and inhibitors, protein phosphorylation, and changes in subcellular localization. GS is phosphorylated on up to 9 residues by a variety of kinases, although site 2 appears to be the most important regulator of hepatic GS (1). In contrast, GP is phosphorylated on a single N-terminal serine residue by phosphorylase kinase, which increases GP activity and its sensitivity to allosteric activators. Both GS and GP are in turn also regulated by protein phosphatases, most notably PP1. Although PP1 is a cytosolic protein, a family of five molecules has been reported that targets the enzyme to glycogen particles (2–7), whereas another two glycogen-targeting subunits have been putatively identified based on sequence homology (8). Published work has indicated that each targeting subunit confers differential regulation of PP1 activity by extracellular hormonal signals and/or intracellular changes in metabolites (9–11).Four PP1-glycogen-targeting proteins are expressed in rodent liver, although G_L and PTG/R5 have been most extensively studied (9, 12–15). G_L is present at higher levels in rat liver than PTG (12), but the expression of both proteins is subject to coordinate regulation by fasting/refeeding and insulin (12, 13). Previous studies indicated that the PTG-PP1 complex is primarily responsible for GP dephosphorylation and regulation of glycogenolysis (13, 16), whereas the G_L-PP1 complex preferentially mediates the activation of GS upon elevation of extracellular glucose (9, 13). However, the molecular mechanisms underlying these differential properties of PTG and G_L have not been completely defined.Both PTG and G_L directly bind to specific PP1 substrates involved in glycogen metabolism, albeit for different physiological reasons. The extreme C-terminal 16 amino acids of G_L comprises a unique, high affinity binding site for phosphorylated GP (GPa (17)), which has been further delineated to two critical tyrosine residues (18, 37). Interaction of PP1 with G_L reduces phosphatase activity against GPa (3). In turn, GPa binding to the G_L-PP1 complex potently inhibits phosphatase activity against GS in vitro (3, 19) and regulates glycogen-targeted PP1 activity in liver cells and extracts (20–22). PTG contains a single substrate-binding site that interacts with GS and GP (5, 23). In contrast to the regulatory role of the GPa binding to G_L, interaction of substrates with PTG increases PP1 activity against these proteins (24). Indeed, disruption of the substrate-binding site by point mutagenesis abrogated the ability of mutant PTG expression to increase cellular glycogen levels (23), indicating an important role for substrate binding to the PTG-PP1 complex.Previous work has comprehensively compared the metabolic impact of PTG versus G_L overexpression in hepatocytes and thus was not the goal of this study (9, 10). Instead, two novel PP1 targeting constructs were generated in which the high affinity GPa-binding site was removed from G_L or added to the C terminus of PTG. The effects of expressing wild-type and mutant constructs on GS and GP activities and on the regulation of glycogen metabolism by extracellular glucose were investigated using primary mouse hepatocytes.     

Interaction of lipogenic substrates in porcine adipose tissue in vitro

• 1.1. Porcine adipose tissue was incubated with radiolabeled glucose, acetate or lactate. Saturation curves indicated that lactate > glucose > acetate in providing two-carbon units for fatty-acid synthesis.
• 2.2. Competition between individual substrates indicated that lactate was the best lipogenic substrate.
• 3.3. Incubation of all three substrates at concentrations observable in serum indicated that at 5.56mM, glucose was the preferred lipogenic substrate in the presence of 0.1 mM acetate and 1.0 mM lactate.
• 4.4. At elevated concentrations (18.52mM glucose, 1.0 mM acetate and 10.0 mM lactate), acetate and lactate were preferred to glucose as lipogenic substrates.