Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Activation of hepatocyte glycogen synthase by metabolic inhibitors

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.     

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P-labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB-2 phosphorylation was increased by all three hormones while CB-1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2+-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase II (PC0.7) and FA/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK-3, is involved or phosphatase activity is regulated, or both.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

EFFECT OF VITAMIN E SUPPLEMENTATION AND PARTIAL SUBSTITUTION OF POLY- WITH MONO-UNSATURATED FATTY ACIDS IN PIG DIETS ON MUSCLE,AND MICROSOME EXTRACT a-TOCOPHEROL CONCENTRATION AND LIPID OXIDATION

The experiment was organized in a 3×2 factorial arrangement with three dietary fat blends and a basal (20 mg kg^?1 diet) or supplemented (220 mg kg^?1) level of α-tocopheryl acetate. Dietary vitamin E and monounsaturated to polyunsaturated fatty acid ratio (dietary MUFA/PUFA) affected muscle α-tocopherol concentration (α-tocopherol [log μg g^?1]=0.18 (±0.105)+0.0034 (±0.0003)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.39 (±0.122)·dietary MUFA/PUFA (P<0.0036)). An interaction between dietary α-tocopherol and dietary MUFA/PUFA exists for microsome α-tocopherol concentration (α-tocopherol [log μg g^?1]=1.14 (±0.169) (P<0.0001)+0.0056 (±0.00099)·dietary α-tocopherol [mg kg^?1 diet] (P<0.0001)+0.54 (±0.206)·dietary MUFA/PUFA (P<0.0131)?0.0033 (±0.0011)·dietary α-tocopherol [mg kg^?1)]×dietary MUFA/PUFA (P<0.0067)), and hexanal concentration in meat (hexanal [ng·g^?1]=14807.9 (±1489.8)?28.8 (±10.6) dietary α-tocopherol [mg·kg^?1] (P<0.01)?8436.6 (±1701.6)·dietary MUFA/PUFA (P<0.001)+24.0 (±11.22)·dietary α-tocopherol·dietary MUFA/PUFA (P<0.0416)). It is concluded that partial substitution of dietary PUFA with MUFA lead to an increase in the concentration of α-tocopherol in muscle and microsome extracts. An interaction between dietary α-tocopherol and fatty acids exists, in which at low level of dietary vitamin E inclusion, a low MUFA/PUFA ratio leads to a reduction in the concentration of α-tocopherol in microsome extracts and a concentration of hexanal in meat above the expected values.     

Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Quirks of dye nomenclature. 6. Malachite green

Malachite green was discovered independently by two researchers in Germany in the 19^th century and found immediate employment as a dye and a pigment. Subsequently, other uses, such as staining biological specimens, emerged. A much later application was the control of fungal and protozoan infections in fish, for which the dye remains popular, although illegal in many countries owing to a variety of toxicity problems. In solution, malachite green can exist as five different species depending on the pH. The location of the positive charge of the colored cation on a carbon atom or a nitrogen atom is still debated. The original names of this dye, and their origins, are briefly surveyed.     

Morphological and agronomical diversity patterns in the Spanish barley core collection

Seven thousand years of barley cultivation under the environmental hardships typical of the Mediterranean climate have generated genetic singularity of the Spanish barleys, consistently reported in the literature. From the Spanish National Collection of 2289 accessions, a core subset with 159 landraces and 16 old varieties was constituted. Twenty-seven characters were evaluated for the core collection, to define the structure of the diversity. Several evaluation trials were carried out in 1999-2000, whereas yield trials were performed in earlier years. Phenotypic diversity was large for most of the characters studied. Comparisons of genetic diversity between the core and the original collections suggested that the core is a good representation of the existing diversity in the BNG. Comparisons with results of studies on Spanish materials from other collections seem to indicate that the Spanish diversity is not well represented in some world collections. Principal component analyses for quantitative and qualitative characters revealed a clear distinction between two- and six-row cultivars, and also between landraces and commercial varieties. Geographical origins of the landraces were correlated with grain yield, heading date, duration of grain filling period, and growth class. In relation to diseases, altitude played an important role on the resistance to powdery mildew and brown rust. For brown rust, all the resistant landraces came from low altitudes. These geographical gradients seemed consistent with prior knowledge about barley adaptation, and would confirm the agreement between passport data and true adaptive origin of these landraces from a geographical point of view.