Adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscle with increased glycogen content. (17/602)

The effect of glycogen content on the activation of glycogen phosphorylase during adrenaline stimulation was investigated in soleus muscles from Wistar rats. Furthermore, adrenergic activation of glycogen phosphorylase in the slow-twitch oxidative soleus muscle was compared to the fast-twitch glycolytic epitrochlearis muscle. The glycogen content was 96.4 +/- 4.4 mmol (kg dw)(-1) in soleus muscles. Three hours of incubation with 10 mU/ml of insulin (and 5.5 mM glucose) increased the glycogen content to 182.2+/-5.9 mmol (kg dw)(-1) which is similar to that of epitrochlearis muscles (175.7+/-6.9 mmol (kg dw)(-1)). Total phosphorylase activity in soleus was independent of glycogen content. Adrenaline (10(-6) M) transformed about 20% and 35% (P < 0.01) of glycogen phosphorylase to the a form in soleus with normal and high glycogen content, respectively. In epitrochlearis, adrenaline stimulation transformed about 80% of glycogen phosphorylase to the a form. Glycogen synthase activation was reduced to low level in soleus muscles with both normal and high glycogen content. In conclusion, adrenaline-mediated glycogen phosphorylase activation is enhanced in rat soleus muscles with increased glycogen content. Glycogen phosphorylase activation during adrenaline stimulation was much higher in epitrochlearis than in soleus muscles with a similar content of glycogen.  (+info)

Calorie restriction in nonhuman primates: mechanisms of reduced morbidity and mortality. (18/602)

Long term chronic calorie restriction (CR) of adult nonhuman primates significantly reduces morbidity and increases median age of death. The present review is focused upon an ongoing study of sustained adult-onset calorie restriction, which has been underway for 15 years. Monkeys, initially calorie restricted at about 10 years of age, are now approximately 25 years old. The median life span of these restricted monkeys is increasing, now exceeding that of ad libitum (AL)-fed monkeys. In our laboratory, maximum life span for AL-fed monkeys appears to be about 40 years. Thus, whether CR can also increase maximal life span, as it does in rodents, cannot be determined for at least another 15 years. The earliest detectable positive benefit on morbidity in these monkeys was previously reported as the prevention of obesity. Current evidence, as reviewed here, suggests that much obesity-associated morbidity is also mitigated by sustained calorie restraint in nonhuman primates. Furthermore, probably because of the prevention of obesity, diabetes has also been prevented. Recent findings include the identification of extraordinary changes in the glycogen synthesis pathway, and on the phosphorylation of glycogen synthase in response to insulin. This calorie restriction-induced prevention of morbidity does not require excessive leanness, but is clearly present when body fat is within the normal range of 10 to 22%, and this is likely to be true in humans as well.  (+info)

Cyclic AMP mediates the elevation of proline by AKH peptides in the cetoniid beetle, Pachnoda sinuata. (19/602)

The role of cyclic nucleotides in the transduction of the hyperprolinaemic and hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata. Flight and injection of Mem-CC into the haemocoel of the beetle induce an increase of cAMP levels in the fat body of the beetle. This increase is tissue-specific and does not occur in brain and flight muscles. An elevation of cAMP levels was also found when in vitro preparations of fat body tissue were subjected to Mem-CC. Elevation of the cAMP concentration after injection of Mem-CC is time- and dose-dependent: the maximum response is measured after 1 min, and a dose of 25 pmol Mem-CC is needed. Injection of cpt-cAMP, a cAMP analogue which penetrates the cell membrane, causes a stimulation of proline synthesis but no mobilisation of carbohydrate reserves. The same is measured when IBMX, an inhibitor of phosphodiesterase, is injected. cGMP seems not to be involved in synthesis of proline nor carbohydrate release, because injection of cpt-cGMP has no influence on the levels of proline, alanine and carbohydrates in the haemolymph. Although glycogen phosphorylase of the fat body is activated by Mem-CC in a time- and dose-dependent manner, it cannot be stimulated by cpt-cAMP. The combined data suggest that cAMP is involved in regulation of proline levels by Mem-CC but not in regulation of carbohydrates. Octopamine has no effect on metabolites in the haemolymph and is not capable of activating glycogen phosphorylase, indicating that it is not involved in the regulation of substrates in this beetle. Furthermore, the requirements of the receptor of Mem-CC are different for eliciting a hypertrehalosaemic and a hyperprolinaemic effect, respectively, suggesting that differentiation in signal transduction begins at the receptor level.  (+info)

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. (20/602)

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.  (+info)

Control of glycogen synthesis is shared between glucose transport and glycogen synthase in skeletal muscle fibers. (21/602)

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.  (+info)

Effect of induced metabolic alkalosis on human skeletal muscle metabolism during exercise. (22/602)

The purpose of the study was to examine the roles of active pyruvate dehydrogenase (PDH(a)), glycogen phosphorylase (Phos), and their regulators in lactate (Lac(-)) metabolism during incremental exercise after ingestion of 0.3 g/kg of either NaHCO(3) [metabolic alkalosis (ALK)] or CaCO(3) [control (CON)]. Subjects (n = 8) were studied at rest, rest postingestion, and during constant rate cycling at three stages (15 min each): 30, 60, 75% of maximal O(2) uptake (VO(2 max)). Radial artery and femoral venous blood samples, leg blood flow, and biopsies of the vastus lateralis were obtained during each power output. ALK resulted in significantly (P < 0.05) higher intramuscular Lac(-) concentration ([Lac(-)]; ALK 72.8 vs. CON 65.2 mmol/kg dry wt), arterial whole blood [Lac(-)] (ALK 8.7 vs. CON 7.0 mmol/l), and leg Lac(-) efflux (ALK 10.0 vs. CON 4.2 mmol/min) at 75% VO(2 max). The increased intramuscular [Lac(-)] resulted from increased pyruvate production due to stimulation of glycogenolysis at the level of Phos a and phosphofructokinase due to allosteric regulation mediated by increased free ADP (ADP(f)), free AMP (AMP(f)), and free P(i) concentrations. PDH(a) increased with ALK at 60% VO(2 max) but was similar to CON at 75% VO(2 max). The increased PDH(a) may have resulted from alterations in the acetyl-CoA, ADP(f), pyruvate, NADH, and H(+) concentrations leading to a lower relative activity of PDH kinase, whereas the similar values at 75% VO(2 max) may have reflected maximal activation. The results demonstrate that imposed metabolic alkalosis in skeletal muscle results in acceleration of glycogenolysis at the level of Phos relative to maximal PDH activation, resulting in a mismatch between the rates of pyruvate production and oxidation resulting in an increase in Lac(-) production.  (+info)

1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP as probes of the activator site of glycogen phosphorylase from rabbit skeletal muscle. (23/602)

Both 1,N6-etheno-AMP and 1,N6-etheno-2'-deoxy-AMP bind at the AMP site of phosphorylase b (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC 2.4.1.1). Etheno-AMP induces the same activation as AMP, about 30-fold higher than the activation induced by etheno-dAMP. The fluorescence of etheno-AMP and etheno-dAMP is associated with the base moiety; therefore, when free in solution, the two derivatives have identical fluorescence properties. However, when bound to phosphorylase, the fluorescence of etheno-AMP is quenched more efficiently than the fluorescence of etheno-dAMP. This difference between the fluorescence properties of the bound nucleotides suggests that a modification in the ribose ring affects the position of the adenine in the AMP site of phosphorylase b. The observed quenching may be due to a stacking interaction between an aromatic residue and the base moiety of the bound nucleotide.  (+info)

The manifold of vitamin B6 dependent enzymes. (24/602)

Pyridoxal-5'-phosphate (vitamin B6) binding enzymes form a large superfamily that contains at least five different folds. The availability of an increasing number of known three-dimensional structures for members of this superfamily has allowed a detailed structural classification. Most progress has been made with the fold type I or aspartate aminotransferase family.  (+info)