Effects of chronic AICAR administration on the metabolic and contractile phenotypes of rat slow- and fast-twitch skeletal muscles

The present study examined the effects of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the oxidative capacity and myosin heavy chain (MHC) based fibre phenotype of rodent fast- and slow-twitch muscles. Sprague-Dawley rats received daily injections for 4 weeks of the known AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or vehicle (control). The AICAR group displayed increases in hexokinase-II (HXK-II) activity, expression, and phosphorylation in fast-twitch muscles (P<0.001) but not in the slow-twitch soleus (SOL). In the AICAR group, citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35) were elevated 1.6- and 2.1-fold (P<0.05), respectively, in fast-twitch medial gastrocnemius (MG), and by 1.2- and 1.4-fold (P<0.05) in the slower-twitch plantaris (PLANT). No changes were observed in the slow-twitch SOL. In contrast, the activity of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) remained unchanged in all muscles. AICAR treatment did not alter the MHC-based fibre type composition in fast- or slow-twitch muscles, as determined by immunohistochemical and electrophoretic analytical methods or by RT-PCR. We conclude that chronic activation of AMPK mimics the metabolic changes associated with chronic exercise training (increased oxidative capacity) in the fast-twitch MG and PLANT, but does not coordinately alter MHC isoform content or mRNA expression.     

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org      

Actin Disruption by Latrunculin B Causes Turgor-Related Changes in Tip Growth of Saprolegnia ferax Hyphae

Hyphae of Saprolegnia ferax growing under normal or low-turgor conditions were exposed to 0.1-10 &mgr;g/ml latrunculin B, an actin inhibitor. In the first 10 s of addition, hyphae with normal turgor levels accelerated while those with low turgor decelerated, consistent with the suggestion that actin restrains or protrudes tips under these respective turgor conditions. Both sets of hyphae then decelerated and eventually ceased extension within 60 s. These changes were reflected in rhodamine-phalloidin staining patterns, which showed that actin caps were disrupted progressively under both conditions in a time-dependent manner. After 60 s, normal-turgored hyphae started to swell rapidly while low-turgored hyphae showed little or no swelling. Swelling was characteristically subapical, which is best explained by tip growth models which incorporate actin-mediated exocytosis.     

Glucose metabolism,H+ production and Na+/H+- exchanger mRNA levels in ischemic hearts from diabetic rats

Glycolysis uncoupled from glucose oxidation is a major reason for the intracellular acidosis that occurs during severe myocardial ischemia. The imbalance between glycolysis and glucose oxidation, and the resultant H⁺ produced from glycolytically derived ATP hydrolysis in the diabetic rat heart is the focus of this study. Isolated working hearts from 6 week streptozotocin diabetic rat hearts were perfused with 11 mM glucose and 1.2 mM palmitate and subjected to a 25 min period of global ischemia. A second series of experiments were also performed in which hearts from control, diabetic, and islet-transplanted diabetic rats were subjected to a 30 min aerobic perfusion, followed by a 60 min period of low-flow ischemia (coronary flow = 0.5 ml/min) and 30 min of aerobic reperfusion. H⁺ production from glucose metabolism was measured throughout the two protocols by simultaneous measurement of glycolysis and glucose oxidation using perfusate labelled with [5-³H/U-¹⁴C]-glucose. Rates of H⁺ production were calculated by measuring the difference between glycolysis and glucose oxidation. The H⁺ production throughout the perfusion was generally lower in diabetic rat hearts compared to control hearts, while islet-transplantation of diabetic rats increased H⁺ production to rates similar to those seen in control hearts. This occurred primarily due to a dramatic increase in the rates of glycolysis. Despite the difference in H⁺ production between control, diabetic and islet-transplanted diabetic rat hearts, no difference in mRNA levels of the cardiac Na⁺/H⁺-exchanger (NHE-1) was seen. This suggests that alterations in the source of protons (i.e. glucose metabolism) are as important as alterations in the fate of protons, when considering diabetes-induced changes in cellular pH. Furthermore, our data suggests that alterations in Na⁺/H⁺-exchange activity in the diabetic rat heart occur at a post-translational level, possibly due to direct alterations in the sarcolemmal membranes.     

Maturation of fatty acid and carbohydrate metabolism in the newborn heart

During fetal life, myocardial ATP is derived predominantly from glycolysis and lactate oxidation. Following birth, a rapid maturational increase in fatty acid oxidation occurs along with a decline in glycolytic and lactate oxidative rates, thus changing the major source of myocardial ATP production. This shift in energy substrate preference occurs in response to changes in the circulating substrate content of newborn plasma with the onset of suckling, and is also due to alterations in circulating levels of hormones, such as insulin and glucagon. Important changes in subcellular regulatory mechanisms of both fatty acid and carbohydrate metabolism in the heart also characterize this response. This review deals with recent advances in the understanding of these subcellular mechanisms which regulate this important shift in myocardial energy metabolism, with particular emphasis on the molecular events occurring in the heart during the transition from fetal to newborn life.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Polarized Growth of Fungal Hyphae Is Defined by an Alkaline pH Gradient

Robson, G. D., Prebble, E., Rickers, A., Hosking, S., Denning, D. W., Trinci, A. P. J., and Robertson, W. 1996. Polarized growth of fungal hyphae is defined by an alkaline pH gradient. Fungal Genetics and Biology 20, 289-298. Polarized cell growth is exhibited by a diverse range of eukaryotic and prokaryotic cells. The events which are responsible for this growth are poorly understood. However, the existence of ion gradients may play an important role in establishing and driving cell polarity. Using a pH-sensitive, ratiometric fluorescent dye to monitor intracellular pH in growing fungal hyphae, we report a gradient at the extending hyphal tip that is up to 1.4 pH units more alkaline than more distal regions. Both the magnitude and the length of the pH gradient were strongly correlated with the rate of hyphal extension and eradication of the gradient-arrested growth. These results suggest that alkaline pH gradients may be integral to hyphal extension in fungi.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.     

Acute insulin withdrawal contributes to ischemic heart failure in spontaneously diabetic BB Wistar rats

The contribution of poor metabolic control to myocardial ischemic failure was determined in isolated working hearts from insulin-dependent BB Wistar rats. Removal of insulin treatment 24 h prior to study (uncontrolled diabetic rats) resulted in significant increases in serum glucose, serum fatty acids, and myocardial triglyceride, compared with animals in which insulin treatment was not withheld (insulin-treated diabetic rats). Isolated working hearts obtained from these two groups were subjected to a 40% reduction in coronary flow in the presence of a maintained metabolic demand (hearts were paced at 200 beats/min and perfused at an 80 mmHg (1 mmHg = 133.3 Pa) left aortic afterload, 11.5 mmHg left atrial preload). Within 15 min of ischemia, a significant deterioration of mechanical function occurred in the uncontrolled diabetic rats, whereas function was maintained in the insulin-treated diabetic rats. Oxygen consumption by the two groups of hearts was similar prior to the onset of ischemia and decreased during ischemia in parallel with the work performed by the hearts. This suggests that the accelerated failure rate in uncontrolled diabetic rat hearts is unlikely a result of an increased oxygen requirement. These data are a direct demonstration that acute changes in metabolic control of the diabetic can contribute to the severity of myocardial ischemic injury.