Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Compositional statistics: An improvement of evolutionary parsimony and its application to deep branches in the tree of life

Summary We present compositional statistics, a new method of phylogenetic inference, which is an extension of evolutionary parsimony. Compositional statistics takes account of the base composition of the compared sequences by using nucleotide positions that evolutionary parsimony ignores. It shares with evolutionary parsimony the features of rate invariance and the fundamental distinction between transitions and transversions. Of the presently available methods of phylogenetic inference, compositional statistics is based on the fewest and mildest assumptions about the mode of DNA sequence evolution. It is therefore applicable to phylogenetic studies of the most distantly related organisms or molecules. This was illustrated by analyzing conservative positions in the DNA sequences of the large subunit of RNA polymerase from three archaebacterial groups, a eubacterium, a chloroplast, and the three eukaryotic polymerases. Internally consistent results, which are in accord with our knowledge of organelle origin and archaebacterial physiology, were achieved.     

31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.     

Hydroquinone: O-glucosyltransferase from cultivated Rauvolfia cells: enrichment and partial amino acid sequences

Plant cell suspension cultures of Rauvolfia are able to produce a high amount of arbutin by glucosylation of exogenously added hydroquinone. A four step purification procedure using anion exchange, hydrophobic interaction, hydroxyapatite-chromatography and chromatofocusing delivered in a yield of 0.5%, an approximately 390 fold enrichment of the involved glucosyltransferase. SDS-PAGE showed a M(r) for the enzyme of 52 kDa. Proteolysis of the pure enzyme with endoproteinase LysC revealed six peptide fragments with 9-23 amino acids which were sequenced. Sequence alignment of the six peptides showed high homologies to glycosyltransferases from other higher plants.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.     

Isolation of RNA from apple skin

A method has been developed for the isolation of RNA from apple skin. The method involves an adaptation of the Manning (1991) method and includes a high-salt extraction step and a final purification step through a CsCl cushion. The RNA isolated was of high quality and produced good hybridization signals in northern blot analyses.     

Monocarboxylate transporters in subsarcolemmal and intermyofibrillar mitochondria

Whether subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria contain monocarboxylate transporters (MCTs) is controversial. We have examined the presence of MCT1, 2, and 4 in highly purified SS and IMF mitochondria. These mitochondria were not contaminated with plasma membrane, sarcoplasmic reticulum or endosomal compartments, as the marker proteins for these sub-cellular compartments (Na+-K+-ATPase, Ca2+-ATPase, and the transferrin receptor) were not present in SS or IMF mitochondria. MCT1, MCT2, and MCT4 were all present at the plasma membrane. However, MCT1 and MCT4 were associated with SS mitochondria. In contrast, the IMF mitochondria were completely devoid of MCT1 and MCT4. However, MCT2 was associated with both SS and IMF mitochondria. These observations suggest that SS and IMF mitochondria have different capacities for metabolizing monocarboxylates. Thus, the controversy as to whether mitochondria can take up and oxidize lactate will need to take account of the different distribution of MCTs between SS and IMF mitochondria.     

Sequence first. Ask questions later

Comparative sequence analyses of eukaryotic genes and genomic regions are beginning to provide a wealth of information that is directly relevant to human biology. Functional changes that set us apart from apes are identifiable, as are functional constraints in proteins and genomic elements that arose in our relatively distant phylogenetic past.