Geoffrey J. Appleby

Insulin-like action of catecholamines and Ca2+ to stimulate glucose transport and GLUT4 translocation in perfused rat heart

The uptake of 2-deoxyglucose by perfused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the alpha-adrenergic combination of epinephrine + propranolol, the beta-adrenergic agonist isoproterenol, high (8 mM) Ca2+ concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 microM) + propranolol (10 microM), isoproterenol (10 microM), high Ca2+, insulin (1 microM) + epinephrine (1 microM) + propranolol (10 microM) and insulin (1 microM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fraction (putatively sarcolemmal). There were significant correlations (r = -0.673, P less than 0.001) between the stimulation of 2-deoxyglucose uptake and the loss of GLUT4 from the intracellular membrane fraction, or the increase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted to insulin.

ß CARBOXYLATION ENZYMES IN MARINE PHYTOPLANKTON AND ISOLATION AND PURIFICATION OF PYRUVATE CARBOXYLASE FROM AMPHIDINIUM CARTERAE (DINOPHYCEAE)1

The diatoms, Phaeodactylum tricornutum Bohlin, Cylindrotheca closterium var. californica (Meres.) Reim. & Lewin, Thalassiosira pseudonana Hasle & Heimdal and the prymnesiophyte Pavlova lutheri (Droop), Green, have been shown to contain phosphoenolpyruvate carboxykinase E.C.4.1.1.49. Another diatom Chaetoceros calcitrans (Paulsen) Takano, the chlorophyte Dunaliella tertiolecta Butcher, a rhodophyte Porphyridium cruentum Naegeli and the cyanophyte Anabaena cylindrica Lemmermann, all possessed phosphoenolpyruvate carboxylases E.G.4.1.1.31, which were stimulated by Mn ions and inhibited by malate and aspartate. Two dinoflagellates, Amphidinium carterae Hulbert and Gymnodinium sp., were shown to contain pyruvate carboxylase E.G.6.4.1.1., not previously reported in plants or marine algae. Pyruvate carboxylase was isolated and purified and found to contain biotin and it was inhibited by avidin and could be distinguished from the other two enzymes by complete inhibition by 5 mM Mn ions. The β carboxylating enzymes account for the anaplerotic formation of amino acids and intermediates of the tricarboxylic acid cycle formed during short‐term fixation of CO2.

α-Adrenergic receptors in rat skeletal muscle

Sarcolemma-enriched preparations from muscles rich in slow oxidative red fibres contained specific binding sites for the alpha 1 antagonist, prazosin (e.g. soleus Kd 0.13 nM, Bmax 29 fmol/mg protein). Binding sites for prazosin were almost absent from white muscle. Displacement of prazosin binding from sarcolemma of soleus muscle (phentolamine greater than phenylephrine greater than idazoxan greater than yohimbine) suggested that the receptors were alpha 1. Binding sites for dihydroalprenolol (beta antagonist) were also more concentrated on red than white muscle and outnumbered prazosin sites by approx. 10:1. Binding sites for idazoxan (alpha 2 antagonist) were undetectable. Contamination of sarcolemma-enriched preparations by endothelial tissue indicated by the activity of angiotensin converting enzyme did not correlate with prazosin binding. It is concluded that post-synaptic alpha 1 adrenergic receptors are present on the sarcolemma of slow oxidative red fibres of rat skeletal muscle. The presence provides the mechanistic basis for apparent alpha-adrenergic effects to increase glucose and oxygen uptake in perfused rat hindquarter.

Reduced glycogen phosphorylase activity in denervated hindlimb muscles of rat is related to muscle atrophy and fibre type.

Changes in the activity of muscle glycogen synthase or phosphorylase (GP) may be responsible for the deregulation of glycogen synthesis and storage which occurs in diabetes mellitus. To clarify the relationship between muscle atrophy, fibre type, insulin-stimulated glucose uptake and GP activity during insulin resistance, we used sciatic nerve severance to induce insulin resistance in rat hindlimb muscles and compared the above parameters in muscles with a range of fibre types. Changes were analysed by comparison with the contralateral hindlimb, which bears more weight due to denervation of the opposing limb, as well as the sham-operated and contralateral limb of a separate rat. Denervation caused a decrease in insulin-stimulated glucose uptake by 1 day after denervation and a decline of GP activity after 7 days in all muscles investigated. GP activity decreased by 73% in soleus, 36% in red gastrocnemius, 35% in tibialis and 13% in white gastrocnemius, which was related to the degree of muscle atrophy and inversely related to the overall GP activity in non-denervated muscles. GP activity in muscles of the contralateral limb from the denervated rat did not differ from either hindlimb of the sham-operated rat. We conclude that the fibre-type related reduction in insulin-stimulated glucose uptake of denervated muscle determines the change in its metabolism and it is this metabolic change which determines the mechanism, rate and degree of muscle atrophy, which is directly related to the decline in GP activity.

Assays of glucose tolerance factor and its mode of action, studied with brewer's yeast.

Glucose tolerance factor (GTF) has usually been assayed by manometric measurement of CO2 evolved when glucose was metabolizing glucose. By using 14C labeled substrates it has been shown that GTF increases the decarboxylation of pyruvate to ethanol and CO2. Thus in addition to measuring CO2 evolution, the enzymatic estimation of the increased ethanol production can be used to assay GTF. A further effect of GTF was to cause increased carboxylation of pyruvate to substrates that are used in the biosynthesis of cell substance. The metabolic sites of action of GTF are discussed.

Purine and pyrimidine nucleotide metabolism of vascular smooth muscle cells in culture

1. Cultures of vascular smooth muscle cells accumulate extracellular breakdown products of purine and pyrimidine nucleotides that, over 9 hr, represent 60 +/- 7 and 78 +/- 17%, respectively, of the intracellular nucleotide content. 2. The accumulation is stimulated during contracture with 20 mM KCl or 70 microM carbachol, consistent with the notion that both pyrimidine and purine nucleotides are involved in the energetics of smooth muscle contracture. 3. Because the intracellular levels of pyrimidine and purine nucleotides remain constant, it appears likely that rates of synthesis match the rates of release. 4. Ectonucleotidases are present that can degrade ATP, UTP, and CTP. High-energy nucleotides may be the primary products released.