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Biomedical subjects

E G Loten

Publications and source records attributed to E G Loten.

At least 37 records · Page 2Linked to original sources
PubMed1987

Phosphorylation of endogenous and exogenous peptides by rat heart sarcolemma.

Rat heart plasma membranes contain a calcium-dependent protein kinase which phosphorylates endogenous protein substrates as well as added histones. The major endogenous protein phosphorylated is of 17 kDa on SDS-polyacrylamide gel electrophoresis. Proteins of 85 kDa and 60 kDa were also phosphorylated. Treatment of a rat heart homogenate with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased the recovery of kinase activity in the sarcolemmal membranes by up to 10-fold. The activity in such membranes was no longer calcium dependent. Although several histones were effective substrates for the enzyme, myosin light chain and phosvitin were not phosphorylated. These membranes contain a very active ATP hydrolysing activity which necessitated very brief incubation times to avoid loss of substrate. The membranes also contain cyclic AMP dependent protein kinase activity which is not active unless cyclic AMP is added to the incubations. The calcium dependent endogenous kinase, which is not inhibited by the heat stable inhibitor protein of cyclic AMP-dependent kinase, or by trifluoperazine, has several properties in common with protein kinase C. Preincubation of the sarcolemmal membranes with a high concentration of insulin caused inhibition of the phosphorylation of the endogenous 17 kDa and 85 kDa bands. There was no effect on the phosphorylation of the 60 kDa peptide. This effect of insulin was specific for the hormone and required preincubation of the hormone with the membranes for 20 min.

Animals↗
PubMed1986

Preparation of rat liver plasma membranes in a high yield.

Existing procedures for the preparation of rat liver plasma membranes are time consuming and generally produce low yields. A method is described in which a rat liver homogenate low-speed pellet is fractionated on a self-forming Percoll gradient. Plasma membranes can be removed from the gradient in a high yield along with much of the DNA in the liver homogenate. A second Percoll step performed in the presence of a low concentration of calcium ions separates the DNA from the plasma membranes. The final membrane fraction has high specific activities of marker enzymes with little contamination with microsomal, mitochondrial, Golgi, or lysosomal markers.

5'-Nucleotidase↗
PubMed1986

Stimulation of low Km cyclic AMP phosphodiesterase by sulphydryl modification.

Low concentrations of the organic mercurials, p-chloromercuribenzoate or p-chloromercuriphenylsulphonate activate the particulate low Km phosphodiesterase from adipose tissue and liver. Higher concentrations are inhibitory. Enzyme which has been activated by treatment of adipocytes with insulin, is not activated by the organic mercurials although inhibition by higher concentrations is seen. Enzyme from non-insulin treated adipocytes is activated and solubilised by mild trypsin treatment. Enzyme activated by either insulin treatment, or by p-chloromercuribenzoate is not further activated by trypsin, but it is solubilised.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

The action of adenosine in relation to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipocytes.

The adenosine-sensitive cyclic AMP phosphodiesterase of rat adipocytes was found to reside in the same subcellular fraction as the enzyme sensitive to insulin. There were several similarities between the action of adenosine and that of insulin on the enzyme. The action of adenosine on the phosphodiesterase is probably like that of insulin, both being receptor-mediated, although different sites or different receptors could be involved. Adenosine analogues with intact ribose but a modified purine moiety elicited a response similar to that of adenosine. Added Ca2+ was also not a requirement for the action of adenosine. The action of adenosine was not synergistic with that of insulin, neither was adenosine essential for insulin action. Insulin stimulated the enzyme even at low cell concentrations and in the presence of adenosine deaminase. Adenosine, however, enhanced the effect of insulin, but only at insulin concentrations that produced submaximal effects. Thus the mechanisms of action could be similar or related. The time-course effect of a suboptimal concentration of insulin was transitory, like that of adenosine, and was influenced by the presence of adenosine, whereas that of a maximally effective concentration of insulin was sustained for at least 20 min and was not affected by the presence of adenosine. Isoprenaline enhanced phosphodiesterase activity stimulated by optimal concentrations of either adenosine or insulin, suggesting that their effects were mediated through different mechanisms of action.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Detergent solubilisation of rat liver particulate cyclic AMP phosphodiesterase.

Detergent solubilisation of particulate rat liver low Km cyclic AMP phosphodiesterase in the presence of protease inhibitors yields a form of the enzyme with a larger molecular weight than the form solubilised by protease treatment. The detergent solubilised enzyme could be partially purified by anion exchange chromatography. It displayed a marked tendency to precipitate from solution when detergent was removed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Evidence for insulin degradation by muscle and fat tissue in an insulin resistant diabetic patient.

The period before, during and after resistance to subcutaneous insulin in a 20-year-old, non-obese insulin-dependent patient with 'brittle' diabetes is documented and clinical observations are correlated with experimental results of insulin degradation in vitro. Treatment with intravenous but not subcutaneous aprotinin markedly reduced subcutaneous insulin requirements. Insulin resistance recurred following cessation of aprotinin. Serum free insulin levels were low during the subcutaneous resistant phase compared with those during the more sensitive phase. Insulin degradation in vitro by adipose tissue and muscle taken from the patient during a resistant phase was increased compared with degradation by tissue taken during a sensitive phase and by tissue from normal subjects. Chromatography of incubation media revealed that during the resistant phase, tissue from the patient degraded insulin to small fragments. It is concluded that, in this patient, insulin resistance was caused by excessive degradation in both muscle and adipose tissue.

Adipose Tissue↗
PubMed1981

The antilipolytic action of insulin on adrenocorticotrophin-stimulated rat adipocytes. The roles of adenosine 3',5'-monophosphate and the protein kinase dependent on adenosine 3',5'-monophosphate.

The extent to which a fall in cellular cyclic AMP could account for the antilipolytic action in rat epididymal adipocytes incubated with adrenocorticotrophic hormone was studied. The antilipolytic effect, measured by suppression of glycerol release, was always associated with a decrease in cyclic AMP, but the magnitude of the fall was modified by several factors. For example, it was greater when the cAMP level was high, as when it is at its peak after hormone stimulation, or when cell concentrations are low. Glucose did not modify appreciably the insulin effect on the nucleotide level. The inhibitory effects of insulin on corticotrophin-stimulated lipolysis and cyclic AMP levels were detectable at the concentrations of 1 microU/ml and were biphasic, with maximal effects at 10-100 microU/ml. Protein kinase activity ratio was similarly affected. Activity of cyclic-AMP-dependent protein kinase conformed closely to the level of cyclic AMP. There was no indication that insulin modified the sensitivity of the kinase to cyclic AMP. Insulin did not alter the relationship of cellular cyclic AMP levels to glycerol when adipocytes were incubated with various concentrations of corticotrophin. This was true, irrespective of whether measurements were made when cyclic AMP was on the upward rise after hormone stimulation, or on the decline. The curves obtained with and without insulin were superimposable. It is concluded that the inhibitory action of insulin on lipolysis in fat cells can be fully accounted for by a decrease in cyclic AMP.

Adipose Tissue↗
PubMed1981

Energy-dependent activation and magnesium--dependent inactivation of hepatocyte hormone-sensitive phosphodiesterase.

Incubation of solubilized hormone-activated phosphodiesterase from isolated hepatocytes, under conditions likely to favour a dephosphorylation reaction, did not cause a loss of the hormone activation. If, however, the enzyme was incubated with Mg2+ (10 mM) while still associated with its membrane, and subsequently solubilized, the activity of the hormone-stimulated enzyme declined to the level seen in control cells. Diminution of hepatocyte ATP levels to about 20% of control values, by incubation with fructose, blunted the effect of glucagon and abolished the effect of insulin on phosphodiesterase. More severe ATP depletion caused by dinitrophenol abolished the stimulation of the enzyme by both hormones. These effects were not considered likely to be due to altered hormone-binding and are consistent with the involvement of an energy-dependent step in the hormonal activation of phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Is extracellular calcium required for insulin action?

Isolated hepatocytes and isolated adipocytes incubated in the absence of added calcium ions respond to insulin with a decrease in tissue cyclic AMP levels, and an increase in low Km phosphodiesterase activity. Isolated hepatocytes also showed a diminution of glucagon stimulated glucose output in response to insulin, while adipocytes responded with increased rates of glucose oxidation, lipid synthesis and decreased glycerol output. These responses to insulin are the same as those seen when the cells are incubated in buffers containing physiological concentrations of calcium ions. When extracellular concentrations of calcium ions were made extremely low by using either gelatine or albumin which had been pretreated to remove calcium, and/or the incubation buffers contained EGTA, both the hepatocytes and adipocytes continued to respond to insulin. These results suggest that extracellular calcium ions are not required for insulin action.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1978

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.

Adenosine↗
PubMed1977

Effect of fasting and streptozotocin in the obese-hyperglycemic (ob/ob) mouse. Apparent lack of a direct relationship between insulin binding and insulin effects.

The decrease of insulin binding to plasma membranes of liver, adipose, and muscle tissues observed in obese-hyperglycemic (ob/ob) mice was reversed towards normal by prolonged fasting or streptozotocin treatment. The extent of this reversal was related to that of the decrease in hyperinsulinemia of the obese mice. In contrast, binding of glucagon to liver plasma membranes was little influenced by fasting or streptozotocin treatment of obese animals. The relationship between insulin binding and metabolic effects of the hormone did not appear to be identical in all tissues. In muscle, insulin binding and insulin effect on glucose uptake and metabolism changed in parallel--i.e., when binding increased, tissue sensitivity to the hormone increased. In the liver, the increase in insulin binding that followed fasting or streptozotocin treatment was not accompanied by any detectable metabolic effect of insulin on hepatic metabolism. A similar situation appeared to prevail in adipose tissue. The varying relationships observed between the state of insulin binding to membranes and the target tissue responsiveness to the hormone probably reflect the multiplicity of the factors operative in these processes and help us to understand why the over-all obese-hyperglycemic syndrome of ob/ob mice cannot be improved simply by decreasing endogenous hyperinsulinemia.

Adipose Tissue↗