Avasthy, Neelima; (1990) Serum inhibitors of insulin action. Doctoral thesis (Ph.D), University of London, Royal Free Hospital School of Medicine.

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Abstract

This work describes an investigation into the presence of inhibitors of insulin and somatomedin C in sera of (i) normal, diabetic and obese humans and (ii) sera of normal and diabetic rats. The effect of whole sera and fractions prepared from sera was tested on (i) basal and insulin stimulated rat adipocyte lipogenesis and glucose oxidation and (ii) basal and serum somatomedin stimulated [3δS] labelled sulphate uptake by porcine cartilage. Fractions were prepared from human sera by using supernatants prepared after the precipitation of larger proteins with ammonium sulphate. Supernatants were further fractionated by ultrafiltration. Inhibitory activity in sera from normal subjects was found in 10-30kDa and 30-50kDa fractions. Sera from diabetic and obese patients had inhibitory activity in the fractions similar to those above and this activity was not significantly different to that found in normal subjects. However sera from obese non-insulin dependent diabetics showed significantly greater inhibitory activity than sera from non-obese noninsulin dependent diabetics. A further small molecular weight inhibitor was identified by acid- ethanol extraction of human sera and during the course of these experiments it also became clear that glucose and insulin have to be excluded from inhibitory preparations and methodology was devised accordingly. If difference was found between the inhibitory activity of these fractions from sera of controls and non-insulin dependent diabetic patients. However, fractions prepared from insulin dependent diabetics appear to have insulin inhibitory factors. Studies to identify the site of action of the inhibitory fractions prepared by ammonium sulphate precipitation of human sera led to a series of experiments to determine a role for protein kinase C activation, calcium influx and calmodulin in basal and insulin stimulated lipogenesis. While verapamil, a calcium channel blocker, H7, a protein kinase C inhibitor and calmidazolium, a calmodulin inhibitor, inhibited basal and insulin stimulated lipogenesis, phorbol esters, known protein kinase C stimulators and calcium ionophore (A23187) stimulated basal and insulin induced lipogenesis. The serum inhibitory fractions (10-30kDa and 30-50kDa) from human sera inhibited not only insulin stimulated but also phorbol ester and A23187 stimulated lipogenesis. Thus the site of action of these inhibitors was distal to that of protein kinase C and calcium entry into the cell. These inhibitors did not affect the glucose transporter activity. Sera from rats with streptozotocin induced diabetes had a marked inhibitory activity as far as C-sulphate uptake by porcine cartilage is concerned. This inhibitory activity was exhibited in the <1kDa, 1-10kDa, 30-50kDa and >300kDa fractions. In the rat lipogenesis bioassay only the <1kDa fraction was found to be inhibitory. It is concluded that normal human sera have insulin and somatomedin inhibitory activity and that these inhibitory fractions do not demonstrate increased activity in either type I or type II diabetes. However, obese patients with type II diabetes have a significantly greater insulin inhibitory activity in their sera. Sera from rats also have somatomedin inhibitory fractions. The inhibitory activity of these fractions increases following induction of diabetes with streptozotocin; this increase in inhibitory activity can be prevented by treating diabetic rats with insulin. The role of the inhibitors in human serum in the pathogenesis of insulin resistance requires further investigation.

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