We have studied the mechanism ofgeneration of insulin mediators by using specific antibodies raised against the oligosaccharide anchor of membrane proteins. These antibodies (i) block the in vitro effects ofpurified insulin mediators and (it) block the insulin-induced stimulation of pyruvate dehydrogenase in intact BC3H1 myocytes but not insulinstimulated glucose uptake, generation of diacylglycerol, or generation of insulin mediators. When added to intact cells in the presence of insulin, these antibodies induce the accumulation of insulin mediator activity in the extracellular medium. We therefore conclude that these anti-inositolglycan antibodies block some of the effects of insulin by inhibiting the uptake of specific insulin mediators generated outside the cell.
Previously, we demonstrated that nondiabetic insulin-resistant monkeys had reduced covalent insulin activation of muscle glycogen synthase (GS) compared to normal monkeys and that covalent insulin activation of adipose tissue GS was absent in these monkeys. Covalent insulin activation of muscle and adipose tissue GS in monkeys with impaired glucose tolerance and noninsulin-dependent diabetes (NIDDM) was also absent. As in humans, monkeys with NIDDM have a lower urinary excretion rate of chiroinositol (CI), a component of a putative mediator of insulin action, compared to normal monkeys. To determine whether the urinary excretion rate of CI was related to insulin resistance, which develops naturally in many obese rhesus monkeys, we examined the relationships between 24-h urinary CI excretion rate and 1) whole body insulin-mediated glucose disposal rates (M) and insulin-mediated changes in 2) the skeletal muscle GS activity ratio (sm delta GSAR), 3) the skeletal muscle glycogen phosphorylase activity ratio, and 4) the adipose tissue GS activity ratio (at delta GSAR) in 27 monkeys ranging from normal (n = 12) to insulin resistant (n = 8) to overtly diabetic (n = 7). The urinary CI excretion rate was significantly correlated with M (r = 0.47; P < 0.02), sm delta GSAR (r = 0.38; P < 0.05), skeletal muscle glycogen phosphorylase activity ratio (r = -0.49; P < 0.01), and at delta GSAR (r = 0.46; P < 0.02). The urinary CI excretion rate was also correlated with glucose tolerance (r = 0.39; P < 0.05). There was a wide range of urinary CI excretion rates (0.42-5.17 mumol/day) in monkeys with normal fasting plasma glucose concentrations. However, of the 7 diabetic monkeys, 6 had a urinary CI excretion rate below 2.0 mumol/day, and in the subgroup of 16 monkeys with a urinary CI excretion rate less than 2.0 mumol/day, the associations of urinary CI with M rate (r = 0.65; P < 0.005), glucose tolerance (r = 0.63; P < 0.01), and sm delta GSAR (r = 0.73; P < 0.001) increased in strength and significance. We propose that the urinary CI excretion rate may be 1) a biochemical indicator of both in vivo and in vitro insulin resistance and 2) a noninvasive diagnostic tool with potential for the identification of those individuals at risk for NIDDM and other related diseases with insulin resistance.
A novel low mol wt inositol phosphoglycan antagonist of insulin action of oxidative glucose metabolism in isolated rat adipocytes was partially purified from normal human plasma and shown to be increased in type II diabetic plasma. It was characterized chemically as a myo-inositol phosphoglycan containing a cyclic 1,2-phosphate. This antagonist, termed fraction V3, is now shown to inhibit the action of an inositol glycan insulin pH 2.0 mediator that stimulates pyruvate dehydrogenase phosphatase in a similar manner to insulin. In addition, fraction V3 inhibits stimulation of the pyruvate dehydrogenase (PDH) phosphatase by Mg2+, the enzyme's required metal, and by spermine, a polyamine. Fraction V3 does not inhibit active PDH itself. The inhibitory effect is dose dependent and apparently noncompetitive or nonsurmountable for the insulin inositol glycan pH 2.0 mediator, thus comparing kinetically with its insulin antagonistic action on intact adipocytes. Its inhibitory action on PDH phosphatase is dose dependent and competitive for Mg2+ stimulation of the phosphatase. Additionally, fraction V3 is shown to inhibit stimulation by Mg2+ of cloned recombinant PDH phosphatase catalytic subunit. Inhibition by fraction V3 of Mg(2+)-stimulated PDH phosphatase and its cloned catalytic subunit helps explain its mechanism of action to inhibit insulin-stimulated oxidative glucose metabolism in adipocytes and its potential clinical significance in insulin resistance.
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