Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 µmol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.

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Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction.

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Propionate (0, 1, 2, 4, 8, 16, 32, and 64 mumol.kg BW-1 x min-1 for 30 min) was infused i.v. to investigate the physiological effects of propionate on insulin and glucagon responses in sheep. An i.v. propionate infusion (32 mumol.kg BW-1 x min-1 for 30 min) with adrenergic and cholinergic blockades was also conducted to clarify the role of autonomic innervation in the control of propionate-induced insulin and glucagon responses. In the experiment in which we studied responses to propionate infusion, the concentrations of plasma insulin and glucagon during propionate infusion increased (P < .05) from the preinfusion concentrations at infusion rates of > 4 and 8 mumol.kg BW-1 x min-1, respectively. The incremental response areas of plasma insulin and glucagon during propionate infusion increased (P < .05) at infusion rates of > 16 and 32 mumol.kg BW-1 x min-1, respectively. In the experiment studying the effects of adrenergic and cholinergic blockades on responses to propionate, the insulin incremental response area during propionate infusion was suppressed (P < .05) by atropine infusion but it was not influenced by phentolamine, propranolol, or hexamethonium infusions. The glucagon response area was suppressed (P < .05) by phentolamine infusion, but it was not influenced by propranolol, atropine, or hexamethonium infusions. It is concluded that in sheep 1) propionate may have a physiological role in stimulating insulin and glucagon responses, 2) the propionate-induced insulin response is partly due to the parasympathetic nervous system through activation of a muscarinic receptor, and 3) the propionate-induced glucagon response is stimulated by adrenergic alpha-receptors.