Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (α, β1, β2, and γ) and novel (δ and ε) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-β2 and -δ and delayed the activation of PKC-β1 and -ε, whereas membrane translocation of PKC-α and -γ was not affected. Treatment with tolrestat prevented phosphorylation of PKC-β2 and -δ. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-γ1 (PLC-γ1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-γ1 and PLC-β2 and -δ. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-γ1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.

01 Dec 1998·Investigative ophthalmology & visual scienceQ2 · MEDICINE

3-FG as substrate for investigating flux through the polyol pathway in dog lens by 19F-NMR spectroscopy.

Q2 · MEDICINE

ArticleOA

Author: Secchi, E F ; Akagi, Y ; Sato, S ; Lee, J W ; Kador, P F ; Kubo, E ; Lizak, M J

PURPOSETo investigate flux through the polyol pathway in the dog lens by 19F-nuclear magnetic resonance (19F-NMR) spectroscopy, using 3-fluoro-3-deoxy-D-glucose (3-FG) as a substrate.METHODS3-FG metabolism was monitored by 19F-NMR analysis. Dog lenses were incubated in Dulbecco's modified Eagle's medium containing 10 mM 3-FG. Enzymatic reductase and dehydrogenase activities were spectrophotometrically determined, whereas the analyses of 3-FG metabolites were conducted by 19F-NMR analysis. Aldose reductase (AR) was immunohistochemically localized in dog lens with antibodies raised against dog kidney AR.RESULTS19F-NMR spectra indicate that incubation of purified dog lenses AR with 3-FG results in the formation of 3-fluoro-3-deoxy-D-sorbitol (3-FS) and that incubation of dog liver sorbitol dehydrogenase (SDH) with 3-FS results in the formation of 3-fluoro-3-deoxy-D-fructose (3-FF). This confirms that 3-FG is metabolized to 3-FF by the polyol pathway enzymes. The affinity (Km) of AR for 3-FG is approximately 20-fold better than that for D-glucose, whereas the Km of SDH for 3-FS was fourfold less than for D-sorbitol. 3-FG in cultured dog lenses is metabolized primarily to 3-FS; however, small amounts of 3-FF and 3-fluoro-3-deoxy-D-gluconic acid (3-FGA) are also formed. 3-FS formation was reduced by the AR inhibitor AL 1576, and 3-FF formation was eliminated by the SDH inhibitor CP-166,572. In dog lens epithelial cells cultured with 3-FG, only 3-FS is formed. Similarly, only 3-FS is formed when lens capsule containing primarily epithelial lens contaminated with superficial epithelial cells was incubated in 3-FG. Similar incubation of the remaining cortex resulted primarily in the formation of 3-FS and 3-FGA. This enzymatic distribution was confirmed by spectrophotometric activity analysis and the immunohistochemical localization of AR.CONCLUSIONSThe data confirm that flux through the polyol pathway primarily results in sorbitol accumulation. The absence of fructose and gluconic acid from cultured lens epithelium suggests that the epithelial cells primarily contain AR, whereas differentiated fiber cells also contain SDH and glucose dehydrogenase.

01 Dec 1998·Journal of Neuropathology and Experimental NeurologyQ3 · MEDICINE

Effect of Sorbitol Dehydrogenase Inhibition on Experimental Diabetic Autonomic Neuropathy

Q3 · MEDICINE

Article

Author: Yasuo Ido ; Joseph R. Williamson ; Denise A. Dorsey ; Santiago B. Plurad ; Lucie N. Beaudet ; Robert E. Schmidt

The polyol pathway and its dependent biochemical pathways are thought to play a role in the pathogenesis of diabetic neuropathy. We have developed an animal model of diabetic autonomic neuropathy characterized by neuroaxonal dystrophy involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozocin-diabetic rats. Our previous studies have shown a salutary effect of aldose reductase inhibitors on experimental autonomic neuropathy, suggesting a role for the polyol pathway in its pathogenesis. In the current studies we have examined the effect of the sorbitol dehydrogenase inhibitor (SDI) CP-166,572, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the polyol pathway) resulting in markedly increased levels of sorbitol in peripheral nerve. Fourteen weeks of treatment with CP-166,572 resulted in a dramatically increased frequency of neuroaxonal dystrophy in ileal mesenteric nerves and SMG. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics than untreated diabetics did, their anatomic distribution and ultrastructural appearance were identical to that previously reported in long-term untreated diabetics. CP-166,572 treatment did not produce neuroaxonal dystrophy in control animals despite the fact that sciatic nerve sorbitol levels were markedly increased, reaching the same levels as untreated diabetic animals. Treatment of diabetic rats for 14 weeks with the aldose reductase inhibitor zopolrestat resulted in a significant decrease in the frequency of neuroaxonal dystrophy compared with untreated diabetics.

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