Diane R. Sawicki

Structural abnormalities do not explain the early functional abnormalities in the peripheral nerves of the streptozotocin diabetic rat.

The streptozotocin (STZ)‐diabetic rat, the most commonly employed model of experimental diabetic neuropathy, is characterised by a reduction in nerve conduction velocity, pain threshold and blood flow. Whether or not structural abnormalities underlie these functional abnormalities is unclear. 10 adult male Sprague–Dawley STZ‐diabetic rats (diabetes duration 27 d) and 10 age‐matched (23 wk) control animals were studied. Motor nerve conduction velocity (m s−1) was significantly reduced in diabetic (41.31±0.8) compared with control (46.15±1.5) animals (P<0.001). The concentration of sciatic nerve glucose (P<0.001), fructose (P<0.001) and sorbitol (P<0.001) was elevated, and myoinositol (P<0.001) was reduced in diabetic compared with control animals. Detailed morphometric studies demonstrated no significant difference in fascicular area, myelinated fibre density, fibre and axon areas as well as unmyelinated fibre density and diameter. Endoneurial capillary density, basement membrane area and endothelial cell profile number did not differ between diabetic and control animals. However, luminal area (P<0.03) was increased and endothelial cell area (P<0.08) was decreased in the diabetic rats. We conclude there is no detectable structural basis for the reduction in nerve conduction velocity, pain threshold or blood flow, observed in the streptozotocin diabetic rat.

Polyol pathway activity in nervous tissues of diabetic and galactose-fed rats: Effect of dietary galactose withdrawal or tolrestat intervention therapy

Enhanced polyol pathway activity resulting in an accumulation of sorbitol and a depletion of myoinositol in nervous tissues has been proposed to be important in development of diabetic neuropathies. This investigation demonstrated that in two models of diabetic complications, streptozocin (STZ)-induced diabetic rats and galactose-fed rats, 5 weeks of disease led to an accumulation of sorbitol or galactitol, respectively, in three cranial nerves (the optic (II), trigeminal (V), and vagus (X) nerves), as well as the sciatic nerve, cerebral cortex, and retina. In both models, the cranial nerves and cerebral cortex contained lower levels of accumulated polyol than the sciatic nerve. In addition, myoinositol depletion was observed in the sciatic nerve only. In a second galactose-fed rat study, returning 5-week galactose-fed rats to a normal diet for 6 weeks led to complete elimination of galactitol from the optic nerve, sciatic nerve, and retina and normalization of the sciatic nerve myoinositol concentration. Similarly, continuing the galactose diet for 6 more weeks (ie, a total of 11 weeks) as well as administration of the aldose reductase inhibitor (ARI) tolrestat (20 and 40 mg/kg/day), caused the sciatic nerve to contain a normal myoinositol concentration and a galactitol concentration that was 95% below the level observed in galactose-fed controls. In the optic nerve and retina, tolrestat was less effective, resulting in 69-78% lower galactitol levels. In conclusion, these findings indicate that sorbitol and galactitol accumulate in cranial nerves, brain, and retina without a concomitant decrease in myoinositol. Either withdrawal of the galactose diet or intervention with tolrestat normalized polyol levels in the sciatic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance is associated with abnormal dephosphorylation of a synthetic phosphopeptide corresponding to the major autophosphorylation sites of the insulin receptor

Insulin resistance in the ob/ob mouse model is associated with a reduction in insulin-induced protein-tyrosine phosphorylation in tissues such as liver. To ascertain whether this decrease in phosphorylation may be due to increased phosphatase activity, protein-tyrosine phosphatase (PTPase) activity was determined in particulate and soluble fractions from livers of 5- to 23-week-old ob/ob mice and age-matched lean littermates. PTPase activity was measured using a synthetic phosphopeptide, TRDIY(P)ETDY(P)Y(P)RK, as the substrate, corresponding to residues 1142 to 1153 of the insulin receptor and containing the major autophosphorylation sites of the regulatory domain. The ob/ob mice were hyperinsulinemic across all age groups, but only the youngest mice (aged 5 to 7 weeks) were hyperglycemic. Most PTPase activity was present in the liver particulate fraction and was 19% to 114% greater in ob/ob mice as compared with controls. PTPase activity in the liver soluble fraction was 26% less than control values in the youngest ob/ob mice (5 to 7 weeks), but increased with age and was 41% and 131% above control values at 21 to 23 and 25 to 27 weeks of age, respectively. Oral administration of the PTPase inhibitor sodium orthovanadate (0.6 mg/mL in drinking water for 2 weeks) to young ob/ob mice caused a significant reduction in the elevated particulate PTPase activity, with concomitant decreases in plasma insulin and plasma glucose. Assessment of PTPase activity with a monophosphate form of the same synthetic peptide, TRDIY(P)ETDYYRK, showed lower PTPase activities as compared with the triphosphate form and no significant differences between ob/ob and control preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatase Activity in Sciatic Nerve Tissue of Streptozocin-Induced Diabetic Rats with and without High Dietary Sucrose: Effect of Aldose Reductase Inhibitors

Abstract The ability of aldose reductase inhibitors to prevent the decline in neural Na+,K+-ATPase activity in diabetic rats has not been confirmed by all laboratories. In this study, the efficacy of two structurally different aldose reductase inhibitors was evaluated under different experimental conditions. Na+,K+-ATPase activity was measured in sciatic nerves from streptozocin-induced diabetic rats fed normal rodent chow or a chow supplemented with 68% sucrose. Nerve homogenates from chow-fed rats were prepared with a Dounce tissue grinder, whereas homogenates from the sucrose-fed rats were prepared with an Ultra-Turrax disperser. In the chow-fed rats, 4 weeks of untreated diabetes resulted in an increase in neural sorbitol and fructose, a decrease in myoinositol, and a 54% decline in Na+,K+-ATPase activity. Sorbinil administration (20 mg/kg/day) completely prevented the rise in sorbitol and fructose and the depletion of myoinositol, but did not prevent the decline in Na+,K+-ATPase activity. In diabetic rats fed the sucrose diet for 4, 6, and 8 weeks, the neural sorbitol and fructose levels were elevated, the myoinositol concentration declined, and the Na+,K+-ATPase activity was 26 to 28% below the control. Prevention or intervention treatment with sorbinil (20 mg/kg/day) or tolrestat (50 mg/kg/day) for 4 to 6 weeks prevented the alterations in sorbitol, fructose, and myoinositol, and also prevented the decline in Na+,K+-ATPase activity. In conclusion, prevention and intervention therapy with aldose reductase inhibitors prevented the decline in Na+,K+-ATPase in sciatic nerves of sucrose-fed streptozocin-diabetic rats that were homogenized with an Ultra-Turrax disperser, but not in sciatic nerves from streptozocin-diabetic rats fed normal rodent chow that were homogenized with a Dounce tissue grinder. These findings indicate that the assessment of aldose reductase inhibitor efficacy is dramatically affected by the type of nerve preparation assayed and/or the diet.

Discovery of [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl]acetic acids as highly potent and selective inhibitors of aldose reductase for treatment of chronic diabetic complications.

Efforts to identify treatments for chronic diabetic complications have resulted in the discovery of a novel series of highly potent and selective [3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl]acetic acid aldose reductase inhibitors. The lead candidate, [6-methyl-3-(4,5,7-trifluoro-benzothiazol-2-ylmethyl)-pyrrolo[2,3-b]pyridin-1-yl]acetic acid example 16, inhibits aldose reductase with an IC50 of 8 nM, while being inactive against aldehyde reductase (IC50>100 microM), a related enzyme involved in the detoxification of reactive aldehydes.

Synthesis and PTP1B Inhibition of Novel 4-Aryl-1-Oxa-9-Thiacyclopenta[b]fluorenes

Novel 4-aryl-1-oxa-9-thiacyclopenta[b]fluorenes were designed, synthesized, and evaluated as inhibitors of the protein tyrosine phosphatase, PTP1B. Compounds 3 (IC50 = 284 nM) and 4 (IC50 = 74 nM), showed nanomolar potency against PTP1B (TRDI(P)YETD(P)Y(P)YRK as substrate). Compound 4 also lowered insulin in the diabetic ob/ob mouse at a dose of 10 mg/kg/day, p.o.