Depletion of glutathione by inhibition of its synthesis by buthionine sulfoximine, an irreversible inhibitor of vglutamylcysteine synthetase, leads to increased sensitivity to (i) irradiation and (ii) oxidative stress. In the present work, an intracellular cysteine delivery system was used to promote glutathione synthesis, and this was found to protect against toxicity. Thus, administration of L-2-oxothiazolidine-4-carboxylate protected against acetaminophen toxicity in mice; the thiazolidine, which is converted to L:cysteine by the enzyme 5-oxo-L-prolinase (present in many animal tissues and in plants) promotes the synthesis of glutathione, which is the actual protectant. The effect of this thiazolidine in increasing the level of glutathione is prevented by administration of buthionine sulfoximine. This thiazolidine may be useful in the treatment of other toxicities and in the treatment of certain diseases. It may also be valuable as a component of amino acid mixtures used in therapy and as a safener in agriculture.Recent studies in this laboratory have shown that it is possible to modulate the synthesis and metabolism of glutathione by administration of selective enzyme inhibitors (1). Thus, inhi-. bition of y-glutamylcysteine synthetase by administration of buthionine sulfoximine to animals (2, 3) or to cells grown in tissue culture (4) leads to a substantial decline in.the intracellular glutathione concentration. Decreased glutathione synthesis has been found to have the following effects: decreased cell viability (4), increased sensitivity of cells to the effects of irradiation (4), increased sensitivity of tumor cells to cytolysis by peroxide (5) (see also ref. 6), decreased synthesis ofprostaglandin E and leukotriene C (7), and selective destruction of trypanosomes in mice (8). Although these effects of glutathione depletion are clearly of interest, the possibility that an increase in tissue or cellular glutathione might lead to potentially. useful effects also needs to be considered. That the glutathione content of tissues may be increased by supplying certain precursors of this tripeptide has been indicated by studies showing that administration of y-glutamylcysteine and related compounds to mice leads to increased levels ofrenal glutathione. (9) and that administration of L-2-oxothiazolidine-4-carboxylate to mice produces a substantial increase in liver glutathione levels (10).In the present work, we have examined the use of L-2-oxothiazolidine-4-carboxylate in a cysteine delivery system that protects mice against the toxic effects of acetaminophen. The biochemical basis of this. effect lies in the fact that this thiazolidine is an excellent substrate ofthe enzyme 5-oxo-L-prolinase, which converts this substrate to S-carboxy-L-cysteine, which spontaneously decarboxylates to yield L-cysteine. The L-cysteine formed in this manner is rapidly utilized for glutathione synthesis. Since 5-oxo-L-prolinase.is found in many animal tissues, the thiazolidine is probably utilized for glutathione synthesis ...

The enzyme glucokinase (GK) plays a central role in glucose homeostasis. Hepatic GK activity is acutely controlled by the action of the GK regulatory protein (GKRP). In vitro evidence suggests that GKRP reversibly binds to GK and inhibits its activity; however, less is known about the in vivo function of GKRP. To further explore the physiological role of GKRP in vivo, we used an E1/E2a/E3-deficient adenoviral vector containing the cDNA encoding human GKRP (Av3hGKRP). High fat diet-induced diabetic mice were administered Av3hGKRP or a control vector lacking a transgene (Av3Null). Surprisingly, the Av3hGKRP-treated mice showed a significant improvement in glucose tolerance and had lower fasting blood glucose levels than Av3Null-treated mice. A coincident decrease in insulin levels indicated that the Av3hGKRP-treated mice had sharply improved insulin sensitivity. These mice also exhibited lower leptin levels, reduced body weight, and decreased liver GK activity. In vitro experiments indicated that GKRP was able to increase both GK protein and enzymatic activity levels, suggesting that another role for GKRP is to stabilize and/or protect GK. These data are the first to indicate the ability of GKRP to treat type 2 diabetes and therefore have significant implications for future therapies of this disease.

Hyperglycemia of diabetes is caused in part by perturbation of hepatic glucose metabolism. Hepatic glucokinase (GK) is an important regulator of glucose storage and disposal in the liver. GK levels are lowered in patients with maturity-onset diabetes of the young and in some diabetic animal models. Here, we explored the adenoviral vector-mediated overexpression of GK in a diet-induced murine model of type 2 diabetes as a treatment for diabetes. Diabetic mice were treated by intravenous administration with an E1/E2a/E3-deleted adenoviral vector encoding human hepatic GK (Av3hGK). Two weeks posttreatment, the Av3hGK-treated diabetic mice displayed normalized fasting blood glucose levels (95 ؎ 4.8 mg/dl; P < 0.001) when compared with Av3Null (135 ؎ 5.9 mg/dl), an analogous vector lacking a transgene, and vehicle-treated diabetic mice (134 ؎ 8 mg/dl). GK treatment also resulted in lowered insulin levels (632 ؎ 399 pg/ml; P < 0.01) compared with the control groups (Av3Null, 1,803 ؎ 291 pg/ml; vehicle, 1,861 ؎ 392 pg/ml), and the glucose tolerance of the Av3hGK-treated diabetic mice was normalized. No significant increase in plasma or hepatic triglycerides, or plasma free fatty acids was observed in the Av3hGK-treated mice. These data suggest that overexpression of GK may have a therapeutic potential for the treatment of type 2 diabetes.

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