Metformin is an effective hypoglycemic drug that lowers blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in skeletal muscle; however, the molecular site of metformin action is not well understood. AMP-activated protein kinase (AMPK) activity increases in response to depletion of cellular energy stores, and this enzyme has been implicated in the stimulation of glucose uptake into skeletal muscle and the inhibition of liver gluconeogenesis. We recently reported that AMPK is activated by metformin in cultured rat hepatocytes, mediating the inhibitory effects of the drug on hepatic glucose production. In the present study, we evaluated whether therapeutic doses of metformin increase AMPK activity in vivo in subjects with type 2 diabetes. Metformin treatment for 10 weeks significantly increased AMPK ␣2 activity in the skeletal muscle, and this was associated with increased phosphorylation of AMPK on Thr172 and decreased acetyl-CoA carboxylase-2 activity. The increase in AMPK ␣2 activity was likely due to a change in muscle energy status because ATP and phosphocreatine concentrations were lower after metformin treatment. Metformin-induced increases in AMPK activity were associated with higher rates of glucose disposal and muscle glycogen concentrations. These findings suggest that the metabolic effects of metformin in subjects with type 2 diabetes may be mediated by the activation of AMPK ␣2.

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 ...

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5-Oxo-L-prolinase, the enzyme that catalyzes the conversion of 5-oxo-L-proline to L-glutamate coupled to the cleavage of ATP to ADP and Pi, also acts on L-2-oxothiazolidine-4-carboxylate (an analog of 5-oxoproline in which the 4-methylene moiety is replaced by sulfur) and ATP to yield cysteine and ADP. The enzyme, which exhibits an affinity for the analog similar to that for the natural substrate, is inhibited by the analog in vitro and in vivo. L-2-Oxothiazolidine-4-carboxylate thus serves as a ptent inhibitor of the y-glutamyl cycle at the step of 5-oxoprolinase. Administration of L-2-oxothiazolidine-4-carboxylate to mice that had been depleted of hepatic glutathione led to restoration of normal hepatic glutathione levels. Since L-2-oxothiazolidine-4-carboxylate is an excellent substrate of the enzyme, it may serve as an intracellular delivery system for cysteine and thus has potential as a therapeutic agent for conditions in which there is depletion of hepatic glutathione.5-Oxo-L-prolinase catalyzes the ATP-dependent hydrolysis of 5-oxo-L-proline according to the reaction given in Fig. 1. The requirement for energy in this reaction is consistent with the position of the equilibrium between 5-oxoproline and glutamate, which markedly favors the cyclic product (1-3). 5-Oxoprolinase activity has been found in a number of animal tissues and has been purified from kidney, a rich source of the enzyme. Earlier work in this laboratory established that 5-oxo-L-proline is a quantitatively significant metabolite of glutathione which is formed in the 'y-glutamyl cycle by the action of 'y-glutamyl cyclotransferase on y-glutamyl amino acids (4). Thus, the enzyme-catalyzed hydrolysis of 5-oxo-L-proline links the reactions involved in the utilization of glutathione (catalyzed by y-glutamyl transpeptidase, y-glutamyl cyclotransferase, and cysteinylglycinase) with those involved in its synthesis (catalyzed by y-glutamylcysteine synthetase and glutathione synthetase). In previous work (5) it was shown that L-2-imidazolidone4-carboxylate, a competitive inhibitor of 5-oxoprolinase, markedly decreases the metabolism of 5-oxoproline in vivo.Here we describe a new heterocyclic substrate of 5-oxoprolinase, L-2-oxothiazolidine-4-carboxylate, which is cleaved by the enzyme according to the scheme given in Fig. 1. In this pathway it is assumed that S-carboxy-cysteine, the initial product of hydrolysis, decarboxylates nonenzymatically. We have found that administration of L-2-oxothiazolidine-4-carboxylate to mice produces marked inhibition of the metabolism of 5-oxoproline but not that of glutamate. Administration of this compound also stimulates formation of glutathione in the liver.EXPERIMENTAL PROCEDURES Materials. The L and D isomers of 2-oxothiazolidine-4-carboxylate were synthesized by the method of Kaneko et al. (6) as modified (7). We are indebted to Sidney Weinhouse for a sample of the L isomer which was used in our initial studies. 5-Oxo-L-prolinase has been purified from rat kidney (3); the enzyme used in the present...