John Kuhlenkamp

Insulin and glucocorticoid dependence of hepatic gamma-glutamylcysteine synthetase and glutathione synthesis in the rat. Studies in cultured hepatocytes and in vivo.

We reported that glucagon and phenylephrine decrease hepatocyte GSH by inhibiting gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis (Lu, S.C., J. Kuhlenkamp, C. Garcia-Ruiz, and N. Kaplowitz. 1991. J. Clin. Invest. 88:260-269). In contrast, we have found that insulin (In, 1 microgram/ml) and hydrocortisone (HC, 50 nM) increased GSH of cultured hepatocytes up to 50-70% (earliest significant change at 6 h) with either methionine or cystine alone as the sole sulfur amino acid in the medium. The effect of In occurred independent of glucose concentration in the medium. Changes in steady-state cellular cysteine levels, cell volume, GSH efflux, or expression of gamma-glutamyl transpeptidase were excluded as possible mechanisms. Both hormones are known to induce cystine/glutamate transport, but this was excluded as the predominant mechanism since the induction in cystine uptake required a lag period of greater than 6 h, and the increase in cell GSH still occurred when cystine uptake was blocked. Assay of GSH synthesis in extracts of detergent-treated cells revealed that In and HC increased the activity of GCS by 45-65% (earliest significant change at 4 h) but not GSH synthetase. In and HC treatment increased the Vmax of GCS by 31-43% with no change in Km. Both the hormone-mediated increase in cell GSH and GCS activity were blocked with either cycloheximide or actinomycin D. Finally, when studied in vivo, streptozotocin-treated diabetic and adrenalectomized rats exhibited lower hepatic GSH levels and GCS activities than respective controls. Both of these abnormalities were prevented with hormone replacement. Thus, both in vitro and in vivo, In and glucocorticoids are required for normal expression of GCS.

Hormone-mediated Down-Regulation of Hepatic Glutathione Synthesis in the Rat

Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.

Direct Protection Against Acetaminophen Hepatotoxicity by Propylthiouracil: IN VIVO AND IN VITRO STUDIES IN RATS AND MICE

Hepatotoxicity caused by acetaminophen can be prevented by enzyme-catalyzed conjugation of its reactive metabolite with glutathione (GSH). Since we have shown in previous studies that 6-N-propyl-2-thiouracil (PTU) can substitute for GSH as a substrate for the GSH S-transferases, we examined the possibility that PTU might also protect against acetaminophen hepatotoxicity by direct chemical interaction with the reactive metabolite of acetaminophen. In an in vitro system consisting of [(3)H]acetaminophen, liver microsomes from phenobarbital-pretreated rats, and an NADPH-generating system, we found that PTU had a dose-dependent additive effect with GSH on inhibition of acetaminophen covalent binding. PTU administration also resulted in a dose-dependent decrease in both GSH depletion and covalent binding in vivo in acetaminophen-treated mice. To examine the possible mechanisms by which PTU exerts its protective effect, we studied the action of PTU on both acetaminophen conjugation and metabolic activation. PTU had no effect upon acetaminophen pharmacokinetics in phenobarbital-pretreated rats, as examined by measuring acetaminophen concentration in bile, urine, and blood after an intraperitoneal dose, nor did it alter the total amount of polar conjugates formed. Microsomes from PTU-treated rats were unaltered in cytochrome P-450 concentrations and p-nitroanisole-O-demethylase, benzo-alpha-pyrene hydroxylase, and cytochrome c-reductase activities. Furthermore PTU did not decrease acetaminophen-GSH adduct formation in vitro, suggesting that there was no reduction in drug activation. However, in bile from [(35)S]PTU and [(3)H]acetaminophen treated rats, as well as in incubates of the two drugs with liver microsomes, a new (35)S- and (3)H-containing product could be identified. By both thin layer chromatography and high pressure liquid chromatography this new product, which co-eluted with [(3)H]acetaminophen, was separated from unreacted [(35)S]PTU. The formation of this product in vitro was a function of PTU concentration and reached a maximum of 0.06 mumol/min per mg protein at 0.5 mM PTU. In vivo, the total biliary excretion of this product over 4 h (116 nmol) equaled the net reduction in acetaminophen metabolite covalent binding in the liver of phenobarbital-pretreated rats (108 nmol). We conclude that PTU, independent of its antithyroid effect, diminishes hepatic macromolecular covalent binding of acetaminophen reactive metabolite both in vivo and in vitro, and it does so by detoxifying the reactive metabolite through direct chemical interaction in a manner similar to GSH. These observations may define the mechanism by which PTU is protective against liver injury caused by acetaminophen.

Changes in the expression of methionine adenosyltransferase genes and S-adenosylmethionine homeostasis during hepatic stellate cell activation†

Hepatic stellate cell (HSC) activation is an essential event during liver fibrogenesis. Methionine adenosyltransferase (MAT) catalyzes biosynthesis of S‐adenosylmethionine (SAMe), the principle methyl donor. SAMe metabolism generates two methylation inhibitors, methylthioadenosine (MTA) and S‐adenosylhomocysteine (SAH). Liver cell proliferation is associated with induction of two nonliver‐specific MATs: MAT2A, which encodes the catalytic subunit α2, and MAT2β, which encodes a regulatory subunit β that modulates the activity of the MAT2A‐encoded isoenzyme MATII. We reported that MAT2A and MAT2β genes are required for liver cancer cell growth that is induced by the profibrogenic factor leptin. Also, MAT2β regulates leptin signaling. The strong association of MAT genes with proliferation and leptin signaling in liver cells led us to examine the role of these genes during HSC activation. MAT2A and MAT2β are induced in culture‐activated primary rat HSCs and HSCs from 10‐day bile duct ligated (BDL) rat livers. HSC activation led to a decline in intracellular SAMe and MTA levels, a drop in the SAMe/SAH ratio, and global DNA hypomethylation. The decrease in SAMe levels was associated with lower MATII activity during activation. MAT2A silencing in primary HSCs and MAT2A or MAT2β silencing in the human stellate cell line LX‐2 resulted in decreased collagen and alpha‐smooth muscle actin (α‐SMA) expression and cell growth and increased apoptosis. MAT2A knockdown decreased intracellular SAMe levels in LX‐2 cells. Activation of extracellular signal‐regulated kinase and phosphatidylinositol‐3‐kinase signaling in LX‐2 cells required the expression of MAT2β but not that of MAT2A. Conclusion: MAT2A and MAT2β genes are induced during HSC activation and are essential for this process. The SAMe level falls, resulting in global DNA hypomethylation. (HEPATOLOGY 2010.)

Identification and purification of a 36 kDa bile acid binder in human hepatic cytosol

We recently purified two closely related 33 kDa proteins from rat hepatic cytosol, designated bile acid binder I and II, which selectively bind bile acids with comparable affinity as glutathione S‐transferase B. This work has now been extended to human liver in which we have identified a similar cytosolic binding activity in the 30–40 kDa fraction from gel filtration. Subsequent chromatofocusing and hydroxyapatite chromatography resulted in the isolation of a homogenous monomeric protein of 36 kDa. The binding affinity of this protein for lithocholate using the displacement of 1‐anilino‐8‐naphthalenesulfonate (ANS) was 0.1 μM, whereas human hepatic glutathione S‐transferases purified from glutathione affinity chromatography demonstrated no competitive displacement of ANS.

Role of Glutathione Status in Protection against Ethanol-Induced Gastric Lesions

The role of glutathione status in gastric mucosal cytoprotection has been a subject of controversy. Cysteamine, an exogenous sulfhydryl agent and diethyl maleate (DEM), an endogenous glutathione (GSH) depletor both appear to protect rats from ethanol-induced gastric lesions. In this study, we used various agents to alter gastric mucosal GSH levels and assessed the effects on susceptibility to ethanol injury. We found that DEM and buthionine sulfoximine both depleted gastric GSH but only DEM protected against ethanol-induced gastric lesions. L-Oxothiazolidine-4-carboxylate (OXT) and N-acetyl-L-cysteine (NAC) both potentiated ethanol-induced gastric lesions even though only NAC significantly raised the GSH level. The depletion of GSH by DEM was reversed by supplying cysteine in the form of OXT or NAC so that the net result was a GSH level close to normal control. The potentiation of ethanol injury by NAC and OXT was still apparent. These experiments show no relation between gastric GSH levels and susceptibility to ethanol injury.

Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver

Sinusoidal membrane vesicles from rat liver were employed to study the characteristics of GSH transport. Saturable concentration dependent uptake was best described by the sum of a high and low Km transport. Preloading with GSH markedly stimulated the initial uptake of GSH. GSH transport was electrogenic; uptake was enhanced by an inwardly directed K+ gradient which could be blocked by the K+-channel blocker, Ba2+. The other cations such as Na+, Li+ were poor substitutes for K+. These results therefore show that net GSH transport involves movement of K+.

Hepatic Glutathione S-Transferases: Identification by Gel Filtration and in Vitro Inhibition by Organic Anions

Summary In the gel filtration of 100,000 g rat liver supernatant, four major gluta-thione S-transferase activities, S-aryl-, S-epoxide-, S-aralkyl-, and S-alkyltransferase, were identified as having an elution volume identical to that of fractions exhibiting either glutathione or sulfobromophthalein sodium binding. The organic anions, sulfobromophthalein sodium, indocyanine green, and bilirubin, were found to be competitive inhibitors of the four glutathione S-transferase activities. These findings indicate that the glutathione S-transferases bind organic anions and, as a group, have a similar molecular weight to a known organic anion-binding protein. It is proposed that these enzymes also serve nonenzymically as a group of binding proteins in the hepatic cytoplasmic transport of organic anions.