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Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of the liver metabolism due to functional deficiency of the peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). AGT deficiency results in overproduction of oxalate which complexes with calcium to form insoluble calcium-oxalate salts in urinary tracts, ultimately leading to end-stage renal disease. Currently, the only curative treatment for PH1 is combined liver-kidney transplantation, which is limited by donor organ shortage and lifelong requirement for immunosuppression. Transplantation of genetically modified autologous hepatocytes is an attractive therapeutic option for PH1. However, the use of fresh primary hepatocytes suffers from limitations such as organ availability, insufficient cell proliferation, loss of function, and the risk of immune rejection. We developed patient-specific induced pluripotent stem cells (PH1-iPSCs) free of reprogramming factors as a source of renewable and genetically defined autologous PH1-hepatocytes. We then investigated additive gene therapy using a lentiviral vector encoding wild-type AGT under the control of the liver-specific transthyretin promoter. Genetically modified PH1-iPSCs successfully provided hepatocyte-like cells (HLCs) that exhibited significant AGT expression at both RNA and protein levels after liver-specific differentiation process. These results pave the way for cell-based therapy of PH1 by transplantation of genetically modified autologous HLCs derived from patient-specific iPSCs., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Rapamycin (Rapa), one of the newer immunosuppressants has been found to control and prevent autoimmune features in animal models. This is the first report describing the successful control of post-transplant autoimmune hepatitis (AIH) with Rapa. Post-transplant AIH is diagnosed in the presence of raised transaminases, elevated immunoglobulin G, presence of autoantibodies and histologic changes consistent with AIH on liver biopsy. It may represent a recurrence of the original AIH that led to transplantation or present as a de novo AIH after liver transplant. Post-transplant AIH has conventionally been treated with Prednisolone (Pred) and Azathioprine (AZA). In this report, tailoring of immunosuppression after diagnosis of post-transplant AIH is described with special emphasis on those treated successfully with Rapa. Fifteen of 21 patients responded to treatment with an increase in dose of Pred and addition of AZA or Mycophenolate Mofetil (MMF) to calcineurin inhibitor. Five non-responders and one other patient with post-transplant AIH were treated with addition of Rapa. All six responded to treatment but drug was withdrawn in one patient. Adverse events were minimal. Rapa may prove to be an important addition in the control of autoimmune liver disease.

Fibrates modify the expression of genes implicated in lipoprotein and fatty acid metabolism via the peroxisome proliferator-activated receptor alpha(PPARalpha), leading to reductions in serum triglycerides and cholesterol. The expression of certain genes regulated by PPARalpha have been shown to be modified in a species dependent manner. Aspartate aminotransferase (AspAT or GOT) and alanine aminotransferase (AlaAT or GPT) are enzymes involved in intermediate metabolism in all cells and in hepatic gluconeogenesis. These enzymes are also widely used as serum markers of possible tissue damage. This study investigated whether fenofibrate could modify the expression of liver AspAT and/or AlaAT and thus possibly alter transaminase levels independently of a cytotoxic effect. In human Hep G2 cells, fenofibrate increased cytosolic AspAT (cAspAT) activity by 40% and AlaAT activity by 100%, as well as both mRNAs. Nuclear run on assays showed that this effect was, at least in part, transcriptional. Increases in mRNA were also observed in human hepatocyte cultures at concentrations of the drug attained in patients. In C57BL/6 mice, fenofibrate decreased cAspAT and cAlaAT mRNA, while these effects were abolished in PPARalpha knock-out mice. In conclusion, fenofibrate has been shown to modify cAspAT and AlaAT gene expression in a species and PPARalpha dependent manner. This is the first demonstration that cAspAT and AlaAT activities may be pharmacologically altered, independently of a toxic phenomenon.

During the preclinical, early clinical, late-stage clinical, and postmarketing phases of the pharmaceutical discovery and development process, one important aspect of drug safety assessment involves monitoring for possible drug-induced hepatic injury. Hepatic injuries vary in nature from direct, intrinsic effects that are observed in most recipients and more than one species to rare idiosyncratic responses seen only in a few clinical subjects. Histological types of injuries vary from hepatocellular to hepatobiliary with multiple cellular effects characteristic of each type. Of the various clinical laboratory markers for hepatic injury, serum transaminases, especially alanine aminotransferase (ALT), are the most universally important indicators for studies ranging from early preclinical animal testing to postmarketing patient monitoring. This review examines the characteristics of hepatic toxicity that result in serum ALT changes, the differences in the etiology of hepatic responses which govern when liver injury is most likely to be detected during the four phases of the drug discovery and development process, and those modulating factors which affect the utility of ALT as a dependable marker of hepatic injury in clinical populations. The paper concludes with a summary of some ancillary methods for early preclinical screening such as in vitro metabolism and toxicity assays, gene and protein expression analysis, and some strategies for enhancing the probability for the early detection of idiosyncratic hepatotoxic responses which are infrequent but significant factors in the safety assessment process., (Copyright 1998 Academic Press.)

The hemin regulation of L-alanine:4,5-dioxovalerate transaminase, the enzyme proposed for an alternate route of delta-aminolevulinic acid (ALA) biosynthesis in mammalian system was studied in different conditions: phenylhydrazine induced anemia, polycythemia by erythropoietin to anemic rats, treatment with cobalt chloride, a porphyrogenic drug. The activity of L- alanine:4,5-dioxovalerate transaminase in liver and kidney is stimulated in phenylhydrazine, whereas, erythropoietin injection to anemic rats prevents such stimulation. Further treatment with cobalt chloride to erythropoietin treated anemic rats stimulates the enzyme activity. Actinomycin D, however, inhibits the stimulation of L-alanine:4,5-dioxovalerate transaminase by phenylhydrazine suggesting that induction is at the level of transcription. Induced level of this enzyme in anemic condition was estimated quantitatively by radial immunodiffusion using antibody raised against L-alanine:4,5-dioxovalerate transaminase. Moreover, our studies reveal that stimulation of L-alanine:4,5-dioxovalerate transaminase in anemic condition is dependent on depletion of heme level. The regulatory role of intracellular heme pool on the induction of this enzyme suggests its physiological importance in heme biosynthesis.

Glucose administration inhibits the induction of ornithine aminotransferase (OAT) in both the whole animal and cultured hepatocytes. We have examined the ability of several hexoses and related molecules to inhibit the cAMP-dependent induction of OAT in primary cultures of adult rat hepatocytes. The hexoses (D-glucose, fructose, sorbitol, sorbose, and mannose) that were effective as inhibitors of OAT induction also resulted in accumulation of lactate in the culture medium, although lactate itself was not effective as an inhibitor. The hexoses and related 6-carbon structures (galactose, L-glucose, 2-deoxyglucose, 3-O-methylglucose, rhamnose, mannitol, and inositol) that were not effective as inhibitors of OAT induction did not result in accumulation of lactate in the culture medium. These results suggest that the carbohydrate repression of hepatic OAT requires metabolism of the carbohydrate by the liver cell. Upon addition to the culture medium of several compounds related to carbohydrate metabolism, many (ribose, xylitol, dihydroxyacetone, and glycerol) exhibited an inhibitory effect, with glycerol exhibiting the greatest effect. Fructose and glycerol inhibit OAT induction in the presence of 2-deoxyglucose, suggesting that the inhibitory effect of nonglucose carbohydrates is not occurring through conversion to glucose. The carbon sources observed to be most effective as inhibitors of OAT induction (glycerol, fructose, sorbitol, and sorbose result in more than 90% inhibition at 25 mM) all enter the glycolytic pathway at the triosephosphate level. The mechanism of the inhibitory effect of simple carbohydrates on OAT induction is not known but may involve an increase in certain glycolytic intermediates. Glucose and the related carbon sources exert their effect by inhibiting the cAMP-dependent increase in OAT synthesis. The cAMP-dependent increase in OAT mRNA was inhibited by fructose. These findings suggest that the carbohydrate inhibition of the cAMP-dependent increase in OAT synthesis occurs at a pretranslational level.

The effectiveness of dietary and hormonal treatments in inducing several pyridoxal phosphate-(PLP)-dependent enzymes has been examined in vitamin B-6 deficient rats. Holo- and apoenzymes of serine dehydratase and ornithine aminotransferase were inducible in both control and deficient rats by feeding them 80% casein diets or by injecting them with glucagon. Holo- and apotyrosine aminotransferase were induced in both control and deficient rats by injecting them with glucagon or with dexamethasone phosphate. Phosphoenolpyruvate carboxykinase, a non-PLP-dependent enzyme, was inducible in both control and deficient rats by glucagon treatment if the rats were fed, but not if they had been starved. The degree of induction of certain enzymes depended upon whether rats were fed ad libitum, starved overnight, or fed a protein-free diet prior to the induction period. Phosphoenolpyruvate carboxykinase activities were about the same in both control and deficient rats. In vitamin B-6 deficient rats, both uninduced and induced activities of serine dehydratase, ornithine aminotransferase, and tyrosine aminotransferase assayed in the prsence of PLP, but not in its absence, either equaled or exceeded control values under most experimental conditions. Synthesis of excess of apoenzyme of PLP-dependent enzymes generally accounted for the high total enzyme activity in deficient rats. Differences between values for control and deficient rats could not be accounted for by differences in liver cyclic AMP concentrations nor were they apparently related to reduced food intake of the deficient rats. High apoenzyme concentration during depletion of coenzyme would tend to prevent depletion of active enzyme.

Hepatic ornithine aminotransferase (EC 2.6.1.13) (OAT) is a mitochondrial matrix enzyme that plays a role in amino acid catabolism and in gluconeogenesis. In rats, the synthesis of hepatic OAT is regulated by glucagon, dietary protein, and glucose. Serum-free primary cultures of adult rat hepatocytes were used to demonstrate that glucagon, cyclic AMP, and glucose are able to alter OAT synthesis by a direct action on hepatocytes. The rates of OAT synthesis were measured by immunoprecipitation of pulse-labeled OAT with an affinity-purified monospecific antibody. Ten hours after cyclic AMP addition to the culture medium, the relative rate of OAT synthesis reached a peak value that was six- to eightfold above the control rate. OAT activity accumulated more slowly, reaching a level that was approximately threefold above the control by 24 h. The inclusion of glucose in the culture medium inhibited the increases in OAT synthesis and activity in a dose-dependent manner. Although synthesized as a precursor (pOAT), no pOAT was detected under control, induced, or carbohydrate-inhibited conditions; this suggests that pOAT processing may not be a regulatory site of OAT expression. By following the loss of labeled OAT, a half-life of 34 h in these cultures under all of the above conditions was observed. Regulation of OAT levels in cultured hepatocytes appears to be achieved primarily through changes in the rate of OAT synthesis.