diphosphate (UDP)-glucuronosyltransferase (UGT) and re-Significant controversy exists regarding the regulaquires UDP-glucuronic acid (UDP-GlcUA), which serves as tion of glucuronidation during the process of hepatic the donor substrate of the conjugating sugar. The active site regeneration. We used a partial hepatectomy rat model of UGT is localized in the lumen of the endoplasmic reticulum to elucidate the effects of hepatic regeneration on the (ER) and is at least partially latent. 1,2 various components of the microsomal glucuronidation The mammalian liver is unique in its ability to proliferate system. Hepatic microsomes were prepared by standard in response to injury, whether this is caused by viruses, toxsucrose density centrifugation, coupled with a modified ins, ischemia, or surgery. Two-thirds hepatectomy is a welltechnique involving Percoll centrifugation. Microestablished model used to study hepatic regeneration, which somal uridine diphosphate (UDP)-glucuronosyltransferis characterized by rapid proliferation of hepatocytes with ase (UGT) protein expression and UGT messenger RNA restoration of the original liver mass within 10 to 14 days. 3,4 (mRNA) levels were measured by Western and Northern
In the course of this rapid proliferation, the structure and blotting. UGT enzyme activity was determined toward composition of the endoplasmic reticulum may undergo moditwo prototypical aglycones, p-nitrophenol and estrone, fication, 5 and the need for regeneration may take priority in intact and digitonin-treated microsomes. Microsomal over the restoration of liver-specific enzyme activity. 5,6 Moreuptake of the cosubstrate for all glucuronidation reacover, recent evidence documents that changes in the exprestions, UDP-glucuronic acid (UDP-GlcUA), was detersion of a variety of genes occur during hepatic regeneramined using a rapid-filtration assay. Microsomal enrichtion. 3,7,8 ment after hepatectomy was preserved only when the Previous studies showed that the activities of several drug-Percoll method was used. Microsomal UGT protein exmetabolizing enzymes are impaired during hepatic regenerapression and UGT mRNA levels were unaltered after tion, although not all enzymes are influenced to the same hepatectomy. UGT enzyme activity toward estrone was extent. 9 Thus, it would appear highly relevant to define in unchanged 1 day posthepatectomy compared with sham what manner glucuronidation, the most important hepatic laparotomy controls. Similarly, p-nitrophenol glucuroconjugation reaction, is affected. However, previous data on nide formation was unaffected by hepatic regeneration UGT activity have yielded conflicting results. One study doc-1, 2, and 5 days posthepatectomy when digitonin-treated umented preservation of UGT activity in liver homogenate microsomes were used. Glucuronidation of p-nitropheafter partial hepatectomy, 9 and two subsequent studies using nol in intact microsomes was increased in partial hepamicrosomes presented findings that contradict this original tectomy compared with sham-operated controls at 1 and report. 10,11
2 days. This increase was not attributable to changes in
To define the effect of hepatic regeneration on glucuronidamicrosomal UDP-GlcUA uptake, which was comparable tion and to explain the discrepancy in the results of the three in both groups. We conclude that microsomal glucuronistudies above, we systematically assessed the different comdation, in contrast to other well characterized hepatic ponents of the hepatic microsomal glucuronidation system metabolic functions, is highly preserved during liver reduring liver regeneration. We examined microsomal UGT generation. (HEPATOLOGY 1996;24:1250-1255.) protein expression, as well as hepatocellular messenger RNA (mRNA) levels because these were not examined by previous investigators. We also measured microsomal UDP-GlcUA up-Glucuronidation is a detoxifying reaction whereby a wide variety of endogenous and exogenous lipophilic sub-take because the availability of UDP-GlcUA to the active site of UGT may be rate-limiting for glucuronidation. [12][13][14][15] In strates(aglycones) are conjugated in the liver with D-glucuronic acid and thus made water soluble for excretion in addition, UGT activity was analyzed using two aglycones, each of which is glucuronidated by a distinct UGT isoform. 16 urine or bile. The process is catalyzed by the enzyme uridine
In our preliminary experiments, when microsomal fractions prepared by crude-sucrose centrifugation were used, we noted a significant decrease in microsomal UDP-GlcUA up-Abbreviations: UDP, uridine diphosphate; UGT, UDP-glucuronosyltransferase; UDP-take as well as UGT protein expression in the regenerating GlcUA, UDP-glucuronic acid; ER, endoplasmic reticulum; CHAPSO, 3-[(3-cholamidopropyl)liver. However, standard tests to validate these microsomes dimethyammonid]-2-hydroxy-1-propanesulfonate; mRNA, messenger RNA; NADPH, nicorevealed them to be significantly less enriched in ER marker tinamide adenine dinucleotide phosphate; Ig, immunoglobulin; cDNA, complementary DNA. From the Division of Gastroenterology, Brigham and Women's Hospital, Harvard Medi-enzymes than microsomes isolated from normal rat liver.