No abstract

This study was designed to characterize insulin receptor substrate-4 (IRS-4) in isolated rat hepatocytes and to examine its role in liver regeneration. Subcellular fractionation revealed that 85% of IRS-4 is located at isolated hepatocyte plasma membranes. The distribution of IRS-4 among intracellular compartments remained unchanged in insulin-stimulated cells. Two bands corresponding to 145 and 138 kd were observed in immunoblotting experiments. Immunoprecipitation of hepatocyte lysates with a highly specific antibody against IRS-4 led to an insulin and insulin-like growth factor 1 (IGF-1)-dependent increase in phosphotyrosine residues of the 145-kd band. IRS-4 was found to be associated with Src homology 2 (SH2) domain-containing proteins (phosphatidylinositol 3-kinase [PI 3-kinase] and Src homology phosphatase [SHP-2]) and with protein kinase C (PKC ). Insulin and IGF-1 elicited a rapid and dose-dependent binding of these 3 proteins to IRS-4. These data suggest that IRS-4 is insulin-/IGF-1-activated by phosphorylation and not by translocation, inducing the recruitment of SH2 domain-containing proteins and PKC to the membrane. To evaluate the possible role of IRS-4 in liver regeneration, we also examined this system after partial hepatectomy (PH). One day after PH, IRS-1 expression increased, consistent with a stimulatory role in the regenerative process, whereas it decreased 7 days after liver resection. This drastic IRS-1 depletion occurred at the expense of increased IRS-2 and IRS-4 expression 7 days after PH. In addition, at this period of time after surgery, the in vivo insulin stimulation of remnant rat livers showed an increase in IRS-4/PI 3-kinase association. Given that 1 and 7 days after PH isolated hepatocytes responded similarly to insulin in terms of induced cell proliferation, a compensatory role is proposed for IRS-2/4 induction. In conclusion, IRS-4 is activated by insulin and IGF-1-like IRS-1 in rat hepatocytes, and the induced expression of IRS-4 is a compensatory mechanism that plays a role in conditions of liver regeneration. T he insulin receptor is a tyrosine kinase that undergoes tyrosine phosphorylation of its ␤-subunit and subsequent kinase activation on insulin binding. The signaling pathways initiated by the insulin receptor depend on the recruitment of signaling proteins that associate with the receptor and become phosphorylated. The main substrates for ␤-subunit kinase activity are insulin receptor substrate (IRS) proteins. 1 Then, the resultant phosphotyrosine motifs in IRS bind proteins containing Src homology 2 (SH2) domains, notably phosphatidylinositol 3-kinase (PI 3-kinase), growth factor receptor binding protein 2, and the protein tyrosine phosphatase Src homology phosphatase (SHP)-2/Syp, activating a variety of biologic effects including mitogenesis, gene expression, glucose transport, and glycogen biosynthesis. 2 To date, 4 members of the IRS family (IRS-1, IRS-2, IRS-3, and IRS-4) have been identified 2 and observed to show significant structural and functional heterogene...

Azathioprine is an immunosuppressant drug widely used. Our purpose was to 1) determine whether its associated hepatotoxicity could be attributable to the induction of a necrotic or apoptotic effect in hepatocytes, and 2) elucidate the mechanism involved. To evaluate cellular responses to azathioprine, we used primary culture of isolated rat hepatocytes. Cell metabolic activity, reduced glutathione, cell proliferation, and lactate dehydrogenase release were assessed. Mitochondria were isolated from rat livers, and swelling and oxygen consumption were measured. Mitogen-activated protein kinase pathways and proteins implicated in cell death were analyzed. Azathioprine decreased the viability of hepatocytes and induced the following events: intracellular reduced glutathione (GSH) depletion, metabolic activity reduction, and lactate dehydrogenase release. However, the cell death was not accompanied by DNA laddering, procaspase-3 cleavage, and cytochrome c release.The negative effects of azathioprine on the viability of hepatocytes were prevented by cotreatment with N-acetyl-L-cysteine. In contrast, 6-mercaptopurine showed no effects on GSH content and metabolic activity. Azathioprine effect on hepatocytes was associated with swelling and increased oxygen consumption of intact isolated rat liver mitochondria. Both effects were cyclosporine A-sensitive, suggesting an involvement of the mitochondrial permeability transition pore in the response to azathioprine. In addition, the drug's effects on hepatocyte viability were partially abrogated by c-Jun N-terminal kinase and p38 kinase inhibitors. In conclusion, our findings suggest that azathioprine effects correlate to mitochondrial dysfunction and activation of stress-activated protein kinase pathways leading to necrotic cell death. These negative effects of the drug could be prevented by coincubation with N-acetyl-L-cysteine.

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

This report examines the effect of FK506 pretreatment on liver insulin receptor expression in partially (70%) hepatectomized rats. FK506 pretreatment led to an increased insulin receptor number 24 hours after hepatectomy, detected by means of insulin binding and crosslinking procedures. This increase was related to enhanced insulin receptor expression determined by in vitro mRNA translation and Western blot techniques. We also tested the functionality of the expressed insulin receptors by [ 3 H] thymidine incorporation into DNA in insulin-stimulated hepatocytes. The results show that FK506 pretreatment elicits an increase in the amount of insulin receptor ␣-subunits as measured by Western blot. Maximum ␣-subunit expression recorded 24 hours after surgery was preceded by increased insulin receptor mRNA levels, which were detected 6 hours after hepatectomy. Moreover, in FK506 -pretreated rat hepatocytes, obtained from remnant livers 24 hours after partial hepatectomy (PH), the increase in insulin receptor number was associated with improved sensitivity to the hormone. However, in both experimental groups (FK506-pretreated and nonpretreated rats), the sensitivity of hepatocytes toward epidermal growth factor (EGF) showed no significant change, which suggests a specific effect of FK506 on insulin receptor expression. In conclusion, our findings suggest that FK506 pretreatment induces insulin receptor expression in regenerating rat liver and promotes liver regeneration in hepatectomized rats. (HEPATOLOGY 2002;36:555-561.) P retreatment with immunosuppressive drugs, such as FK506 and cyclosporin A (CsA), increases the liver regenerative response after partial hepatectomy (PH) in rats. 1 Several research efforts have tried to determine the factors involved in the proliferative response associated with drug treatment. FK506 or CsA could modulate the liver response by (1) increasing the expression of local mitogens such as hepatocyte growth factor 2 or insulin-like growth factor I 3 ; (2) decreasing the production of inhibitory cytokines such as transforming growth factor ␤ 1 2 or interleukin 2 4 ; (3) inhibiting natural killer activity 4 ; or (4) predisposing the liver to regenerative signals. This last hypothesis has been suggested but not shown. Insulin is considered a secondary mitogen that enhances liver sensitivity to mitogens during its regeneration. 5 Administering insulin to 70% PH rats increases [ 3 H] thymidine labeling of DNA in hepatic parenchyma cells. 6 Insulin induces a specific peak in [ 3 H] thymidine incorporation 24 hours after PH, which is not observed in insulin-untreated or glucagon-treated animals. 6 This 24-hour period after PH also coincides with maximal effects of FK506 1 or CsA 2 on the liver regenerative response. Moreover, the role of insulin in liver regeneration is supported by its blunting of the immediate-early gene response after PH in diabetic animals. 7 In a very recent study performed in 70% PH/50% pancreatectomized rats, it was shown that liver mass recovery in these animals was del...