Activation of autophagy during normothermic machine perfusion of discarded livers is associated with improved hepatocellular function.

Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function.NEW & NOTEWORTHY We demonstrate that ischemia-reperfusion injury occurs in all livers during NMP, though there are notable differences in gene expression between functional and nonfunctional livers. We further demonstrate that activation of the liver's repair and homeostasis mechanisms through autophagy plays a vital role in the graft's response to injury and may impact liver function. These findings indicate that liver autophagy might be a key therapeutic target for rehabilitating the function of severely injured or untransplantable livers.

Mitochondrial genotype alters the impact of rapamycin on the transcriptional response to nutrients in Drosophila.

BACKGROUND: In addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood. RESULTS: Using a Drosophila model in which alternative mtDNAs are present on a common nuclear background, we studied the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status. Adult flies with the 'native' and 'disrupted' genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment. CONCLUSIONS: Many of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.

Transcriptional changes during hepatic ischemia-reperfusion in the rat.

There are few effective targeted strategies to reduce hepatic ischemia-reperfusion (IR) injury, a contributor to poor outcomes in liver transplantation recipients. It has been proposed that IR injury is driven by the generation of reactive oxygen species (ROS). However, recent studies implicate other mediators of the injury response, including mitochondrial metabolic dysfunction. We examined changes in global gene expression after transient hepatic ischemia and at several early reperfusion times to identify potential targets that could be used to protect against IR injury. Male Wistar rats were subjected to 30 minutes of 70% partial warm ischemia followed by 0, 0.5, 2, or 6 hours of reperfusion. RNA was extracted from the reperfused and non-ischemic lobes at each time point for microarray analysis. Identification of differentially expressed genes and pathway analysis were used to characterize IR-induced changes in the hepatic transcriptome. Changes in the reperfused lobes were specific to the various reperfusion times. We made the unexpected observation that many of these changes were also present in tissue from the paired non-ischemic lobes. However, the earliest reperfusion time, 30 minutes, showed a marked increase in the expression of a set of immediate-early genes (c-Fos, c-Jun, Atf3, Egr1) that was exclusive to the reperfused lobe. We interpreted these results as indicating that this early response represented a tissue autonomous response to reperfusion. In contrast, the changes that occurred in both the reperfused and non-ischemic lobes were interpreted as indicating a non-autonomous response resulting from hemodynamic changes and/or circulating factors. These tissue autonomous and non-autonomous responses may serve as targets to ameliorate IR injury.

Stability of histone post-translational modifications in samples derived from liver tissue and primary hepatic cells.

Chromatin structure, a key contributor to the regulation of gene expression, is modulated by a broad array of histone post-translational modifications (PTMs). Taken together, these "histone marks" comprise what is often referred to as the "histone code". The quantitative analysis of histone PTMs by mass spectrometry (MS) offers the ability to examine the response of the histone code to physiological signals. However, few studies have examined the stability of histone PTMs through the process of isolating and culturing primary cells. To address this, we used bottom-up, MS-based analysis of histone PTMs in liver, freshly isolated hepatocytes, and cultured hepatocytes from adult male Fisher F344 rats. Correlations between liver, freshly isolated cells, and primary cultures were generally high, with R2 values exceeding 0.9. However, a number of acetylation marks, including those on H2A K9, H2A1 K13, H3 K4, H3 K14, H4 K8, H4 K12 and H4 K16 differed significantly among the three sources. Inducing proliferation of primary adult hepatocytes in culture affected several marks on histones H3.1/3.2 and H4. We conclude that hepatocyte isolation, culturing and cell cycle status all contribute to steady-state changes in the levels of a number of histone PTMs, indicating changes in histone marks that are rapidly induced in response to alterations in the cellular milieu. This has implications for studies aimed at assigning biological significance to histone modifications in tumors versus cancer cells, the developmental behavior of stem cells, and the attribution of changes in histone PTMs to altered cell metabolism.

Hepatic Gene Expression During the Perinatal Transition in the Rat.

During the immediate postnatal (PN) period, the liver, with its role in energy metabolism and macromolecule synthesis, plays a central role in the perinatal transition. Using RNA microarrays and several complementary computational analyses, we characterized changes in hepatic gene expression in the rat across a developmental period starting with the late gestation fetus (embryonic day 21), and including 30 min PN, 4 h PN, 12 h PN, 1 day PN, and 1 week after birth. Following subtle changes in gene expression at the earliest PN time point, there were marked changes that occurred between 4 and 12 h after birth. These reflected changes in multiple metabolic pathways, with expression of enzymes involved in glycolysis and cholesterol synthesis showing the greatest change. Over 50% of nuclear-encoded mitochondrial genes changed in the first 7 days of PN life, with 25% changing within the first 24 h. We also observed changes coinciding with a transient period of synchronous hepatocyte proliferation that we had observed previously, which occurs during the first PN week. Analysis for upstream regulators of gene expression indicated multiple initiating factors, including cell stress, hormones, and cytokines. Also implicated were multiple canonical transcription factor networks. We conclude that changes in gene expression during the early phases of the perinatal transition involve a complex, choreographed network of signaling pathways that respond to a variety of environmental stimuli. This transcriptomic response during the immediate PN period reflects a complex metabolic adaptive response that incorporates a panoply of signaling pathways and transcriptional regulators.

Engraftment and Repopulation Potential of Late Gestation Fetal Rat Hepatocytes.

BACKGROUND: The limited availability of donor organs has led to a search for alternatives to liver transplantation to restore liver function and bridge patients to transplantation. We have shown that the proliferation of late gestation (embryonic day 19) fetal rat hepatocytes is mitogen-independent and that mechanisms regulating mRNA translation, cell cycle progression, and gene expression differ from those of adult rat hepatocytes. In the present study, we investigated whether E19 fetal hepatocytes can engraft and repopulate an injured adult liver. METHODS: Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP- fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. RESULTS: More than a third of LAP+ fetal hepatocytes expressed ductal markers. Transcriptomic analysis revealed that these dual-expressing cells represent a population of less well-differentiated hepatocytes. Upon transplantation, LAP+ late gestation fetal hepatocytes formed hepatic, endothelial, and ductal colonies within 1 month. By 10 months, colonies derived from LAP+ cells increased so that up to 35% of the liver was repopulated by donor-derived cells. CONCLUSIONS: Late gestation fetal hepatocytes, despite being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well-differentiated phenotype.

Regulation of fetal liver growth in a model of diet restriction in the pregnant rat.

Limited nutrient availability is a cause of intrauterine growth restriction (IUGR), a condition that has important implications for the well being of the offspring. Using the established IUGR model of maternal fasting in the rat, we investigated mechanisms that control gene expression and mRNA translation in late-gestation fetal liver. Maternal fasting for 48 h during the last one-third of gestation was associated with a 10-15% reduction in fetal body weight and a disproportionate one-third reduction in total fetal liver protein. The fetal liver transcriptome showed only subtle changes consistent with reduced cell proliferation and enhanced differentiation in IUGR. Effects on the transcriptome could not be attributed to specific transcription factors. We purified translating polysomes to profile the population of mRNAs undergoing active translation. Microarray analysis of the fetal liver translatome indicated a global reduction of translation. The only targeted effect was enhanced translation of mitochondrial ribosomal proteins in IUGR, consistent with enhanced mitochondrial biogenesis. There was no evidence for attenuated signaling through the mammalian target of rapamycin (mTOR). Western blot analysis showed no changes in fetal liver mTOR signaling. However, eukaryotic initiation factor 2α (eIF2α) phosphorylation was increased in livers from IUGR fetuses, consistent with a role in global translation control. Our data indicate that IUGR-associated changes in hepatic gene expression and mRNA translation likely involve a network of complex regulatory mechanisms, some of which are novel and distinct from those that mediate the response of the liver to nutrient restriction in the adult rat.

Profiling of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR).

The mechanistic target of rapamycin (mTOR) integrates growth factor signaling, nutrient abundance, cell growth, and proliferation. On the basis of our interest in somatic growth in the late gestation fetus, we characterized the role of mTOR in the regulation of hepatic gene expression and translation initiation in fetal and adult rats. Our strategy was to manipulate mTOR signaling in vivo and then characterize the transcriptome and translating mRNA in liver tissue. In adult rats, we used the nonproliferative growth model of refeeding after a period of fasting and the proliferative model of liver regeneration following partial hepatectomy. We also studied livers from preterm fetal rats (embryonic day 19) in which fetal hepatocytes are asynchronously proliferating. All three models employed rapamycin to inhibit mTOR signaling. Analysis of the transcriptome in fasted-refed animals showed rapamycin-mediated induction of genes associated with oxidative phosphorylation. Genes associated with RNA processing were downregulated. In liver regeneration, rapamycin induced genes associated with lysosomal metabolism, steroid metabolism, and the acute phase response. In fetal animals, rapamycin inhibited expression of genes in several functional categories that were unrelated to effects in the adult animals. Translation control showed marked fetal-adult differences. In both adult models, rapamycin inhibited the translation of genes with complex 5' untranslated regions, including those encoding ribosomal proteins. Fetal translation was resistant to the effects of rapamycin. We conclude that the mTOR pathway in liver serves distinct physiological roles in the adult and fetus, with the latter representing a condition of rapamycin resistance.

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.

Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2).

The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [Rictor-knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo. Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2-mediated events. Hepatic mTORC2 regulated a complex gene expression and post-translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell-autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2-related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.

Phosphoproteomic profiling of in vivo signaling in liver by the mammalian target of rapamycin complex 1 (mTORC1).

BACKGROUND: Our understanding of signal transduction networks in the physiological context of an organism remains limited, partly due to the technical challenge of identifying serine/threonine phosphorylated peptides from complex tissue samples. In the present study, we focused on signaling through the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which is at the center of a nutrient- and growth factor-responsive cell signaling network. Though studied extensively, the mechanisms involved in many mTORC1 biological functions remain poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We developed a phosphoproteomic strategy to purify, enrich and identify phosphopeptides from rat liver homogenates. Using the anticancer drug rapamycin, the only known target of which is mTORC1, we characterized signaling in liver from rats in which the complex was maximally activated by refeeding following 48 hr of starvation. Using protein and peptide fractionation methods, TiO(2) affinity purification of phosphopeptides and mass spectrometry, we reproducibly identified and quantified over four thousand phosphopeptides. Along with 5 known rapamycin-sensitive phosphorylation events, we identified 62 new rapamycin-responsive candidate phosphorylation sites. Among these were PRAS40, gephyrin, and AMP kinase 2. We observed similar proportions of increased and reduced phosphorylation in response to rapamycin. Gene ontology analysis revealed over-representation of mTOR pathway components among rapamycin-sensitive phosphopeptide candidates. CONCLUSIONS/SIGNIFICANCE: In addition to identifying potential new mTORC1-mediated phosphorylation events, and providing information relevant to the biology of this signaling network, our experimental and analytical approaches indicate the feasibility of large-scale phosphoproteomic profiling of tissue samples to study physiological signaling events in vivo.

The physiology and pathophysiology of rapamycin resistance: implications for cancer.

Rapamycin is an inhibitor of the mammalian Target of Rapamycin, mTOR, a nutrient-sensing signaling kinase and a key regulator of cell growth and proliferation. While rapamycin and related compounds have anti-tumor activity, a prevalent characteristic of cancer cells is resistance to their anti-proliferative effects. Our studies on nutrient regulation of fetal development showed that hepatocyte proliferation in the late gestation fetal rat is resistant to rapamycin. Extension of these studies to other tissues in the fetal and neonatal rat indicated that rapamycin resistance is a characteristic of normal cell proliferation in the growing organism. In hepatic cells, ribosomal biogenesis and cap-dependent protein translation were found to be relatively insensitive to the drug even though mTOR signaling was highly sensitive. Cell cycle progression was also resistant at the level of cyclin E-dependent kinase activity. Studies on the effect of rapamycin on gene expression in vitro and in vivo demonstrated that mTOR-mediated regulation of gene expression is independent of effects on cell proliferation and cannot be accounted for by functional regulation of identifiable transcription factors. Genes involved in cell metabolism were overrepresented among rapamycin-sensitive genes. We conclude that normal cellular proliferation in the context of a developing organism can be independent of mTOR signaling, that cyclin E-containing complexes are a critical locus for rapamycin sensitivity, and that mTOR functions as a modulator of metabolic gene expression in cells that are resistant to the anti-proliferative effects of the drug.

Resveratrol inhibits protein translation in hepatic cells.

Resveratrol is a plant-derived polyphenol that extends lifespan and healthspan in model organism. Despite extensive investigation, the biological processes mediating resveratrol's effects have yet to be elucidated. Because repression of translation shares many of resveratrol's beneficial effects, we hypothesized that resveratrol was a modulator of protein synthesis. We studied the effect of the drug on the H4-II-E rat hepatoma cell line. Initial studies showed that resveratrol inhibited global protein synthesis. Given the role of the mammalian Target of Rapamycin (mTOR) in regulating protein synthesis, we examined the effect of resveratrol on mTOR signaling. Resveratrol inhibited mTOR self-phosphorylation and the phosphorylation of mTOR targets S6K1 and eIF4E-BP1. It attenuated the formation of the translation initiation complex eIF4F and increased the phosphorylation of eIF2α. The latter event, also a mechanism for translation inhibition, was not recapitulated by mTOR inhibitors. The effects on mTOR signaling were independent of effects on AMP-activated kinase or AKT. We conclude that resveratrol is an inhibitor of global protein synthesis, and that this effect is mediated through modulation of mTOR-dependent and independent signaling.

Rapamycin response in tumorigenic and non-tumorigenic hepatic cell lines.

BACKGROUND: The mTOR inhibitor rapamycin has anti-tumor activity across a variety of human cancers, including hepatocellular carcinoma. However, resistance to its growth inhibitory effects is common. We hypothesized that hepatic cell lines with varying rapamycin responsiveness would show common characteristics accounting for resistance to the drug. METHODOLOGY/PRINCIPAL FINDINGS: We profiled a total of 13 cell lines for rapamycin-induced growth inhibition. The non-tumorigenic rat liver epithelial cell line WB-F344 was highly sensitive while the tumorigenic WB311 cell line, originally derived from the WB-F344 line, was highly resistant. The other 11 cell lines showed a wide range of sensitivities. Rapamycin induced inhibition of cyclin E-dependent kinase activity in some cell lines, but the ability to do so did not correlate with sensitivity. Inhibition of cyclin E-dependent kinase activity was related to incorporation of p27(Kip1) into cyclin E-containing complexes in some but not all cell lines. Similarly, sensitivity of global protein synthesis to rapamycin did not correlate with its anti-proliferative effect. However, rapamycin potently inhibited phosphorylation of two key substrates, ribosomal protein S6 and 4E-BP1, in all cases, indicating that the locus of rapamycin resistance was downstream from inhibition of mTOR Complex 1. Microarray analysis did not disclose a unifying mechanism for rapamycin resistance, although the glycolytic pathway was downregulated in all four cell lines studied. CONCLUSIONS/SIGNIFICANCE: We conclude that the mechanisms of rapamycin resistance in hepatic cells involve alterations of signaling downstream from mTOR and that the mechanisms are highly heterogeneous, thus predicting that maintaining or promoting sensitivity will be highly challenging.

The alpha4-containing form of protein phosphatase 2A in liver and hepatic cells.

The Ser/Thr phosphatase PP2A is a set of multisubunit enzymes that regulate many cellular processes. In yeast, the PP2A regulatory subunit Tap42 forms part of the target of rapamycin (TOR) signaling pathway that links nutrient and energy availability to cell growth. The physiological intersection between the mammalian orthologs of Tap42 and TOR, alpha4 and mTOR, has not been fully characterized. We used two in vivo models of liver growth in the rat, late gestation fetal development and regeneration after partial hepatectomy, to explore the regulation of the alpha4-containing form of PP2A. The alpha4/PP2A catalytic subunit (alpha4/PP2A-C) complex was present in both fetal and adult liver extracts. There was a trend towards higher levels of alpha4 protein in fetal liver, but the complex was more abundant in adult liver. Fractionation of extracts by ion exchange chromatography and transient transfection of the AML12 mouse hepatic cell line indicated that alpha4 associates with PP2A-C but that these complexes have low catalytic activity with both peptide and protein substrates. alpha4 was able to associate with forms of PP2A-C that were both methylated and non-methylated at the carboxy-terminus. The mTOR inhibitor rapamycin did not block the formation of alpha4/PP2A-C in liver or hepatic cells, nor did it appear to modulate PP2A activity. Furthermore, sensitivity to the growth inhibitory effects of rapamycin among a panel of hepatic cell lines did not correlate with levels of alpha4 or alpha4/PP2A-C. Our results indicate that the yeast Tap42/TOR paradigm is not conserved in hepatic cells.

Hepatic translation control in the late-gestation fetal rat.

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4A1 and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin.

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A).

The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), and A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit in adult liver. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.

D-type cyclins and G1 progression during liver development in the rat.

Initiation and progression through G1 requires the activity of signaling complexes containing cyclins (D- or E-type) and cyclin-dependent kinases (CDK4/6 and CDK2, respectively). We set out to identify the G1-phase cyclins and CDKs that are operative during late gestation liver development in the rat. This is a period during which hepatocytes show a high rate of proliferation that is, at least in part, independent of the mitogenic signaling pathways that are functional in mature hepatocytes. RNase protection assay and Western immunoblotting indicated that cyclin D1 is expressed at similar levels in fetal and adult liver. When cyclin D1 was induced after partial hepatectomy, its predominant CDK-binding partner was CDK4. In contrast, cyclins D2 and D3 predominated in fetal liver and were complexed with both CDK4 and CDK6. Little CDK6 protein was expressed in quiescent or regenerating adult liver. Cyclins E1 and E2 were both transcriptionally up-regulated in fetal liver. Activity of complexes containing cyclins E1 and E2 was higher in fetal liver, as was content of the cell cycle regulator, Rb. In fetal liver, Rb was highly phosphorylated at both cyclin D- and cyclin E-dependent sites. In conclusion, liver development is associated with a switch from cyclin D2/D3-containing complexes to cyclin D1:CDK4 complexes. We speculate that the switch in D-type cyclins may be associated with the dependence on mitogenic signaling that develops as hepatocytes mature.

Effects of maternal starvation on hepatocyte proliferation in the late gestation fetal rat.

Fetal growth retardation, a common end point for a variety of conditions affecting mother and fetus, is associated with reduced liver mass. We have performed studies to determine the mechanism for decreased liver mass in a maternal starvation model of fetal growth restriction in the rat. Pregnant dams were deprived of food for 48 h before delivery on embryonic day 19 (E19). Fetal body weight was not affected. However, fetal liver weight was reduced by approximately 15%. Immunostaining of fetal liver for proliferating cell nuclear antigen and flow cytometry on isolated fetal hepatocytes showed G1 cell cycle arrest in samples from starved dams. Based on our prior studies showing attenuated hepatic insulin signaling in the late gestation fetal rat, we tested the hypothesis that G1 arrest in our model might be due to altered nutrient signaling. Fetal plasma amino acid analyses showed no decrease in branched-chain amino acids, but arginine concentrations were decreased in fetuses of fasted mothers. Reduced arginine in E19 fetal hepatocyte culture media was associated with decreased DNA synthesis. Whereas levels of cyclins D and E were unchanged in fetal hepatocytes exposed to low arginine, cyclin E-dependent kinase activity was reduced. Low arginine also induced changes in the translational machinery, indicative of impaired signaling through the nutrient sensing kinase mammalian target of rapamycin. Our results are consistent with the hypothesis that restricted nutrient availability signals to the hepatocyte cell cycle in fetuses of fasted mothers, thereby accounting for decreased hepatocyte proliferation and liver mass.

Insulin receptor substrate-1 is present in hepatocyte nuclei from intact rats.

Insulin receptor substrate-1 (IRS-1), the primary substrate for the insulin receptor tyrosine kinase in most cells and tissues, is a key component of the insulin signaling network. Numerous studies have documented the trafficking of IRS-1 from the cell membrane to intracellular, extranuclear compartments. During the course of our previous studies aimed at defining the ontogeny of insulin signaling in the rat, Western immunoblotting showed minimal IRS-1 content in late gestation fetal liver. Immunohistochemical analyses, aimed at corroborating these Western immunoblotting results, showed hepatocyte nuclear staining for IRS-1 in adult liver but not fetal liver. Further analysis of fixed tissue as well as immunofluorescent staining of liver cryosections confirmed the localization of IRS-1 to the nuclear matrix and nucleoli of adult hepatocytes within intact liver. Tissue fractionation and Western immunoblotting also showed nuclear localization of IRS-1, with this fraction accounting for approximately 25% of total liver IRS-1. Administration of insulin to intact animals did not stimulate nuclear translocation of hepatic IRS-1 or the p85 regulatory subunit of phosphatidylinositol-3 kinase. However, insulin did activate IRS-1-associated phosphatidylinositol-3 kinase in nuclear extracts. Our results indicate that insulin signaling, which terminates in an array of nuclear events, may originate at the immediate postreceptor level with IRS-1 activation within the nucleus of normal hepatocytes.