Aberrant Expression of Rest4 Gene in Low-Functioning Pancreatic Beta Cell Line.

Repressor element-1 silencing transcription factor (Rest) is not expressed in pancreatic beta cells and neuronal cells. However, Rest4, a truncated form of Rest, is expressed in high passaged MIN6 (HP-MIN6) cells, a pancreatic beta cell line that lost glucose-responsive insulin secretion. Rest4 is also expressed in injured MIN6 cells and isolated islets. Herein, the forced expression of dominant negative form of Rest in HP-MIN6 cells was subjected to microarray analysis of gene expression to investigate the role of Rest4 gene in MIN6 cells. Furthermore, the forced expression of Rest4 gene in MIN6 cells was subjected to microarray analysis of gene expression to investigate the function of Rest4 in normal insulin-producing cells. The results showed that Rest4 inhibits cell proliferation and DNA and RNA metabolism and stimulates secretory mechanisms and nervous system gene expression. These findings suggest that Rest4 may act defensively against cellular injury in pancreatic beta cells.

High dose of histone deacetylase inhibitors affects insulin secretory mechanism of pancreatic beta cell line.

OBJECTIVE: Histone deacytylase inhibitors (HDACis) inhibit the deacetylation of the lysine residue of proteins, including histones, and regulate the transcription of a variety of genes. Recently, HDACis have been used clinically as anti-cancer drugs and possible anti-diabetic drugs. Even though HDACis have been proven to protect the cytokine-induced damage of pancreatic beta cells, evidence also shows that high doses of HDACis are cytotoxic. In the present study, we, therefore, investigated the eff ect of HDACis on insulin secretion in a pancreatic beta cell line. METHODS: Pancreatic beta cells MIN6 were treated with selected HDACis (trichostatin A, TSA; valproic acid, VPA; and sodium butyrate, NaB) in medium supplemented with 25 mM glucose and 13% heat-inactivated fetal bovine serum (FBS) for indicated time intervals. Protein expression of Pdx1 and Mafa in MIN6 cells was demonstrated by immunohistochemistry and immunocytochemistry, expression of Pdx1 and Mafa genes was measured by quantitative RT-PCR method. Insulin release from MIN6 cells and insulin cell content were estimated by ELISA kit. Superoxide production in MIN6 cells was measured using a Total ROS/Superoxide Detection System. RESULTS: TSA, VPA, and NaB inhibited the expression of Pdx1 and Mafa genes and their products. TSA treatment led to beta cell malfunction, characterized by enhanced insulin secretion at 3 and 9 mM glucose, but impaired insulin secretion at 15 and 25 mM glucose. Th us, TSA induced dysregulation of the insulin secretion mechanism. TSA also enhanced reactive oxygen species production in pancreatic beta cells. CONCLUSIONS: Our results showed that HDACis caused failure to suppress insulin secretion at low glucose concentrations and enhance insulin secretion at high glucose concentrations. In other words, when these HDACis are used clinically, high doses of HDACis may cause hypoglycemia in the fasting state and hyperglycemia in the fed state. When using HDACis, physicians should, therefore, be aware of the capacity of these drugs to modulate the insulin secretory capacity of pancreatic beta cells.

Functional Analysis of Novel Candidate Regulators of Insulin Secretion in the MIN6 Mouse Pancreatic ß Cell Line.

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic ß cells is important for understanding and treating diabetes. The pancreatic ß cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion.

Microarray analysis of novel candidate genes responsible for glucose-stimulated insulin secretion in mouse pancreatic ß cell line MIN6.

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet ß cells is important for understanding and treating diabetes. MIN6 cells, a transformed ß-cell line derived from a mouse insulinoma, retain GSIS and are a popular in vitro model for insulin secretion. However, in long-term culture, MIN6 cells' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as "responder cells." In a DNA microarray analysis, we identified a group of genes with high expression in responder cells ("responder genes"), but extremely low expression in the Pr-HP cells. Another group of genes ("non-responder genes") was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including Chrebp, Scgn, and Syt7. Among the non-responder genes were Car2, Maf, and Gcg, which are not normally expressed in islet ß cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.

Acinar-to-ductal metaplasia induced by adenovirus-mediated pancreatic expression of Isl1.

Tubular complexes (TCs) are aggregates of duct-like monolayered cells in the developing and regenerating pancreas. Recent studies showed that TCs have regenerative potential, including islet neogenesis. We previously delivered adenovirus vector (AdV) into exocrine cells of the pancreas by intra-common bile ductal (ICBD) injection, and found that AdV expressing Pdx1, a pancreas-specific transcription factor, causes TC formation and islet neogenesis. We also established RTF-Pdx1-EGFP mice, which ubiquitously express Pdx1 when tetracycline is removed from the drinking water. However, exogenous Pdx1 expression in adult RTF-Pdx1-EGFP mice did not cause any pathological changes in the pancreas during three weeks of observation after tetracycline withdrawal. To examine whether the host immune response induced by AdV was involved in TC formation, we delivered AdVs expressing pancreas-related transcription factors or an irrelevant protein into the pancreas of RTF-Pdx1-EGFP mice. Histological analyses showed that both AdV injection and Pdx1 expression are required for TC formation. We also analyzed the effects of these ICBD-injected AdVs. AdV expressing Isl1, a proendocrine transcription factor, effectively induced TC formation through acinar-to-ductal metaplasia, and exogenous Pdx1 expression facilitated this process. Considering the regenerative potential of TCs, a strategy that efficiently induces TC formation may lead to novel therapies for diabetes.

Analysis of Foxo1-regulated genes using Foxo1-deficient pancreatic ß cells.

Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic ß-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient ß cells. To elucidate Foxo1's function in ß cells, we produced a ß-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a ß-cell line from an insulinoma induced in this knockout mouse by the ß-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced ß-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to ß-cell function. These cells should be useful for further studies on Foxo1's roles in ß-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus.

Analysis of the transcription factor cascade that induces endocrine and exocrine cell lineages from pancreatic progenitor cells using a polyoma-based episomal vector system.

UNLABELLED: Aims/Introduction: We recently established a strategy for isolating multipotential duct-like cells, called pdx-1-positive pancreatic cell-derived (PPPD) cells, from the pancreas. To analyze the molecular mechanisms of pancreatic cell differentiation, we introduced a polyoma-based episomal vector system into PPPD cells. MATERIALS AND METHODS: PPPD cells were stably transfected with a polyoma large T (PLT)-expressing plasmid vector, which included the polyoma origin of replication, to generate PLT-PPPD cells. Various cDNA for pancreas-related transcription factors were subcloned into the expression plasmid pPyCAG, which included the polyoma origin of replication. PLT-PPPD cells were stably transfected with the resulting plasmid vectors and then subjected to gene and protein expression analyses. RESULTS: The coexpression of Mafa, Neurod1 and Ipf1 induced Ins1 and Ins2 expression in PLT-PPPD cells. The forced expression of Pax6 alone induced the expression of glucagon. The coexpression of Neurod1 and Isl1 induced Ins2 and Sst expression. In contrast, the expression of Ptf1a and Foxa2 induced the expression of exocrine markers Cpa1 and Amy2. Transfections with multiple transcription factors showed that Isl1 is required for the differentiation of both insulin-positive cells and somatostatin-positive cells. In addition, Foxa2 induced the differentiation of glucagon-positive cells and inhibited the differentiation of insulin-positive and somatostatin-positive cells. PLT-PPPD cells allow episomal vector-based gene expression and should be useful for studying the transcription factor cascades involved in the differentiation of pancreatic cell types in vitro. CONCLUSIONS: Our coexpression study showed novel critical roles for Isl1 and Foxa2 in the differentiation of PPPD cells into endocrine cells. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00136.x, 2012).

Nuclear hormone retinoid X receptor (RXR) negatively regulates the glucose-stimulated insulin secretion of pancreatic ß-cells.

OBJECTIVE: Retinoid X receptors (RXRs) are members of the nuclear hormone receptor superfamily and are thought to be key regulators in differentiation, cellular growth, and gene expression. Although several experiments using pancreatic ß-cell lines have shown that the ligands of nuclear hormone receptors modulate insulin secretion, it is not clear whether RXRs have any role in insulin secretion. RESEARCH DESIGN AND METHODS: To elucidate the function of RXRs in pancreatic ß-cells, we generated a double-transgenic mouse in which a dominant-negative form of RXRß was inducibly expressed in pancreatic ß-cells using the Tet-On system. We also established a pancreatic ß-cell line from an insulinoma caused by the ß-cell-specific expression of simian virus 40 T antigen in the above transgenic mouse. RESULTS: In the transgenic mouse, expression of the dominant-negative RXR enhanced the insulin secretion with high glucose stimulation. In the pancreatic ß-cell line, the suppression of RXRs also enhanced glucose-stimulated insulin secretion at a high glucose concentration, while 9-cis-retinoic acid, an RXR agonist, repressed it. High-density oligonucleotide microarray analysis showed that expression of the dominant-negative RXR affected the expression levels of a number of genes, some of which have been implicated in the function and/or differentiation of ß-cells. CONCLUSIONS: These results suggest that endogenous RXR negatively regulates the glucose-stimulated insulin secretion. Given these findings, we propose that the modulation of endogenous RXR in ß-cells may be a new therapeutic approach for improving impaired insulin secretion in type 2 diabetes.

Maternal-effect gene Ces5/Ooep/Moep19/Floped is essential for oocyte cytoplasmic lattice formation and embryonic development at the maternal-zygotic stage transition.

In a search for genes specifically expressed in mouse embryonic stem cells, we identified one we called Ces5. We found that it corresponded to the Ooep gene, which was recently reported to be expressed specifically in oocytes. Mouse Ces5/Ooep, also called Moep19 or Floped, encoded a 164-amino acid protein, which was detected in the cytoplasm of developing and mature oocytes and in embryos throughout the preimplantation period. To examine its function, we carried out targeted disruption of this gene. The Ces5/Ooep-null mice were grossly normal, but the females were infertile. Although the ovaries and ovulation appeared normal, the embryos from Ces5/Ooep-null females mated with wild-type males showed developmental arrest at the two- or four-cell stage. In addition, their first cleavage was considerably delayed and often asymmetrical. Thus, Ces5/Ooep is a maternal-effect gene. By electron microscopy, we found that the eggs from Ces5/Ooep-null females lacked oocyte cytoplasmic lattices (CPLs), which have long been predicted to function as a storage form for components that are maternally contributed to the early embryo. Further analysis showed that CES5/OOEP was directly associated with the CPLs. These results indicate that CES5/OOEP is an essential component of the CPLs and is required for embryonic development at the maternal-zygotic stage transition.

Gtsf1/Cue110, a gene encoding a protein with two copies of a CHHC Zn-finger motif, is involved in spermatogenesis and retrotransposon suppression in murine testes.

We recently reported that the Gtsf1/Cue110 gene, a member of the evolutionarily conserved UPF0224 family, is expressed predominantly in male germ cells, and that the GTSF1/CUE110 protein is localized to the cytoplasm of these cells in the adult testis. Here, to analyze the roles of the Gtsf1/Cue110 gene in spermatogenesis, we produced Gtsf1/Cue110-null mice by gene targeting. The Gtsf1/Cue110-null mice grew normally and appeared healthy; however, the males were sterile due to massive apoptotic death of their germ cells after postnatal day 14. In contrast, the null females were fertile. Detailed analyses revealed that the Gtsf1/Cue110-null male meiocytes ceased meiotic progression before the zygotene stage. Thus, the Gtsf1/Cue110 gene is essential for spermatogenesis beyond the early meiotic phase. Furthermore, the loss of the Gtsf1/Cue110 gene caused increased transcription of the long interspersed nucleotide element (Line-1) and the intracisternal A-particle (IAP) retrotransposons, accompanied by demethylation of their promoter regions. These observations indicate that Gtsf1/Cue110 is required for spermatogenesis and involved in retrotransposon suppression in male germ cells.

Sohlh2 affects differentiation of KIT positive oocytes and spermatogonia.

The differentiation programs of spermatogenesis and oogenesis are largely independent. In the early stages, however, the mechanisms partly overlap. Here we demonstrated that a germ-cell-specific basic helix-loop-helix (bHLH) transcription factor gene, Sohlh2, is required for early spermatogenesis and oogenesis. SOHLH2 was expressed in mouse spermatogonia from the undifferentiated stage through differentiation and in primordial-to-primary oocytes. Sohlh2-null mice, produced by gene targeting, showed both male and female sterility, owing to the disrupted differentiation of mature (KIT(+)) spermatogonia and oocytes. The Sohlh2-null mice also showed the downregulation of genes involved in spermatogenesis and oogenesis, including the Sohlh1 gene, which is essential for these processes. Furthermore, we showed that SOHLH2 and SOHLH1 could form heterodimers. These observations suggested that SOHLH2 might coordinate with SOHLH1 to control spermatogonial and oocyte genes, including Sohlh1, to promote the differentiation of KIT(+) germ cells in vivo. This study lays the foundation for further dissection of the bHLH network that regulates early spermatogenesis and oogenesis.

Transgenic expression of antioxidant protein thioredoxin in pancreatic beta cells prevents progression of type 2 diabetes mellitus.

The authors previously established a transgenic mouse line in the type 1 diabetes model, NOD mouse, in which thioredoxin (TRX), a redox protein, is overexpressed in pancreatic beta cells, and found that TRX overexpression slows the progression of type 1 diabetes. Recent reports on type 2 diabetes suggest that oxidative stress also degrades the function of beta cells. To elucidate whether TRX overexpression can prevent progressive beta cell failure from oxidative stress in type 2 diabetes, the authors transferred the TRX transgene from the NOD mouse onto a mouse model of type 2 diabetes, the db/db mouse. The progression of hyperglycemia and the reduction of body weight gain and insulin content of the db/db mouse were significantly suppressed by the TRX expression. Furthermore, TRX suppressed the reduction of Pdx-1 and MafA expression in the beta cells, which may be one of the cellular mechanisms for protecting beta cells from losing their insulin-secreting capacity. These results showed that TRX can protect beta cells from destruction not only in type 1 but also in type 2 diabetes, and that they provide evidence that oxidative stress plays a crucial role in the deterioration of beta cell function during the progression of type 2 diabetes.

Molecular scanning of the gene for thioredoxin, an antioxidative and antiapoptotic protein, and genetic susceptibility to type 1 diabetes.

Oxidative stress has been implicated in the destruction of beta cells in type 1 diabetes (T1D). Thioredoxin has been shown to protect cells from oxidative stress and apoptosis. In this study, we screened for sequence variants of the human thioredoxin gene (TXN), and studied the association of the variants in persons with T1D in Japanese. The frequency of the A allele of the G/A SNP in the 3' flanking region was highest in T1D (8.4%), followed by type 2 diabetes (6.8%), and the lowest in the controls (5.9%), suggesting the contribution of TXN polymorphism to susceptibility to T1D.

Gene expression pattern of Cue110: a member of the uncharacterized UPF0224 gene family preferentially expressed in germ cells.

The large number of expressed sequence tags (ESTs) now available in databases has enabled the analysis of gene expression profiles in silico. We searched public databases for uncharacterized transcripts specifically expressed in germ cells, in an attempt to identify genes involved in gametogenesis. We found a transcript that is expressed in unfertilized eggs, ovaries, and testes of the mouse. It has an open reading frame (ORF) encoding a 167-amino acid protein belonging to the UPF0224 (unknown protein family 0224) family. We called the novel gene Cue110. We examined the Pfam database for other members of the UPF0224 family, and found a conserved N-terminal portion among members of various species. To study the cellular localization of the Cue110 transcript and protein, we performed in situ hybridization and immunohistochemical analysis of the adult mouse ovary and testis. In the testis, specific hybridization signals were observed weakly in preleptotene spermatocytes but maximally in late round spermatids. Immunostaining showed that Cue110 protein was present predominantly in the cytoplasm of pachytene spermatocytes and round spermatids. In the ovary, weak hybridization signals were observed in primary oocytes in the primordial, primary, and secondary follicles, but Cue110 protein was not detected in oocytes by immunostaining. We next examined the developmental expression pattern of the Cue110 gene using RT-PCR and western blotting, and found its increasing expression coincided with the appearance of spermatocytes. Thus, the Cue110 gene is expressed predominantly in male germ cells at stages from the pachytene spermatocytes to round spermatids.

Both Pdx-1 and NeuroD1 genes are requisite for the maintenance of insulin gene expression in ES-derived differentiated cells.

Embryonic stem (ES) cells can differentiate into many cell types. Recent reports have shown that ES cells can differentiate into insulin-producing cells. We have established an ES cell line in which exogenous Pdx-1 expression was precisely regulated by the Tet-off system integrated into the ROSA26 locus and succeeded to produce insulin-producing cells. The Pdx-1 expressing final differentiated insulin-positive cells can be maintained for more than 2 months. However, in spite of their induced expression of Pdx-1, the repeated passages of cells lost their capacity to express insulin and NeuroD1 gene. Forced expression of NeuroD1 gene by adenoviral vector in these cells restored the expression of insulin. These results suggested that maintenance of the property of insulin-producing cells derived from ES cells could be achieved by synergistic expression of Pdx-1 and NeuroD1.

Protection against CCl4-induced injury in liver by adenovirally introduced thioredoxin gene.

Antioxidation therapy is a promising strategy for treating or preventing oxidative stress-related liver diseases. The human thioredoxin (TRX) gene was inserted into an adenovirus vector (Adv-TRX), which was administered to mice. The mice were treated with 1 ml/kg CCl4 48 h after the infection. Blood samples were taken and the liver was excised 24 h after the CCl4 treatment. Serum ammonia, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were determined, and liver sections were stained with hematoxylin and eosin. RT-PCR analysis showed that the introduced TRX gene was expressed only in the liver. Adv-TRX decreased the serum ammonia, AST, and ALT levels. Hematoxylin-eosin staining indicated that the CCl4-induced injury was significantly prevented by the Adv-TRX infection. The gene delivery of TRX, which plays a central role in intracellular redox control, was shown to be effective in protecting the liver against oxidative stress-induced injury.

CXCL10 DNA vaccination prevents spontaneous diabetes through enhanced beta cell proliferation in NOD mice.

CXCL10, a chemokine for Th1 cells, is involved in the pathogenesis of various Th1-dominant autoimmune diseases. Type 1 diabetes is considered to be a Th1-dominant autoimmune disease, and a suppressive effect of CXCL10 neutralization on diabetes development has been reported in a cyclophosphamide-induced accelerated diabetes model through induction of beta cell proliferation. However, intervention in a diabetes model might bring about opposite effects, depending on the timing, amount, or method of treatment. In the present study, we examined the effect of CXCL10 neutralization in a "spontaneous diabetes" model of NOD mice, using CXCL10 DNA vaccination (pCAGGS-CXCL10). pCAGGS-CXCL10 treatment in young NOD mice induced the production of anti-CXCL10 Ab in vivo and suppressed the incidence of spontaneous diabetes, although this treatment did not inhibit insulitis or alter the immunological response. pCAGGS-CXCL10 treatment enhanced the proliferation of pancreatic beta cells, resulting in an increase of beta cell mass in this spontaneous diabetes model as well. Therefore, CXCL10 neutralization is suggested to be useful for maintaining beta cell mass at any stage of autoimmune diabetes.

Development of a single-cassette system for spatiotemporal gene regulation in mice.

The tetracycline-regulated gene expression system has been widely used in mice to turn a transgene on and off in a target organ, but with only limited success. We developed an advanced system in which a Tet-off regulation unit was integrated into the ROSA26 locus and became active after Cre-mediated excision of the neo(r) gene. We examined the utility of this system through regulable expression of the homeodomain transcription factor pdx-1 and enhanced green fluorescent protein. The resulting mice showed strict tetracycline-regulable gene expression in all the organs where the neo(r) gene had been removed. When combined with organ-specific Cre recombinase transgenic mice, our system allows us to manipulate the gene expression in an organ-specific and temporal manner. This Tet-off system should serve as an efficient tool to analyze the roles of genes in complex biological systems, such as embryogenesis, metabolism, immune system, etc.

Stimulation of hepatocyte survival and suppression of CCl4-induced liver injury by the adenovirally introduced C/EBPbeta gene.

Gene therapy has attracted attention as a potentially effective alternative to liver transplantation for the treatment of hepatic failure. We chose the C/EBPbeta gene, which plays vital roles in liver regeneration, as a candidate for gene therapy, and examined its effect on hepatocyte survival and the suppression of liver inflammation. C/EBPbeta gene overexpression significantly maintained hepatocyte viability during 12 days of the culture. Urea synthesis ability, which is a liver-specific function, in Adv-C/EBPbeta-infected hepatocytes was stably maintained during the culture, but the activity per cell was significantly lower than that in non-infected cells. On the contrary, DNA synthesis activity in Adv-C/EBPbeta-infected hepatocytes was significantly higher than that in non-infected cells. COX-2 was induced in Adv-C/EBPbeta-infected hepatocytes, and the addition of NS398, a specific inhibitor of COX-2, suppressed the viability-maintenance effect. COX-2 was thus shown to be involved in the survival effect of C/EBPbeta gene. The introduction of the C/EBPbeta gene into liver-damaged mice significantly suppressed the serum AST and ALT activities. These results indicate that C/EBPbeta appears to be a survival factor under stressful conditions, and the introduction of the gene has therapeutic function against liver injury.