Regulation of the glyoxalase pathway in human brain microvascular endothelium: effects of troglitazone and tertiary butylhydroperoxide.

The glyoxalase system, comprised of glyoxalase-I and glyoxalase-II with glutathione as the cofactor, plays an important role in the detoxification of methylglyoxal and other alpha-oxo-aldehydes. Such aldehydes, which increase with hyperglycemia, give rise to advanced glycation end products. The objective of this research was to examine the glyoxalase system in human cerebromicrovascular cells. The hypothesis tested was that this pathway would be regulated by phase 2 enzyme inducers such as t-butylhydroquinone and modulated by the insulin-sensitizing drug troglitazone. Human cerebromicrovascular endothelial cells were cultured and exposed to varying concentrations of t-butylhydroquinone or troglitazone. The activity of glyoxylase-I in human endothelial cells was similar to the activity present in hepatocytes. The phase 2 enzyme inducer t-butylhydroquinone had no effect on the glyoxalase enzymes activities but significantly increased glutathione levels and glutathione reductase activity, indicating that phase 2 enzyme inducers might promote alpha-oxo-aldehyde scavenging. Troglitazone decreased the activities of glyoxalase-I and -II and decreased glyoxalase-I mRNA. Troglitazone had no effect on glutathione levels or on the activity of glutathione reductase or glutathione peroxidase. We conclude that phase 2 enzyme inducers may promote scavenging of alpha-oxoaldehydes in endothelial cells.

Troglitazone selectively inhibits glyoxalase I gene expression.

AIMS/HYPOTHESIS: The hyperglycaemia associated with diabetes causes excessive production of cytotoxic methylglyoxal, an alpha-oxo-aldehyde. The glyoxalase system, composed of glyoxalase I and glyoxalase II, with glutathione (GSH) as the cofactor, plays an important role in the detoxification of alpha-oxo-aldehydes. We tested the hypothesis that troglitazone, an insulin-sensitizing drug previously used in the treatment of Type II (non-insulin-dependent) diabetes mellitus, up-regulates the glyoxalase system either by increasing phase 2 enzyme activities and thereby increasing cellular GSH, or, by inducing glyoxalase enzyme activities. METHODS: Human astroglial cells, rat hepatocytes and cardiac myocytes were cultured and exposed to either troglitazone, or tertiary-butylhydroquinone (tBHQ, a phase 2 enzyme inducer). Glutathione content, advanced glycation end products (AGEs) and enzyme (glyoxalase I, glyoxalase II as well as the phase 2 enzymes, glutathione S-transferase and thioredoxin reductase) activities were determined. Glyoxalase I mRNA was also measured. RESULTS: Troglitazone had no effect on cellular GSH nor phase 2 enzyme activities but significantly reduced the activities of glyoxalase I and II; this inhibitory effect was concentration-dependent and time-dependent and was associated with reduced mRNA contents and increased AGEs formation. Rosiglitazone had no effect on glyoxalase I gene expression. tBHQ, a classic phase 2 enzyme inducer, had no effect on the glyoxalase system but did increase glutathione contents and the activities of glutathione S-transferase and thioredoxin reductase. CONCLUSION/INTERPRETATION: Our study shows that troglitazone is a selective inhibitor of the glyoxalase system. This inhibition of the glyoxalase system could contribute to troglitazone's hepatotoxic action which has previously been reported in a small percentage of individuals.

C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4.

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a strong inhibitor of cell proliferation. We found that C/EBPalpha directly interacts with cdk2 and cdk4 and arrests cell proliferation by inhibiting these kinases. We mapped a short growth inhibitory region of C/EBPalpha between amino acids 175 and 187. This portion of C/EBPalpha is responsible for direct inhibition of cyclin-dependent kinases and causes growth arrest in cultured cells. C/EBPalpha inhibits cdk2 activity by blocking the association of cdk2 with cyclins. Importantly, the activities of cdk4 and cdk2 are increased in C/EBPalpha knockout livers, leading to increased proliferation. Our data demonstrate that the liver-specific transcription factor C/EBPalpha brings about growth arrest through direct inhibition of cdk2 and cdk4.

Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by troglitazone: a peroxisome proliferator-activated receptor-gamma (PPARgamma)-independent, antioxidant-related mechanism.

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis. Enhanced expression of the PEPCK gene in liver is present in most models of diabetes, and is thought to contribute to the increased hepatic glucose output seen in this disease. Recently, we showed that troglitazone, the first thiazolidinedione (TZD) used clinically, inhibits expression of the PEPCK gene in isolated hepatocytes. We have pursued the molecular mechanism whereby troglitazone exerts this inhibition. TZDs are known to bind and activate peroxisome proliferator-activated receptor-gamma (PPARgamma), a nuclear receptor, which regulates expression of target genes. Initially, we examined the abilities of three other TZDs (rosiglitazone, englitazone, and ciglitazone) to inhibit expression of the PEPCK gene. Despite the fact that these agents are ligands for PPARgamma, they displayed little if any inhibitory activity on the expression of this gene. GW1929 [N-(2-benzoyl phenyl)-l-tyrosine], another potent PPARgamma ligand that is unrelated structurally to TZDs, had no inhibitory effect on PEPCK gene expression, while a natural PPARgamma ligand, the prostaglandin metabolite 15-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), displayed only modest inhibitory activity. Treatment of hepatocytes with ligands for other isoforms of PPAR also had no significant effect on PEPCK gene expression. Troglitazone has an alpha-tocopherol (vitamin E) moiety that is not present in other TZDs, and treatment of hepatocytes with vitamin E led to an inhibition of PEPCK gene expression. These observations support the conclusion that troglitazone inhibits the expression of the PEPCK gene by a PPARgamma-independent, antioxidant-related mechanism.

Characterization of domains in C/EBPalpha that mediate its constitutive and cAMP-inducible activities.

Structure/function analysis of CCAAT/enhancer binding proteins (C/EBP) alpha and beta have shown that they possess both constitutive and cAMP inducible activities. Three regions conserved between C/EBPalpha and beta were identified which lie within the cAMP inducible domains of each protein. Deletion analysis of these conserved regions within C/EBPalpha show that conserved region 2 plays a particularly critical role in mediating the PKA inducible activity of the protein, however, the constitutive activity of conserved region 2 depends on promoter context. This data supports previous findings that constitutive and cAMP responsiveness are mediated by domains of the protein that do not directly overlap, suggesting that they occur through distinct mechanisms.

Modelling plasmid instability in batch and continuous fermentors.

The probability of complete loss of plasmid material from plasmid bearing cells undergoing active growth has been modelled and incorporated into predictions for the dynamic concentrations of plasmid bearing cells in both batch and continuous flow, stirred tank bioreactors. The new model is based on an extension of the well-used model of Imanaka and Aiba [Ann. New York Acad. Sci. 369 (1981) 1] and is relatively easy to use compared to complex structured models. The new model predicts that both accelerating and decelerating rates of plasmid loss occur in both batch and continuous flow operation, and agrees well with data collected here and published earlier by others.

The role of C/EBP in nutrient and hormonal regulation of gene expression.

C/EBPs are a family of transcription factors that play important roles in energy metabolism. Although initially thought to be constitutive regulators of transcription, an increasing amount of evidence indicates that their transactivating capacity within the cell can be modulated by nutrients and hormones. There are several mechanisms whereby this occurs. First, hormones/nutrients are known to directly alter the expression of C/EBPs. Second, hormones/nutrients may cause an alteration in the phosphorylation state of C/EBPs, which can affect their DNA-binding activity or transactivating capacity. Third, C/EBPs can function as accessory factors on gene promoters within a hormone response unit, interacting with other transcription factors to enhance the degree of responsiveness to specific hormones. Given their role in regulating genes involved in a wide variety of metabolic events, advancing our understanding of the molecular mechanism of action of C/EBPs will undoubtedly further our appreciation for the role these transcription factors play in both health and disease.

What is a cAMP response unit?

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting enzyme of gluconeogenesis, and most, if not all, of the regulation of its activity is exerted at the level of gene expression, with transcriptional regulation being the most predominant. A number of hormones regulate transcription of this gene, but in a defined, tissue-specific fashion. For example, cAMP strongly induces PEPCK gene transcription in liver, but provides only a weak response in kidney. Results from a number of different studies indicate that cAMP responsiveness of this gene is mediated by a 'cAMP response unit' (CRU), consisting of five cis-elements. All five sequences are required for maximal responsiveness and, potentially, four of these are binding sites for a CCAAT/enhancer binding protein (C/EBP). Since alpha- and beta-isoforms of C/EBP are liver-enriched, this may provide the molecular basis for the liver-specific responsiveness to cAMP. A curiosity of this promoter is that one of the cis-elements present in the CRU is a cAMP response element (CRE), which typically acts as a binding site for CRE binding protein (CREB). However, the non-consensus CRE in the PEPCK promoter also binds C/EBP proteins with high affinity, and C/EBPalpha can functionally substitute for CREB in this cAMP response unit while C/EBPbeta cannot. The available data suggest that the PEPCK promoter can exist in altered states of cAMP responsivity, depending on which transcription factors occupy specific cis-elements in the CRU.

Hormonal regulation of the phosphoenolpyruvate carboxykinase gene. Role of specific CCAAT/enhancer-binding protein isoforms.

The CCAAT/enhancer-binding protein alpha (C/EBP) is a transcription factor that trans-activates a number of metabolically important genes. Previous work has demonstrated that C/EBPalpha and C/EBPbeta have the potential to mediate the cAMP responsiveness of phosphoenolpyruvate carboxykinase (PEPCK) in liver cells. However, these studies used GAL4 fusion proteins and artificial promoter-reporter gene vectors in transfection experiments; as a result, these studies only indicated that both isoforms had the potential to mediate the hormonal response and not which isoform actually participated in vivo. To address this issue, we produced hepatoma cell lines that stably expressed either a dominant negative inhibitor or antisense RNA for these two main liver C/EBP isoforms. Inhibition of all C/EBP isoforms via expression of the dominant negative protein eliminated cAMP responsiveness, and reduced glucocorticoid responsiveness, of the endogenous PEPCK gene in hepatoma cells. Antisense directed against C/EBPalpha mRNA, which reduced C/EBPalpha protein levels by nearly 80%, also significantly reduced the cAMP responsiveness of the endogenous PEPCK promoter, whereas antisense directed against C/EBPbeta was without effect. There was no major alteration in cAMP signaling in the C/EBPalpha antisense cells, as cAMP induction of the C/EBPbeta gene was similar to that in wild-type H4IIE cells. These data suggest that the alpha-isoform of C/EBP is specifically utilized for mediating the cAMP responsiveness of the PEPCK gene.

M-DNA: A complex between divalent metal ions and DNA which behaves as a molecular wire.

M-DNA is a complex of DNA with divalent metal ions (Zn(2+), Co(2+), or Ni(2+)) which forms at pH conditions above 8. Upon addition of these metal ions to B-DNA at pH 8.5, the pH decreases such that one proton is released per base-pair per metal ion. Together with previous NMR data, this result demonstrated that the imino proton in each base-pair of the duplex was substituted by a metal ion and that M-DNA might possess unusual conductive properties. Duplexes of 20 base-pairs were constructed with fluorescein (donor) at one end and rhodamine (acceptor) at the other. Upon formation of M-DNA (with Zn(2+)) the fluorescence of the donor was 95 % quenched. Fluorescence lifetime measurements showed the presence of a very fast component in the decay kinetics with tau

Troglitazone inhibits expression of the phosphoenolpyruvate carboxykinase gene by an insulin-independent mechanism.

Troglitazone is an oral insulin-sensitizing drug used to treat patients with type 2 diabetes. A major feature of this hyperglycemic state is the presence of increased rates of hepatic gluconeogenesis, which troglitazone is able to ameliorate. In this study, we examined the molecular basis for this property of troglitazone by exploring the effects of this compound on the expression of the two genes encoding the major regulatory enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in primary cultures of rat hepatocytes. Insulin is able to inhibit expression of both of these genes, which was verified in our model system. Troglitazone significantly reduced mRNA levels of PEPCK and G6Pase in rat hepatocytes isolated from normal and Zucker-diabetic rats, but to a lesser extent than that observed with insulin. Interestingly, troglitazone was unable to reduce cAMP-induced levels of PEPCK mRNA, suggesting that the molecular mechanism whereby troglitazone exerted its effects on gene expression differed from that of insulin. This was further supported by the observation that troglitazone was able to reduce PEPCK mRNA levels in the presence of the insulin signaling pathway inhibitors wortmannin, rapamycin, and PD98059. These results indicate that troglitazone can regulate the expression of specific genes in an insulin-independent manner, and that genes encoding gluconeogenic enzymes are targets for the inhibitory effects of this drug.

Role of CCAAT enhancer-binding protein beta in the thyroid hormone and cAMP induction of phosphoenolpyruvate carboxykinase gene transcription.

Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by thyroid hormone (T3) and cAMP. Two DNA elements in the PEPCK promoter are required for T3 responsiveness including a thyroid hormone response element and a binding site called P3(I) for the CCAAT enhancer-binding protein (C/EBP). Both the alpha and beta isoforms of C/EBP are highly expressed in the liver. C/EBPalpha contributes to the liver-specific expression and cAMP responsiveness of the PEPCK gene. In this study, we examined the ability of C/EBPbeta when bound to the P3(I) site to regulate PEPCK gene expression. We report that C/EBPbeta can stimulate basal expression and participate in the induction of PEPCK gene transcription by T3 and cAMP. The cAMP-responsive element-binding protein and AP1 proteins that contribute to the induction by cAMP are not involved in the stimulation by T3. A small region of the transactivation domain of C/EBPbeta is sufficient for the stimulation of basal expression and cAMP responsiveness. Our results suggest that C/EBPalpha and C/EBPbeta are functionally interchangeable when bound to the P3(I) site of the PEPCK promoter.

Troglitazone induces expression of PPARgamma in liver.

Troglitazone is an insulin sensitizer which affects a number of target tissues. It is believed to exert these effects primarily by binding to and activating the y-isoform of peroxisome proliferator-activated receptor (PPARgamma), which in turn regulates the expression of specific genes. However, in a number of target organs, such as liver, the levels of PPARgamma are low and other isoforms predominate. In the present study, we examined whether troglitazone induces the expression of PPARgamma, thereby sensitizing cells for the action of this drug. Treatment of isolated rat hepatocytes with troglitazone induced both the mRNA and protein levels of PPARgamma in a dose-dependent fashion, with maximal levels of induction being three- to fourfold. This induction was also observed using the 15-deoxy-delta12,14-prostaglandin J2, a known natural ligand for PPARgamma, whereas ligands specific for PPARalpha were without effect. The induction of PPARgamma expression by troglitazone was also observed in livers from rats fed a diet containing troglitazone. Troglitazone had no effect on the expression of the alpha- or beta-isoforms of PPAR, the more predominant liver isoforms. These results indicate that troglitazone produces a reprogramming of PPAR isoform content in liver, which may in part underlie the mechanism whereby troglitazone sensitizes the liver to the action of insulin and/or ameliorates hyperglycemia.

Nuclear factor I regulates expression of the gene for phosphoenolpyruvate carboxykinase (GTP).

Nuclear factor-I (NFI) binds to the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter immediately 5' to the cAMP regulatory element (CRE). This suggests an interaction between NFI and factors that bind the CRE. Of the four NFI isoforms expressed in mammalian tissues, NFI-A and -B stimulate basal transcription from the PEPCK gene promoter in HepG2 cells, while NFI-C and -X are slightly inhibitory. All four NFI isoforms abrogate the 20-fold protein kinase Ac (PKAc)-mediated induction of transcription from the PEPCK gene promoter. Normal PKAc-mediated induction was noted when the CRE was moved 10 base pairs 3' of its original location. However if the CRE was moved 5 base pairs 3', placing it out of phase with the other elements in the promoter, or moved 5' to -285 (the P3(I) site in the promoter), some PKA-mediated stimulation was lost. The NFI-C isoform effectively inhibited PKAc induction regardless of the relative positions of the CRE and the NFI binding sites. NFI-C also abrogated cAMP regulatory element-binding protein (CREB)-induced activity of wild type and mutant PEPCK promoters. There was some cooperativity in the binding of CREB and NFI to their respective binding sites but this did not appear to be physiologically important.

Characterization of CCAAT/enhancer-binding protein alpha as a cyclic AMP-responsive nuclear regulator.

The alpha isoform of CCAAT/enhancer-binding protein (C/EBPalpha) is a transcription factor that regulates expression of genes linked to adipose differentiation and hepatic nutrient metabolism. Recently, our laboratory has characterized a role for C/EBPalpha in mediating hormonal responsiveness. For example, the cAMP responsiveness of the phosphoenolpyruvate carboxykinase gene promoter in liver requires synergism among the cAMP response element-binding protein (CREB), C/EBPalpha, and activator protein-1. In the present study, we show that C/EBPalpha can functionally substitute for CREB in this cAMP response unit, i.e. cAMP responsiveness can occur in the absence of CREB. This observation is physiologically relevant since both CREB and C/EBPalpha have been shown to bind with high affinity to the cAMP response element in this particular promoter. Structure/function analysis of C/EBPalpha identified specific mutations that differentially affected its constitutive and protein kinase A-inducible activities. This finding suggests that the mechanism whereby C/EBPalpha mediates constitutive transactivation is distinct from that whereby it mediates cAMP responsiveness. These data support the hypothesis that C/EBPalpha plays a critical role in metabolism, in part by participating in the hormonal regulation of expression of metabolically important genes.

Hormone response units: one plus one equals more than two.

The transcription rate of many genes, and particularly those which code for metabolically important proteins, is regulated by various hormones. Detailed analysis of the promoters of these genes has shown that, while functional 'Hormone response elements' exist, the hormonal responsiveness of many promoters is often synergistically mediated by several cis-elements, collectively referred to as a hormone response unit. The utilization of a hormone response unit to mediate a response offers several regulatory advantages, including an expansion of the range of transcriptional responses and modulation of the response by tissue- and developmental-specific cues. Furthermore, the presence of Hormone Response Units may provide a mechanism for the coordination of information from two or more signaling pathways into a single, integrated and exquisitely controlled transcriptional response. The protein-protein interactions that likely mediate many of the synergistic functional characteristics of Hormone Response Units may provide unique targets for therapeutic intervention.

Insulin stimulates cAMP-response element binding protein activity in HepG2 and 3T3-L1 cell lines.

Earlier studies from our laboratory demonstrated an insulin-mediated increase in cAMP-response element binding protein (CREB) phosphorylation. In this report, we show that insulin stimulates both CREB phosphorylation and transcriptional activation in HepG2 and 3T3-L1 cell lines, models of insulin-sensitive tissues. Insulin stimulated the phosphorylation of CREB at serine 133, the protein kinase A site, and mutation of serine 133 to alanine blocked the insulin effect. Many of the signaling pathways known to be activated by insulin have been implicated in CREB phosphorylation and activation. The ability of insulin to induce CREB phosphorylation and activity was efficiently blocked by PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK1), but not significantly by rapamycin or wortmannin. Likewise, expression of dominant negative forms of Ras or Raf-1 completely blocked insulin-stimulated CREB transcriptional activity. Finally, we demonstrate an essential role for CREB in insulin activation of fatty-acid synthase and fatty acid binding protein (FABP) indicating the potential physiologic relevance of insulin regulation of CREB. In summary, insulin regulates CREB transcriptional activity in insulin-sensitive tissues via the Raf --> MEK pathway and has an impact on physiologically relevant genes in these cells.

CCAAT-enhancer-binding protein alpha (C/EBP alpha) is required for the thyroid hormone but not the retinoic acid induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription.

Transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK) is stimulated by cAMP, the thyroid hormone tri-iodothyronine (T3) and retinoic acid (RA). Regulation of PEPCK transcription by T3 involves two sites in the promoter including a thyroid-hormone-response element (TRE) and a CCAAT-enhancer-binding protein (C/EBP) binding site called P3I. Mutation of either the TRE or P3I eliminates the T3 response. In this study, we examined the role of C/EBPs in the induction of PEPCK transcription by T3 and RA. PEPCK-CAT vectors were transfected into HepG2 cells. Co-transfection of a dominant negative C/EBP eliminated the T3 stimulation indicating that a member of the C/EBP family is required. To determine which C/EBP isoform was required, Gal4 fusion proteins were created that contained the Gal4 DNA-binding domain ligated to the transcriptional activation domain of C/EBP alpha, C/EBP beta or the cAMP-responsive-element-binding protein. A Gal4 DNA-binding site was introduced into the P3(I) site of the PEPCK-CAT vector. Only co-transfection of the Gal4-C/EBP alpha vector was able to restore T3 responsiveness to the PEPCK-CAT vector. The T3 and RA receptors are members of the nuclear receptor superfamily and bind to repeats of the AGGTCA motif. We found that the RA receptor can bind to sequences within the PEPCK-TRE and contribute to RA responsiveness of the PEPCK gene. However, the RA induction of PEPCK transcription was found to be independent of C/EBPs, further demonstrating the specificity of the involvement of C/EBP alpha in the T3 effect.

Hepatic expression of CCAAT/enhancer binding protein alpha: hormonal and metabolic regulation in rats.

There is a significant body of evidence which suggests that the alpha-isoform of the CCAAT/enhancer binding protein (C/EBP alpha) plays a central regulatory role in energy metabolism in the liver. However, there is little information available regarding regulation of its expression in this tissue. In this study, we examined the effect of hormones and diabetes on its expression in rat H4IIE hepatoma cells and in rat liver. Treatment of H4IIE cells with dexamethasone led to a threefold increase in C/EBP alpha mRNA within 4 h. Insulin treatment produced a bi-phasic response, initially reducing mRNA levels up to the 4 h time point, but after 8 h a twofold increase in C/EBP alpha mRNA was observed. Treatment with 8-chlorophenylthio-cAMP produced a twofold induction of C/EBP alpha mRNA after 8 h. Western analysis indicated that the changes in mRNA in response to hormonal treatment generally resulted in corresponding alterations in C/EBP alpha protein levels. Finally, we observed an inhibition of C/EBP alpha gene expression in streptozotocin-diabetic rat liver, reflected by a decrease in both mRNA and protein levels that were partially reversed by insulin treatment. These results indicate that the expression of C/EBP alpha in liver is under complex control by both hormonal and metabolic signals, which is consistent with its role as a trans -regulator of genes which play a role in energy metabolism.