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Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.

Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys(588) and Lys(591) of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.

The Koletsky (SHROB) strain of rats is spontaneously hypertensive and displays insulin resistance, hyperglucagonemia and hypertriglyceridemia but is normoglycemic under fasting conditions. The aim of this study was to unravel the pattern of expression of genes encoding key regulatory enzymes involved in carbohydrate metabolism in the liver and kidney that may be impacted in this strain. We found that SHROB animals have decreased beta-adrenergic receptor density and, consequently, blunted increases in cAMP levels in response to beta-adrenergic agonists. They also have lower levels of hepatic as well as renal phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) mRNA and protein than their lean littermates. Expression of the genes for glycogen phosphorylase and glycogen synthase was also decreased. Hepatocytes from the SHROB animals exhibited glycogen depletion of only 50% compared to 86% by hepatocytes from lean littermates when challenged with either glucagon or forskolin to stimulate adenylyl cyclase. The expression of C/EBPalpha and C/EBPbeta, two key transcription factors that are essential for the coordinated expression of genes involved in glucose homeostasis, was depressed in livers of the SHROB rats, as were levels of HNF-4alpha, PPARalpha and PGC-1alpha. We conclude that overproduction of glucose is prevented in the SHROB rats by decreased expression of the genes for glycogen phosphorylase and the gluconeogenic enzymes PEPCK and G6Pase, which may prevent progression to diabetes in this model.

Fatty acids and glucose are strong modulators of the expression of glucose-6-phosphatase (Glc-6-Pase), an enzyme that plays a key role in glucose homeostasis. PUFA inhibit, whereas SFA and monounsaturated fatty acids induce the expression of the Glc-6-Pase gene. Palmitate and oleate are the most abundant fatty acid species in circulation during food deprivation in mammals. Although dietary fats have been shown to modulate the expression of genes involved in both lipid and carbohydrate metabolism in liver, little is known regarding the molecular mechanism of transcriptional response of the Glc-6-Pase gene to long-chain fatty acids. Using H4IIE hepatoma cells and hepatocytes from adult rats, we investigated the mechanism of the induction of this gene by palmitate and oleate. Both of these fatty acids stimulated Glc-6-Pase gene transcription but did not affect the stability of its mRNA. In transient transfection assays, transcription from the Glc-6-Pase gene promoter was markedly enhanced by both palmitate and oleate but not by arachidonate. Chromatin immunoprecipitation analysis was used to show that palmitate induced the recruitment of an array of transcription factors viz hepatic nuclear factor(NF)-4alpha, CAAT/enhancer binding proteinbeta, PPARalpha, chicken ovalbumin upstream promoter transcription factor (COUP-TF), cAMP regulatory element binding protein, and NF-kappaB to this gene promoter. Although it is presently unclear how these various transcription factors interact at this promoter, the data are consistent with the view that multiple regulatory elements in the Glc-6-Pase gene promoter are responsible for the modulation of gene transcription by fatty acids.

Some metabolic processes such as glycolysis, gluconeogenesis, and lipogenesis are readily understood because they are circumscribed in metabolic pathways that have clearly identifiable beginning points, end products, and other features. Other metabolic pathways that do not appear to be straightforward pose difficulties for students. In part I of this two-part series, one such metabolic process, the purine nucleotide cycle, was discussed (I. J. Arinze (2005) Biochem. Mol. Biol. Educ. 33, 165–168). In the present article, the focus is on the metabolism of one-carbon fragments, the so-called one-carbon pool. As with the first article in this series, the intent of this article is to facilitate teaching and learning of this rather complex area of metabolism.

Some metabolic processes are readily understood because they are circumscribed in metabolic pathways that have clearly identifiable beginning points, end products, and other features. Other metabolic pathways that do not appear to be straightforward pose difficulties for students. One such metabolic process, the purine nucleotide cycle, is discussed here.

Very little is known regarding molecular mechanism(s) underlying transcriptional regulation of any G-protein gene despite the importance of G-protein expression in modulating cellular processes. Here we show that phorbol myristate acetate (PMA) and tert-butylhydroquinone (tBHQ), which induce oxidative stress in cells, up-regulate transcription of Galpha(i2) in K562 cells. Redox-sensing chemicals abrogated this transcriptional effect. A dominant negative I-kappaB double mutant (S32A/S36A) suppressed PMA-induced transcription by 54-62%, suggesting involvement of nuclear factor-kappaB (NF-kappaB). SN50, a cell-permeable peptide that inhibits nuclear import of stress-responsive transcription factors (such as NF-kappaB), inhibited PMA- and tBHQ-induced transcription. Deletion of an NF-kappaB-binding motif that maps at +10/+19 in the promoter resulted in 55-60% suppression of PMA-induced transcription, and 81% suppression of tBHQ-induced transcription. Mutation of an antioxidant response element (ARE) that maps at -84/-76 in the promoter resulted in 51 and 86% decrease in PMA- and tBHQ-induced transcription, respectively. In electrophoretic mobility shift assays, this element formed complexes with the transcription factors NF-E2p45 and Nrf2 that are prototypic for binding to the ARE, as well as with c-Fos, which can also interact with the ARE. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter. Exogenously transfected Nrf2 transactivated the Galpha(i2) gene promoter; the cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together, the present studies reveal that transcription factors that bind NF-kappaB and/or antioxidant response elements play an activating role in the transcription of the human Galpha(i2) gene.

Mechanisms underlying dietary nutrient regulation of glucose-6-phosphatase (Glc-6-Pase) gene expression are not well understood. Here we investigated the effects of short-chain fatty acids on the expression of this gene in primary cultures of rat hepatocytes and H4IIE hepatoma cells. Propionate, butyrate, valerate, and caproate induced severalfold increases in the expression of Glc-6-Pase mRNA. In reporter gene assays, propionate, valerate, caproate, and also octanoate increased Glc-6-Pase promoter activity by 6-16-fold. Butyrate, by itself, had little or no effect on promoter activity, but it induced a robust increase (45-fold) in promoter activity in cells co-transfected with a plasmid expressing the transcription factor HNF-4α (α isoforms of hepatic nuclear factor 4). HNF-4α also enhanced promoter activity induced by other short-chain fatty acids. A dominant negative form of HNF-4α abrogated the fatty acid-induced promoter activity, a finding that accentuates a role for HNF-4α in the transcription process studied here. In cells transfected with HNF-4α, short-chain fatty acids and trichostatin A, an inhibitor of histone deacetylase, synergistically enhanced promoter activity, suggesting that hyperacetylation of histones is an important component of the transactivation of the Glc-6-Pase gene promoter by HNF-4α. Region-751/-466 of this promoter contains seven putative HNF-4α-binding motifs. Binding of HNF-4α to this region was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays, indicating that HNF-4α is recruited to the Glc-6-Pase gene promoter during short-chain fatty acid-induced transcription from this promoter.

Valproic acid-induced gene expression has been attributed to the DNA-binding activity of the transcription factor activator protein 1 (AP-1). Using K562 cells, we have studied valproic acid-induced transcription from the human Galpha(i2) gene promoter, which lacks AP-1-binding motifs. We find that valproic acid-induced expression of Galpha(i2) is inhibited by mithramycin A, a compound that interferes with Sp1 binding to GC boxes in DNA. Three Sp1-binding sequences, located at +68/+75, -50/-36, and -92/-85 in the promoter, accounted for about 60% of this transcriptional effect, as judged by transient transfection assays. Electrophoretic mobility shift assays indicated that these sites bind members of the Sp family of transcription factors. Binding to DNA was inhibited by mithramycin A and was greater in nuclear extracts from cells treated with valproic acid than in control cells. Okadaic acid, calyculin A, and fostriecin, which are potent inhibitors of protein phosphatase, suppressed the transcriptional response to valproic acid. This inhibitory effect was not observed when promoter constructs containing mutations in the referenced Sp1-binding sites were used for transfections. In nuclear extracts from cells cultured in the presence of these inhibitors, the binding of Sp1/Sp3 to DNA probes was much less than in control cells. Alkaline phosphatase treatment of nuclear extracts resulted in enhanced binding of Sp proteins to the DNA probes. These results are consistent with the idea that dephosphorylating conditions enhanced Sp binding to the DNA probes as well as Sp-mediated transcription induced by valproic acid. This study demonstrates that the gene expression-inducing effect of valproic acid occurs, in part, through the Sp family of transcription factors.

The cellular abundance of the transcription factor Nrf2 is largely controlled by Keap1, an inhibitory protein that functions as a substrate adaptor for ubiquitylation of Nrf2 by the Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex, thereby targeting Nrf2 for proteasome-dependent degradation in the cytoplasm. It is becoming increasingly apparent that degradation of Nrf2 can also occur by Keap1-independent mechanisms. Recently, we showed that Nrf2 is a SUMO-modifiable protein and that polysumoylated Nrf2 can be polyubiquitylated by the poly-SUMO-specific E3 ubiquitin ligase known as RING finger protein 4 (RNF4) and subsequently degraded in PML-NBs. The role of sumoylation in the turnover of Nrf2, independent of Keap1, is a novel aspect of the biology of Nrf2. Here, we explored whether sumoylated Nrf2 is a substrate for Arkadia, another polySUMO-specific E3 ubiquitin ligase (also known as RING finger protein 111 [RNF111]). Our results indicate that Arkadia can ubiquitylate polysumoylated Nrf2 in PML...

Using a cellular fractionation approach, we have shown that the transcription factor Nrf2 can be degraded in the nucleus [Malloy et al. (2013) J. Biol. Chem. 288, 14569-14583]. The use of photoconv...

The transcription factor Nrf2 induces transcription by binding to the antioxidant response element(s) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. To act as a transcription factor, Nrf2 must be separated from its inhibitory protein Keap1. Post-translational modifications (PTMs) that mediate separation of Nrf2 from Keap1 have been extensively studied but PTMs that impact its biology after this separation are just beginning to emerge. Using in vitro sumoylation assay and other methods, we demonstrated that Nrf2 is a SUMO-targeted protein [Malloy et al. (2013) JBC 288, 14569-14583]. Here, we have used three SUMO site-predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to locate putative SUMO-binding sites in mouse Nrf2. Three of the fifteen putative sumoylation sites identified by these algorithms have high probability scores on all three prediction algorithms. Using site-directed mutagenesis, Co-IP, and in vitro...

Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G alpha(i2) levels in differentiating K562 cells. Here we show that sodium butyrate induces G alpha(i2) gene transcription via sequences at -50/-36 and -92/-85 in the G alpha(i2) gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G alpha(i2) gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at -50/-36 and -92/-85 in the G alpha(i2) gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate- and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.

Fifty percent of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein beta (C/EBP beta-/- mice; B phenotype) die within 1 to 2 h after birth of hypoglycemia. They do not mobilize their hepatic glycogen or induce the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK). Administration of cAMP resulted in mobilization of glycogen, induction of PEPCK mRNA, and a normal blood glucose; these mice survived beyond 2 h postpartum. Adult C/EBP beta-/- mice (A phenotype) also had difficulty in maintaining blood glucose levels during starvation. Fasting these mice for 16 or 30 h resulted in lower levels of hepatic PEPCK mRNA, blood glucose, beta-hydroxybutyrate, blood urea nitrogen, and gluconeogenesis when compared with control mice. The concentration of hepatic cAMP in these mice was 50% of controls, but injection of theophylline, together with glucagon, resulted in a normal cAMP levels. Agonists (glucagon, epinephrine, and isoproterenol) and other effectors of activation of adenylyl cyclase were the same in liver membranes isolated from C/EBP beta-/- mice and littermates. The hepatic activity of cAMP-dependent protein kinase was 80% of wild type mice. There was a 79% increase in the concentration of RI alpha and 27% increase in RII alpha in the particulate fraction of the livers of C/EBP beta-/- mice relative to wild type mice, with no change in the catalytic subunit (C alpha). Thus, a 45% increase in hepatic cAMP (relative to the wild type) would be required in C/EBP beta-/- mice to activate protein kinase A by 50%. In addition, the total activity of phosphodiesterase in the livers of C/EBP beta-/- mice, as well as the concentration of mRNA for phosphodiesterase 3A (PDE3A) and PDE3B was approximately 25% higher than in control animals, suggesting accelerated degradation of cAMP. C/EBP beta influences the regulation of carbohydrate metabolism by altering the level of hepatic cAMP and the activity of protein kinase A.

The chronic myelogenous leukaemia cell line K562 can be triggered in culture to differentiate along the erythrocytic pathway in response to a variety of stimulatory agents. In the presence of sodium butyrate, these cells differentiate to erythroblasts and acquire the capability to synthesize haemoglobin. We used this cell system to study alterations in the levels of several G-protein subunits during the cell differentiation programme and to assess the involvement of G(i)alpha2 in this process. Western immunoblot analysis revealed the presence of G(s)alpha1, G(s)alpha2, G(i)alpha2, G(q)alpha, Galpha(12), Gbeta1 and Gbeta2 in K562 cells. G(o)alpha, G(z)alpha, Galpha(13) and Galpha(16) were not detected. Although the levels of several G-protein subunits were altered after treatment with sodium butyrate, the most striking change was the robust increase in the levels of G(i)alpha2, which was accompanied by an increase in the mRNA for G(i)alpha2. Inactivation of G(i)alpha2 by adding Bordetella pertussis toxin to the cultures inhibited erythroblastic differentiation by as much as 62%, as measured by haemoglobin accumulation. Furthermore, the addition of an oligonucleotide anti-sense to G(i)alpha2 inhibited the sodium butyrate-induced robust increase in G(i)alpha2 levels, decreasing it to the basal levels seen in control cells; this treatment decreased the erythroblastic differentiation of the cells (as measured by haemoglobin expression) by 50%. Taken together, these findings imply that increased levels of G(i)alpha2 contribute to the sodium butyrate-induced erythroblastic differentiation of K562 cells.

The G-protein Gαi2 mediates signaling in a variety of processes. Induced expression of Gαi2 by butyrate and various transcription factors has been established, but transcriptional suppression has not previously been explored. Using HepG2 and K562 cells in culture, we show here that whereas both C/EBPα and C/EBPβ induced transcription from the Gαi2 gene promoter, C/EBPα, but not C/EBPβ, inhibited butyrate-induced Gαi2 expression. Because the transcriptional effect of butyrate on this gene promoter is largely mediated by the transcription factor Sp1, we investigated whether C/EBPα influenced Sp1-induced Gαi2 gene transcription. Binding of C/EBPα to a C/EBP response element in Gαi2 gene promoter inhibited Sp1-induced promoter activity. ChIP analysis showed decreased butyrate-induced recruitment of Sp1 to the Gαi2 gene promoter in response to C/EBPα treatment. Incubating cells with acetate or transfecting them with expression plasmid for either the acetyltransferase p300 or CREB-binding protein (CBP) reversed the antagonistic effect of C/EBPα on Sp1-dependent gene transcription, suggesting that the mechanistic basis for the antagonism is related to the squelching of co-activator acetyltransferase(s) by C/EBPα or the acetylation of Sp1 and/or C/EBPα. This work reveals that C/EBPα plays a dual role as an activator and as a repressor of Gαi2 gene transcription.

Among various G-protein subunits identified in atrial and ventricular membranes from newborn and adult rabbits, the most remarkable developmental and tissue-specific differences were observed in the amounts of the alpha-subunit of Go (Go alpha) and the beta-subunit (G beta). Go alpha was abundant in atrial membranes especially from newborn rabbits but was barely detectable in ventricles. In contrast, G beta was present in both atrial and ventricular membranes. In both tissues the level of G beta-2 was much higher than that of G beta-1. Starting just after birth, the high levels of Go alpha in atria at term decreased gradually to about half of the amount in term animals, at 30 days of age, while the amount of G beta remained relatively constant until 26 days after birth. The levels of G beta in ventricles slowly declined during neonatal development. Similar developmental patterns were also observed when these parameters were studied in membranes isolated from cultured myocytes. Northern blot analysis showed the presence of mRNA for Go alpha-1A isoform in RNA samples from atria but not those from ventricles. The developmental expression of Go alpha-1A mRNA was somewhat different from that of Go alpha protein, whereas the time course of G beta mRNA expression was similar to that of G beta protein. Among other G-protein subunits tested, the amount of the splice variant of Gs protein, Gs alpha-1, in atrial as well as ventricular membranes increased with age of animals, while the amounts of Gs alpha-2, Gi alpha 2 and G alpha q/11 did not change significantly during development. These results indicate differential localization and tissue-specific developmental expression of G-protein subunits in rabbit heart.

The mRNA levels for Gs alpha and G beta in liver from various age groups of rabbits were assessed by Northern and dot-blot hybridization assays using cDNA and oligonucleotide probes. The mRNA levels for both Gs alpha and G beta exhibited a transient 30-35% decrease at 3-6 h after birth, followed by a 3- to 3.5-fold increase which peaked at 2 days after birth, then gradually declined to adult levels at 4-6 weeks. The changes in mRNA levels paralleled the changes in protein levels [previously measured by immunoblotting (Kawai and Arinze, 1991)] in the first 2 days after birth, but not in the later periods of development. Beyond day 3, the increase in protein levels persists, reaching maximal levels at 4 weeks and equalling adult levels while their mRNA levels decrease to about 50% of the levels at day 2. In vivo administration of dexamethasone to neonatal rabbits increased hepatic mRNA levels for both Gs alpha and G beta by about 50%; the effect was moderate compared to the 2- to 3-fold increase in corresponding protein levels. The transcription rate for the Gs alpha gene increased by only 32% at day 2 after birth compared to term. A 68% increase in transcription rate for this gene was observed after the dexamethasone treatment. Taken together, these data indicate that the developmental and glucocorticoid-regulated expression of Gs alpha- and G beta-subunits in neonatal liver is modulated at the transcriptional level.

The effect of dexamethasone administration in vivo on the steady-state levels of G-protein subunits in liver of neonatal rabbits was investigated using specific antibodies to each subunit as well as bacterial toxin-mediated ADP-ribosylation assays. Parallel measurements were also made of the activity of adenylyl cyclase, as influenced by a variety of activators. Dexamethasone administration modulated the levels of G-protein subunits in liver in an age-dependent and subunit-specific manner but not in 24-h-old newborns. The inductive effect of dexamethasone was observed in animals older than 24 h, the greatest effect being on 2- to 3-day-old neonates. In 48-h-old animals the alpha-subunits Gs alpha-1, Gs alpha-2, Gi alpha and the beta-subunit G beta increased 2.0-, 2.1-, 4.3- and 2.8-fold, respectively, compared to the control. The increases were much less for older animals. Dexamethasone treatment also modulated effector-mediated stimulation of adenylyl cyclase activity in vitro and mimicked its effects on G-protein levels; the greatest increase (approximately 2-fold) in the activation of adenylyl cyclase occurred in membranes isolated from 2- to 3-day-old animals. In older animals there was either no effect of dexamethasone or a decrease in activity. The degree of change in enzyme activity paralleled the change in the amount of Gs alpha rather than of Gi alpha or G beta. These results suggest development-dependent regulation of hepatic G-proteins by glucocorticoids.

Ontogeny of trimeric GTP-binding regulatory proteins (G-proteins) and their subunits in rabbit liver during neonatal development was studied, by using bacterial-toxin-catalysed ADP-ribosylation of membrane proteins, immunoblot analysis to quantify the alpha-subunit (alpha s and alpha i) of stimulatory (Gs) and inhibitory (Gi) G-protein and the beta-subunit, and reconstitution assay with cyc- membranes (from Gs-deficient variant of S49 lymphoma cell) to measure Gs activity. Under optimal conditions of ADP-ribosylation, little cholera-toxin substrate (alpha s) was detected in membranes from liver of neonatal animals up to 24 h of age. Thereafter ribosylatable alpha s proteins, i.e. 45 kDa (alpha s-1) and 52 kDa (alpha s-2) proteins, were increasingly evident, reaching maximal levels in membranes from animals aged 4-6 weeks. The concentrations of alpha s-1 and alpha s-2, as determined by immunoblotting, were 6.1 +/- 0.8 and 2.7 +/- 0.4 pmol/mg of protein respectively at birth, and did not change during 0-24 h after birth. Thereafter they gradually increased to maximal levels of 22.1 +/- 1.3 and 10.5 +/- 0.7 pmol/mg of protein for alpha s-1 and alpha s-2 respectively, within 6 weeks. The beta-subunit also showed a similar 3-4-fold increase during the same age span. In contrast, the pertussis-toxin substrate (alpha i) was clearly evident even in membranes from term animals and in all age groups studied. Its developmental pattern, as assessed by ADP-ribosylation, was the same as that determined by immunoblot analysis. The functional activity of Gs in cholate extracts of membranes exhibited similar developmental pattern to that of cholera-toxin-mediated labelling. This activity also paralleled the concentrations of alpha s as measured by immunoblotting. These results suggest differential expression of G-protein subunits in liver during neonatal development.

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42–53) and the other (residues 587–593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494–511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins α5 and β1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin α-β heterodimer nuclear import receptor system plays a critical role in the import process.

Valproic acid (VPA) is a widely used anticonvulsive agent that has profound antiproliferative effects in many cell types, as well as inductive effects on a number of genes. The mechanism of its gene-inducing effect has been reported to involve transcription factors, Sp1 and activator protein-1. Using two well-characterized antioxidant response element (ARE)–driven gene promoters, i.e., mouse heme oxygenase-1 and human NAD(P)H:quinone oxidoreductase 1 genes as tools to monitor the transcriptional response to VPA, we show here that VPA-induced gene transcription was abrogated by antioxidants. With the human Gαi2 gene promoter, which was previously used to establish the involvement of Sp1 in the transcriptional action of VPA, we found that VPA-induced gene transcription was also blocked by antioxidants. Mutation of the ARE (5′-TGACtggGC-3′) in this promoter abrogated the transcriptional response to VPA. With such mutants, the NADPH oxidase inhibitor, diphenyleneiodonium, had no effect on VPA-induced transcription. In gel mobility shift assays, VPA-induced binding of nuclear proteins to a DNA probe containing the relevant ARE sequence in the Gαi2 gene promoter was decreased in nuclear extracts from cells pretreated with antioxidants. Chromatin immunoprecipitation assays showed that the prototype redox-sensitive transcription factors, Nrf2, small Maf protein(s), and c-Fos, were recruited to this promoter in VPA-treated cells. Overall, this study reveals that the mechanism of the transcriptional response to VPA includes VPA-induced production of reactive oxygen species which induce the activation of redox-sensitive transcription factors that interact with the ARE. (Cancer Res 2006; 66(13): 6563-9)

In mammals a variety of metabolic processes are initiated after birth to enable the newborn to adjust to living in an extra-uterine environment. Such metabolic processes include, but are not limited to, gluconeogenesis, glycogenolysis, fatty acid oxidation, ketogenesis, ketone body utilization, and myelination. Other than myelination, the majority of the metabolic processes in this list occur in the liver, albeit with different time frames. Thus, the liver is a key organ for metabolic homeostasis in mammalian newborns. This organ accumulates glycogen as well as triglycerides prior to birth (1–5) such that at birth the concentration of glycogen in various species is generally twofold higher than the level of glycogen found in liver of the adult of the same species (5,6). At least two decades of research dating back to the work of Shelley (5) have delineated enzymatic and metabolite profiles which engender initial synthesis of liver glycogen prior to birth and its breakdown postnatally. This information is available in numerous reviews (7–12).

1. Guinea-pig hepatocytes were prepared by collagenase digestion of the perfused liver. 2. The highest rates of gluconeogenesis were obtained from fructose, followed by pyruvate, xylitol and lactate, glycerol and propionate in that order. Maximum rates of gluconeogenesis were attained at 6–10mm substrate. 3. An initial 15-min lag period occurred during gluconeogenesis from lactate. This lag was abolished by preincubating the cells or by preincubation plus the addition of NH4Cl or lysine. 4. The lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were increased during the lag and adjusted to values favouring rapid gluconeogenesis from lactate after 15min. 5. The data suggest that the low glucose synthesis during the lag resulted from a limitation of the glutamate–aspartate shuttle and from the unusual redox state of the NAD+ couple prevailing during this period. 6. At 0.1mm, amino-oxyacetate, a transaminase inhibitor, decreased gluconeogenesis from lactate by 80%, but had a negligible effect on glucose production from pyruvate. Gluconeogenesis from lactate was also inhibited (20%) by 10mm-dl-3-hydroxybutyrate.

1. 1. At mid-gestation the activities of guinea-pig liver pyruvate carboxylase, glucose-6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase were less than 0.4 unit/g, whereas the activity of fructose-1,6-diphosphatase was 1.6 unit/g. Throughout gestation the activity of cytosolic phosphoenolpyruvate carboxykinase remained unchanged while all other enzymes exhibited a 2- to 3-fold increase over the levels at mid-gestation, beginning at 45–50 days. The activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase in the foetus were largely mitochondrial. 2. 2. No increases in enzyme activities were evident in the first 2–3 hr following birth. Subsequently, increases were noted for all enzymes examined, with an overshoot occurring between 7 and 14 days post partum for pyruvate carboxylase, glucose-6-phosphatase, and fructose-1,6-diphosphatase. 3. 3. The increases in enzyme activities following birth could be prevented by treating the newborns with actinomycin D. 4. 4. In the immediate postnatal period, the degree of hypoglycaemia was not pronounced, the initial 40% drop in blood glucose level 1 hr after birth being reduced to less than 20% at 2hr. 5. 5. Livers taken from near-term foetuses and perfused immediately after Caesarean delivery synthesized appreciable amounts of glucose from lactate and glycerol. 6. 6. The data show that the capacity for hepatic gluconeogenesis in guinea-pigs is acquired before birth.

Mitochondria of cultured normal human fibroblast cells were found to contain the enzyme phosphoenolpyruvate carboxykinase. The activity of this enzyme in these cells is increased 2- to 3-fold by addition of 5 · 10−4 M dibutyryl cyclic AMP, or 1.5- to 2-fold by the addition of dexamethasone (2 · 10−7 M) or hydrocortisone (1.38 · 10−6 M). These increases in enzyme activity were inhibited by cycloheximide and actinomycin D, suggesting they are dependent upon de novo protein synthesis. Cultured human fibroblasts may thus provide a useful system for studying the regulation of mitochondrial phosphoenolpyruvate carboxykinase.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.

Glycogenolysis in hepatocytes isolated from fed guinea pigs was much more enhanced by the beta-agonist, isoproterenol, than by equimolar concentrations of the alpha-agonists, phenylephrine and norepinephrine. The stimulatory effects of catecholamines occurred with the following order of potency: isoproterenol greater than epinephrine greater than norepinephrine. This order of potency is characteristic of beta 2-adrenergic receptors. That beta 2-receptors are responsible for mediating catecholamine stimulation of glycogenolysis in guinea-pig hepatocytes was further deduced from the inhibition of agonist-stimulated glycogenolysis by beta-receptor sub-type-selective antagonists. Thus, IPS 339, a beta-antagonist which has higher affinity at beta 2-sites than at beta 1-sites, was three orders of magnitude more potent in inhibiting isoproterenol-stimulated glycogenolysis than either atenolol or practolol, both of which are beta 1-selective antagonists. The beta 2-agonists zinterol and procaterol also stimulated glycogenolysis in hepatocytes and their effects were inhibited by propranolol and IPS 339, but not by practolol. Furthermore, activation of phosphorylase in these hepatocytes by isoproterenol, epinephrine, and norepinephrine also occurred with the potency order expected for beta 2-receptors. These results are in sharp contrast to those obtained with rat hepatocytes and emphasize that species differences occur in the regulation of hepatic glycogenolysis by catecholamines.

beta-Adrenergic receptors in guinea-pig liver plasma membranes were characterized by radioligand binding, using l-[3H]dihydroalprenolol ([3H]DHA), l-3-[125I]iodocyanopindolol ([125I]CYP) and dl-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2- one hydrochloride [( 3H]CGP-12177). The binding of both [125I]CYP and [3H]CGP-12177 to membranes exhibited high affinity (Kd = 3.5 +/- 0.2 pM for [125I]CYP and 0.75 +/- 0.10 nM for [3H]CGP-12177) and stereospecificity; the maximal binding sites were 130 +/- 15 and 137 +/- 8 fmoles/mg protein respectively. Catecholaminergic agonists competed for these binding sites in the order l-isoproterenol greater than l-epinephrine greater than l-norepinephrine, which is typical for beta 2-adrenergic receptors. The binding data are supported by parallel experiments on adenylate cyclase activation by catecholamines, and on antagonism of this activation by beta 1- and beta 2-selective blockers. The binding of [3H]DHA was excessive (Bmax = 21.4 pmoles/mg protein), exhibited low affinity (Kd = 34.6 nM), and lacked stereospecificity. When liver membranes were incubated at 50 degrees for 40 min in the presence of an agonist, l-isoproterenol, the binding of [3H]DHA to the heat-treated membranes exhibited high affinity (Kd = 1.07 +/- 0.17 nM) and the Bmax was reduced to 139 +/- 22 fmoles/mg protein. In such membranes, as opposed to native membranes, stereospecificity was evident and catecholaminergic agonists competed for the binding sites in the order typical for beta 2-adrenergic receptors. However, agonist competition of the binding to the heat-treated membranes could not be modulated by guanine nucleotides, indicating a loss of communication between the receptor and the guanine nucleotide regulatory protein.

The significance of mitochondrial phosphoenolpyruvate formation for gluconeogenesis was evaluated in isolated, perfused livers from rats, guinea pigs, and rabbits. In guinea pig liver, 0.2 mm octanoate decreased by 40 and 30% the rate of gluconeogenesis from lactate and alanine, respectively, but not from pyruvate. In rat liver, gluconeogenesis from all three substrates was increased. The inhibition of gluconeogenesis in guinea pig liver was associated with an increased level of reducing equivalents in the mitochondria as indicated by the NAD+:NADH ratios in freeze-clamped livers before and during infusion of octanoate. Infusion of β-hydroxybutyrate also increased gluconeogenesis from pyruvate and lactate in rat liver but inhibited glucose formation from lactate in guinea pig liver. Low concentrations of the artificial electron acceptor, phenazine methosulfate, reversed the inhibitory effects of excess mitochondrial reducing equivalents. Aminooxyacetate, which inhibits aminotransferases, totally abolished glucose formation from lactate (but not from pyruvate) in perfused rat liver but reduced gluconeogenesis by only 50 to 60% in guinea pig liver. This inhibition was further increased by β-hydroxybutyrate infusion. In livers from fed rabbits, aminooxyacetate had no effect, but in livers from fasted rabbits in which the activity of cytosolic P-enolpyruvate carboxykinase is induced, the inhibition by aminooxyacetate was quantitatively similar to that in the guinea pig liver. The data show that in the guinea pig and rabbit liver both the cytosolic and mitochondrial forms of P-enolpyruvate carboxykinase actively function during gluconeogenesis from lactate and alanine. The gluconeogenic flux via the mitochondrial enzyme accounts for at least one-half of the over-all rate of glucose formation from lactate. It is concluded that the regulation of gluconeogenesis in species containing a mitochondrial activity of P-enolpyruvate carboxykinase is significantly different from that operating in rat liver and reflect to a large extent the compartmentation of P-enol-pyruvate carboxykinase.