HNF4α is required for Tkfc promoter activation by ChREBP.

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we showed that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of 2 hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and 1 carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in the livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.

ChREBP deficiency prevents high sucrose diet-induced obesity through reducing sucrase expression.

Obesity appears to be a major contributing factor for many health problems. Effective treatments for reducing weight gain, other than caloric restriction and exercise, are limited. The consumption of sugars is a major factor in the development of obesity in part by stimulating the transcription factor, carbohydrate response element binding protein (ChREBP), a process that is driven by de novo lipogenesis. Therefore, we hypothesized that inhibiting the action of ChREBP would be a promising strategy for alleviating these diseases. Using ChREBP deficient mice, the effect of a high intake of sucrose on body weight and blood glucose levels were investigated. Unlike wild type mice, ChREBP deficient mice did not gain much weight and their blood glucose and cholesterol levels remained relatively constant. In tracing it's cause, we found that the levels of expression of sucrase, an enzyme that digests sucrose, and both Glut2 and Glut5, a transporter of glucose and fructose, were not induced by feeding a high sucrose diet in the small intestine of ChREBP deficient mice. Our findings suggest that the inhibition of ChREBP could suppress weight gain even on a high sucrose diet.

A lack of ChREBP inhibits mitochondrial cristae formation in brown adipose tissue.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that increases the transcription of multiple genes. ChREBP is highly localized in the liver, where it upregulates the expression of genes that code for glycolytic and lipogenic enzymes, resulting in the conversion of excess carbohydrate into storage fat. ChREBP knockout (KO) mice display an anti-obese phenotype. However, at this time, role of ChREBP in adipose tissue remains unclear. Therefore, the energy metabolism and morphology of mitochondrial brown adipose tissue (BAT) in ChREBP KO mice was examined. We found increased expression levels of electron transport system proteins including the mitochondrial uncoupling protein (UCP1), and mitochondrial structural alterations such as dysplasia of the cristae and the presence of small mitochondria in BAT of ChREBP KO mice. Mass spectrometry analyses revealed that fatty acid synthase was absent in the BAT of ChREBP KO mice, which probably led to a reduction in fatty acids and cardiolipin, a regulator of various mitochondrial events. Our study clarified the new role of ChREBP in adipose tissue and its involvement in mitochondrial function. A clearer understanding of ChREBP in mitochondria could pave the way for improvements in obesity management.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR.

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

The Ile35 Residue of the ALS-Associated Mutant SOD1 Plays a Crucial Role in the Intracellular Aggregation of the Molecule.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an unknown pathogenesis. It has been reported that mutations in the gene for Cu/Zn superoxide dismutase (SOD1) cause familial ALS. Mutant SOD1 undergoes aggregation and forms amyloid more easily, and SOD1-immunopositive inclusions have been observed in the spinal cords of ALS patients. Because of this, SOD1 aggregation is thought to be related to the pathogenesis of ALS. Some core regions of amyloid have been identified, but the issue of whether these regions form aggregates in living cells remains unclear, and the mechanism responsible for intracellular SOD1 aggregation also remains unclear. The findings reported in this study indicate that the aggregation of the ALS-linked mutant SOD1-EGFP was significantly enhanced when the BioID2 gene was fused to the N-terminus of the mutant SOD1-EGFP plasmid for cellular expression. Expression of a series of BioID2-(C-terminal deletion peptides of SOD1)-EGFP permitted us to identify 1-35 as a minimal N-terminal sequence and Ile35 as an essential amino acid residue that contributes to the intracellular aggregation of SOD1. The findings also showed that an additional substitution of Ile35 with Ser into the ALS mutant SOD1 resulted in the significant suppression of aggregate formation. The fact that no Ile35 mutations have been reported to date in ALS patients indicates that all ALS mutant SOD1s contain Ile35. Taken together, we propose that Ile35 plays a pivotal role in the aggregation of the ALS-linked SOD1 and that this study will contribute to our understanding of the mechanism responsible for SOD1 aggregation.

Iron deficiency aggravates DMNQ-induced cytotoxicity via redox cycling in kidney-derived cells.

Iron, an essential element for most of living organisms, participates in many biological functions. Since iron is redox-active transition metal, it is known that excessive levels stimulate the formation of reactive oxygen species (ROS) and exacerbate cytotoxicity. An iron deficiency is the most common nutritional deficiency disorder in the world (about 30% of the population) and is more common than cases of iron overload. However, the effects of iron deficiency on ROS-induced cytotoxicity and the maintenance of intracellular redox homeostasis are not fully understood. The present study reports on an evaluation of the effects of iron deficiency on cytotoxicity induced by several ROS generators. In contrast to hydrogen peroxide and erastin, the cytotoxicity of 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a redox cycling agent that induces intracellular superoxide anion formation, was exacerbated by iron deficiency. Cytochrome b5 reductase was identified as a candidate enzyme responsible for the redox cycling of DMNQ under conditions of iron depletion. Moreover, the DMNQ-induced intracellular accumulation of ROS and a decrease in NADH/NAD+ ratios were enhanced by an iron deficiency. These negative changes were found to be ameliorated by overexpressing NAD(P)H:quinone oxidoreductase 1 (NQO1) in kidney-derived cells that originally showed a very low expression of NQO1. These results indicate that NQO1 plays a protective role against redox cycling quinone-mediated cytotoxicity under iron-depleted conditions. This is because NQO1 generates less-toxic hydroquinones via the two-electron reduction of quinones. The collective findings reported herein demonstrate that not only an iron overload but also an iron deficiency exacerbates ROS-mediated cytotoxicity.

Introduction of team-based learning improves understanding of glucose metabolism in biochemistry among undergraduate students.

Team-based learning (TBL) is an active learning method used in many educational institutions. However, there are few examples of its use in basic medicine, such as biochemistry in medical schools. This study used TBL to teach glucose metabolism to first-year medical students. The process was in four phases: preclass preparation, readiness assurance tests, advanced questions, and a TBL test, with peer evaluation and a questionnaire. There were positive correlations between the TBL test, peer evaluation, and individual readiness test performance. Tests were taken immediately after learning and 2 weeks later, and scores decreased significantly less with TBL than traditional lectures (-2.3% vs. -17.5%). This suggests that TBL was more effective than traditional lectures in supporting knowledge retention. We used a Moodle system to facilitate communication between students and teachers, and this was evaluated positively by both groups. It was particularly useful for managing TBL. These findings suggest that TBL could be used to improve student performance in biochemistry.

Activation of the mitogen-activated protein kinase ERK1/2 signaling pathway suppresses the expression of ChREBPα and ß in HepG2 cells.

The carbohydrate response element-binding protein (ChREBP), a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis, exists as two isoforms: ChREBPα and ChREBPß. However, the mechanism responsible for regulating the expression of both ChREBPα and ß, as well as the mechanism that determines which specific isoform is more responsive to different stimuli, remains unclear. To address this issue, we compared the effects of several stimuli, including oxidative stress, on the mRNA and protein expression levels of ChREBPα and ß in the hepatocyte cell line, HepG2. We found that H2 O2 stimulation suppressed the expression of both mRNA and protein in HepG2 cells, but the mRNA expression level of ChREBPß was < 1% of that for ChREBPα levels. In addition, the reduction in both ChREBPα and ß mRNA levels was reversed by PD98059, a selective and cell permeable inhibitor of the MEK/ERK pathway. Additionally, the administration of 12-O-tetradecanoylphorbol 13-acetate (TPA) and staurosporine (STS), activators of extracellular-signal-regulated kinase (ERK) signaling, also resulted in a decrease in the levels of both ChREBPα and ß mRNA in HepG2 cells through ERK signaling. These collective data suggest that oxidative stress, including STS treatment, suppresses the expression of ChREBPα and ß via the activation of ERK signaling in HepG2 cells. Such a decrease in the levels of expression of ChREBPα and ß could result in the suppression of hepatic glycolysis and lipogenesis, and this would be expected to prevent further oxidative stress.

The role of O-linked GlcNAc modification on the glucose response of ChREBP.

The carbohydrate response element-binding protein (ChREBP) functions as a transcription factor in mediating the glucose-activated gene expression of multiple liver enzymes, which are responsible for converting excess carbohydrate to storage fat. ChREBP is translocated into the nucleus in response to high glucose levels, and then up-regulates transcriptional activity. Although this glucose activation of ChREBP is generally observed only in liver cells, overexpression of wild type max-like protein X (Mlx), but not an inactive mutant Mlx, resulted in the exhibition of the ChREBP functions also in a human kidney cell line. Because high glucose conditions induce the glycosylation of cellular proteins, the effect of O-linked GlcNAc modification on ChREBP functions was examined. Treatment with an O-GlcNAcase inhibitor (PUGNAc), which increases the O-linked GlcNAc modification of cellular proteins, caused an increase in the glucose response of ChREBP. In contrast, treatment with a glutamine fructose amidotransferase inhibitor (DON), which decreases O-GlcNAcylation by inhibiting the hexosamine biosynthetic pathway, completely blocked the glucose response of ChREBP. These results suggest that the O-linked glycosylation of ChREBP itself or other proteins that regulate ChREBP is essential for the production of functional ChREBP.

Basic skills examination in a biochemical practical training program for undergraduate students.

University lectures are mainly passive in nature, and there are few subjects in which students need to learn and function independently. Tutorial education and related activities at universities that specialize in medical and pharmaceutical training have been actively carried out, and lectures in conjunction with practical skills are gradually being developed, although progress has been slow in this area. In past years, our biochemistry practice classes have been evaluated in reports dealing with experiments and written examinations, as is done in other universities. However, using this methodology, we are not able to evaluate the extent to which students master biochemical experimental skills. To address this, we introduced a basic skill test to our biochemical curriculum for the first time. Our exams contributed to a deeper understanding of student skills and could be good tools for evaluating the degree of understanding of the students. The students understood the contents of the training well and felt interested in research in the field of basic medicine. Thus, we conclude that introducing practical testing to biochemical practice was effective for medical students in the field of biochemistry. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):279-287, 2019.

Caffeine-stimulated muscle IL-6 mediates alleviation of non-alcoholic fatty liver disease.

Caffeine intake is associated with a reduced risk developing non-alcoholic fatty liver disease (NAFLD), but the underlying molecular mechanisms remain to be fully elucidated. We report here that caffeine markedly improved high fat diet-induced NAFLD in mice resulting in a 10-fold increase in circulating IL-6 levels, leading to STAT3 activation in the liver. Interestingly, the expression of IL-6 mRNA was not increased in the liver, but increased substantially in the muscles of caffeine-treated mice. Caffeine was found to stimulate IL-6 production in cultured myotubes but not in hepatocytes, adipocytes, or macrophages. The inhibition of p38/MAPK abrogated caffeine-induced IL-6 production in muscle cells. Caffeine failed to improve NAFLD in IL-6 and hepatocyte-specific STAT3 knockout mice, indicating that the IL-6/STAT3 pathway is vital for the hepatoprotective effects of caffeine in NAFLD. The possibility that IL-6/STAT3-mediated hepatic autophagosome induction and hepatocytic oxygen consumption are involved in the anti-NAFLD effects of caffeine cannot be excluded, based on the findings presented here. Our results reveal that caffeine ameliorates NAFLD via crosstalk between muscle IL-6 production and liver STAT3 activation.

Cu/Zn-superoxide dismutase forms fibrillar hydrogels in a pH-dependent manner via a water-rich extended intermediate state.

Under certain conditions, amyloid-like fibrils can develop into three-dimensional networks and form hydrogels by a self-assembly process. When Cu/Zn superoxide dismutase (SOD1), an anti-oxidative enzyme, undergoes misfolding, fibrillar aggregates are formed, which are a hallmark of a certain form of familial amyotrophic lateral sclerosis (ALS). However, the issue of whether SOD1 fibrils can be assembled into hydrogels remains to be tested. Here, we show that the SOD1 polypeptides undergo hydrogelation accompanied by the formation of thioflavin T-positive fibrils at pH 3.0 and 4.0, but not at pH 5.0 where precipitates are formed. The results of viscoelastic analyses indicate that the properties of SOD1 hydrogels (2%) were similar to and slightly more fragile than a 0.25% agarose gel. In addition, monitoring by a quartz crystal microbalance with admittance analysis showed that the denaturing of immobilized SOD1 on a sensor under the hydrogelation conditions at pH 3.0 and 4.0 resulted in an increase in the effective acoustic thickness from ~3.3 nm (a folded rigid form) to ~50 and ~100 nm (an extended water-rich state), respectively. In contrast, when SOD1 was denatured under the same conditions at pH 5.0, a compact water-poor state with an effective acoustic thickness of ~10 nm was formed. The addition of physiological concentrations of NaCl to the pH 4.0 sample induced a further extension of the SOD1 with larger amounts of water molecules (with an effective acoustic thickness of ~200 nm) but suppressed hydrogel formation. These results suggest that different denatured intermediate states of the protein before self-assembly play a major role in determining the characteristics of the resulting aggregates and that a conformational change to a suitable level of extended water-rich intermediate state before and/or during intermolecular assembling is required for fibrillation and hydrogelation in the case of globular proteins.

The internalization and metabolism of 3-deoxyglucosone in human umbilical vein endothelial cells.

3-Deoxyglucosone (3-DG), a dicarbonyl compound produced by glycation, plays a role in the modification and cross-linking of long-lived proteins. We synthesized [3H]3-DG from [3H]glucose and developed an internalization assay system using HPLC to examine its cellular metabolism. When smooth muscle cells or human umbilical vein endothelial cells were incubated with [3H]3-DG, it was found that [3H]3-DG was internalized by cells in a time dependent manner. The rate of internalization was reduced when the cells were incubated at 4 degrees C or treated with phenylarsine oxide (PAO). By monitoring [3H]3-DG taken up by cells, it was confirmed that 3-DG is reduced to 3-deoxyfructose (3-DF) and that this reaction was inhibited by an aldo-keto reductase inhibitor (ARI). The presence of 3-DG led to an increase in reactive oxygen species levels in the cells and subsequent apoptosis, and the effect was enhanced by pretreatment with ARI. These results suggest that 3-DG is internalized by cells and reduced to 3-DF by aldo-keto reductases, and that the internalized 3-DG is responsible for the production of intracellular oxidative stress.

Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in Fut8-/- mice.

alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine residue of N-glycans. Here we report that the loss of core fucosylation impairs the function of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), a multifunctional scavenger and signaling receptor, resulting in a reduction in the endocytosis of insulin like growth factor (IGF)-binding protein-3 (IGFBP-3) in the cells derived from Fut8-null (Fut8-/-) mice. The reduced endocytosis was restored by the re-introduction of Fut8. Serum levels of IGFBP-3 were markedly upregulated in Fut8-/- mice. These data clearly indicate that core fucosylation is crucial for the scavenging activity of LRP-1 in vivo.

Cu,Zn-SOD deficiency induces the accumulation of hepatic collagen.

Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic diseases, and results in the development of fibrosis. Oxidative stress is thought to be one of the underlying causes of NAFLD. Copper/zinc superoxide dismutase (SOD1) is a primary antioxidative enzyme that scavenges superoxide anion radicals. Although SOD1 knockout (KO) mice have been reported to develop fatty livers, it is not known whether this lack of SOD1 leads to the development of fibrosis. Since the accumulation of collagen typically precedes liver fibrosis, we assessed the balance between the synthesis and degradation of collagen in liver tissue from SOD1 KO mice. We found a higher accumulation of collagen in the livers of SOD1 KO mice compared to wild type mice. The level of expression of HSP47, a chaperone of collagen, and a tissue inhibitor (TIMP1) of matrix metalloproteinases (a collagen degradating enzyme) was also increased in SOD1 KO mice livers. These results indicate that collagen synthesis is increased but that its degradation is inhibited in SOD1 KO mice livers. Moreover, SOD1 KO mice liver sections were extensively modified by advanced glycation end products (AGEs), which suggest that collagen in SOD1 KO mice liver might be also modified with AGEs and then would be more resistant to the action of collagen degrading enzymes. These findings clearly show that oxidative stress plays an important role in the progression of liver fibrosis.

The absence of the SOD1 gene causes abnormal monoaminergic neurotransmission and motivational impairment-like behavior in mice.

Copper/zinc superoxide dismutase (SOD1), a primary anti-oxidative enzyme, protects cells against oxidative stress. We report herein on a comparison of behavioral and neurobiological changes between SOD1 knockout (KO) and wild-type mice, in an attempt to assess the role of SOD1 in brain functions. SOD1 KO mice exhibited impaired motivational behavior in both shuttle-box learning and three-chamber social interaction tests. High levels of dopamine transporter protein and an acceleration of serotonin turnover were also detected in the cerebrums of the SOD1 KO mice. These findings suggest that SOD1 deficiency disturbs monoaminergic neurotransmission leading to a decrease in motivational behavior.

A common pathway for intracellular reactive oxygen species production by glycoxidative and nitroxidative stress in vascular endothelial cells and smooth muscle cells.

A large body of evidence suggests that carbonyl compounds induce intracellular signaling by increasing oxidative stress in the cell; however, the mechanisms involved have not been fully described. The focus of our research is on the pathway in which antioxidative enzymes are modified and inactivated by carbonyl compounds, resulting in the accumulation of active oxygen species in the cell. A common pathway appears to exist for cellular signaling evoked by nitroxidative stress. It could be concluded that some glycoxidative stress and nitroxidative stress cause intracellular signaling via similar mechanisms. The elucidation of the pathway for extracellular stress-induced reactive oxygen species (ROS) production would be important for our understanding of the role of ROS as signaling molecules.

Glycation proceeds faster in mutated Cu, Zn-superoxide dismutases related to familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. It has been shown that 15-20% of patients with familial ALS (FALS) have defects in the Sod1 gene that encodes Cu, Zn-superoxide dismutase (SOD). To elucidate the pathological role of mutated Cu, Zn-SODs in FALS, the susceptibility of mutants to glycation was examined. Mutated Cu, Zn-SODs (G37R, G93A, and I113T) related to FALS and wild type were produced in a baculovirus/insect cell expression system. Glycated and nonglycated proteins were separated on a boronate column, and the nonglycated fraction was then incubated with glucose. The mutated Cu, Zn-SODs were found to be highly susceptible to glycation compared with the wild-type enzyme as estimated by Western blot analysis using an anti-hexitol lysine antibody. The mutated Cu, Zn-SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild-type enzyme. Mutated Cu, Zn-SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type. These results suggest that high susceptibility of mutated Cu, Zn-SODs to glycation could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.

Acrolein induces Hsp72 via both PKCdelta/JNK and calcium signaling pathways in human umbilical vein endothelial cells.

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehydes to which humans are exposed in a variety of environment situations and is also a product of lipid peroxidation. Increased levels of unsaturated aldehydes play an important role in the pathogenesis of a number of human diseases such as Alzheimer's disease, atherosclerosis and diabetes. A number of studies have reported that acrolein evokes downstream signaling via an elevation in cellular oxidative stress. Here, we report that low concentrations of acrolein induce Hsp72 in human umbilical vein endothelial cells (HUVEC) and that both the PKCdelta/JNK pathway and calcium pathway were involved in the induction. The findings confirm that the production of reactive oxygen species (ROS) is not directly involved in the pathway. The induction of Hsp72 was not observed in other cells such as smooth muscle cells (SMC) or COS-1 cells. The results suggest that HUVEC have a unique defense system against cell damage by acrolein in which Hsp72 is induced via activation of both the PKCd/JNK and the calcium pathway.