[show abstract][hide abstract] ABSTRACT: Postprandial insulin plays a critical role in suppressing hepatic glucose production to maintain euglycemia in mammals. Insulin-dependent activation of protein kinase B (Akt) regulates this process, in part, by inhibiting FoxO1-dependent hepatic gluconeogenesis by direct phosphorylation and subsequent cytoplasmic exclusion. Previously, it was demonstrated that protein arginine methyltransferase 1 (PRMT1)-dependent arginine modification of FoxO1 interferes with Akt-dependent phosphorylation, both in cancer cells and in the Caenorhabditis elegans model, suggesting that this additional modification of FoxO1 might be critical in its transcriptional activity. In this study, we attempted to directly test the effect of arginine methylation of FoxO1 on hepatic glucose metabolism. The ectopic expression of PRMT1 enhanced messenger RNA levels of FoxO1 target genes in gluconeogenesis, resulting in increased glucose production from primary hepatocytes. Phosphorylation of FoxO1 at serine 253 was reduced with PRMT1 expression, without affecting the serine 473 phosphorylation of Akt. Conversely, knockdown of PRMT1 promoted an inhibition of FoxO1 activity and hepatic gluconeogenesis by enhancing the phosphorylation of FoxO1. In addition, genetic haploinsufficiency of Prmt1 reduced hepatic gluconeogenesis and blood-glucose levels in mouse models, underscoring the importance of this factor in hepatic glucose metabolism in vivo. Finally, we were able to observe an amelioration of the hyperglycemic phenotype of db/db mice with PRMT1 knockdown, showing a potential importance of this protein as a therapeutic target for the treatment of diabetes. Conclusion: Our data strongly suggest that the PRMT1-dependent regulation of FoxO1 is critical in hepatic glucose metabolism in vivo. (HEPATOLOGY 2012).
[show abstract][hide abstract] ABSTRACT: There is increasing evidence that the retinoic acid receptor-related orphan receptor α (RORα) plays an important role in the regulation of metabolic pathways, particularly of fatty acid and cholesterol metabolism; however, the role of RORα in the regulation of hepatic lipogenesis has not been studied. Here, we report that RORα attenuates hepatic steatosis, probably via activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and repression of the liver X receptor α (LXRα). First, RORα and its activator, cholesterol sulfate (CS), induced phosphorylation of AMPK, which was accompanied by the activation of serine-threonine kinase liver kinase B1 (LKB1). Second, the activation of RORα, either by transient transfection or CS treatment, decreased the TO901317-induced transcriptional expression of LXRα and its downstream target genes, such as the sterol regulatory element binding protein-1 (SREBP-1) and fatty acid synthase. RORα interacted physically with LXRα and inhibited the LXRα response element in the promoter of LXRα, indicating that RORα interrupts the autoregulatory activation loop of LXRα. Third, infection with adenovirus encoding RORα suppressed the lipid accumulation that had been induced by a free-fatty-acid mixture in cultured cells. Furthermore, we observed that the level of expression of the RORα protein was decreased in the liver of mice that were fed a high-fat diet. Restoration of RORα via tail-vein injection of adenovirus (Ad)-RORα decreased the high-fat-diet-induced hepatic steatosis. Finally, we synthesized thiourea derivatives that activated RORα, thereby inducing activation of AMPK and repression of LXRα. These compounds decreased hepatic triglyceride levels and lipid droplets in the high-fat-diet-fed mice. CONCLUSION: We found that RORα induced activation of AMPK and inhibition of the lipogenic function of LXRα, which may be key phenomena that provide the beneficial effects of RORα against hepatic steatosis.
[show abstract][hide abstract] ABSTRACT: Although mitochondrial impairment has often been implicated in carcinogenesis, the mechanisms of its development in cancer remain unknown. We report here that autophagy triggered by oncogenic K-Ras mediates functional loss of mitochondria during cell transformation to overcome an energy deficit resulting from glucose deficiency. When Rat2 cells were infected with a retrovirus harboring constitutively active K-Ras (V12) , mitochondrial respiration significantly declined in parallel with the acquisition of transformation characteristics. Decreased respiration was not related to mitochondrial biogenesis but was inversely associated with the increased formation of acidic vesicles enclosing mitochondria, during which autophagy-related proteins such as Beclin 1, Atg5, LC3-II and vacuolar ATPases were induced. Interestingly, blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes recovered respiratory protein expression and respiratory activity; JNK was involved in these phenomena as an upstream regulator. The cells transformed by K-Ras (V12) maintained cellular ATP level mainly through glycolytic ATP production without induction of GLUT1, the low Km glucose transporter. Finally, K-Ras (V12) -triggered LC3-II formation was modulated by extracellular glucose levels, and LC3-II formation increased only in hepatocellular carcinoma tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, our observations suggest that mitochondrial functional loss may be mediated by oncogenic K-Ras-induced mitophagy during early tumorigenesis even in the absence of hypoxia, and that this mitophagic process may be an important strategy to overcome the cellular energy deficit triggered by insufficient glucose.
[show abstract][hide abstract] ABSTRACT: Diet-induced obesity (DIO) is linked to peripheral insulin resistance-a major predicament in type 2 diabetes. This study aims to identify the molecular mechanism by which DIO-triggered endoplasmic reticulum (ER) stress promotes hepatic insulin resistance in mouse models.
C57BL/6 mice and primary hepatocytes were used to evaluate the role of LIPIN2 in ER stress-induced hepatic insulin resistance. Tunicamycin, thapsigargin, and lipopolysaccharide were used to invoke acute ER stress conditions. To promote chronic ER stress, mice were fed with a high-fat diet for 8-12 weeks. To verify the role of LIPIN2 in hepatic insulin signaling, adenoviruses expressing wild-type or mutant LIPIN2, and shRNA for LIPIN2 were used in animal studies. Plasma glucose, insulin levels as well as hepatic free fatty acids, diacylglycerol (DAG), and triacylglycerol were assessed. Additionally, glucose tolerance, insulin tolerance, and pyruvate tolerance tests were performed to evaluate the metabolic phenotype of these mice.
LIPIN2 expression was enhanced in mouse livers by acute ER stress-inducers or by high-fat feeding. Transcriptional activation of LIPIN2 by ER stress is mediated by activating transcription factor 4, as demonstrated by LIPIN2 promoter assays, Western blot analyses, and chromatin immunoprecipitation assays. Knockdown of hepatic LIPIN2 in DIO mice reduced fasting hyperglycemia and improved hepatic insulin signaling. Conversely, overexpression of LIPIN2 impaired hepatic insulin signaling in a phosphatidic acid phosphatase activity-dependent manner.
These results demonstrate that ER stress-induced LIPIN2 would contribute to the perturbation of hepatic insulin signaling via a DAG-protein kinase C ε-dependent manner in DIO mice.
[show abstract][hide abstract] ABSTRACT: Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved in the regulation of hepatic glucose metabolism in mice. Expression of hepatic SMEK1/2 is up-regulated during fasting or in mouse models of insulin-resistant conditions in a Peroxisome Proliferator-Activated Receptor-gamma Coactivator 1α (PGC-1α)-dependent manner. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas depletion of the SMEK proteins reduces hyperglycemia and enhances CRTC2 phosphorylation; the effect is blunted by S171A CRTC2, which is refractory to salt-inducible kinase (SIK)-dependent inhibition. Taken together, we would propose that mammalian SMEK/PP4C proteins are involved in the regulation of hepatic glucose metabolism through dephosphorylation of CRTC2.
Proceedings of the National Academy of Sciences 09/2010; 107(41):17704-9. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Endoplasmic reticulum (ER)-bound transcription factor families are shown to be involved in the control of various metabolic pathways. Here, we report a critical function of ER-bound transcription factor, CREBH, in the regulation of hepatic gluconeogenesis. Expression of CREBH is markedly induced by fasting or in the insulin-resistant state in rodents in a dexamethasone- and PGC-1alpha-dependent manner, which results in the accumulation of active nuclear form of CREBH (CREBH-N). Overexpression of constitutively active CREBH activates transcription of PEPCK-C or G6Pase by binding to its enhancer site that is distinct from the well-characterized CREB/CRTC2 regulatory sequences in vivo. Of interest, knockdown of CREBH in liver significantly reduces blood glucose levels without altering expression of genes involved in the ER stress signaling cascades in mice. These data suggest a crucial role for CREBH in the regulation of hepatic glucose metabolism in mammals.
[show abstract][hide abstract] ABSTRACT: DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenital critical region on X chromosome, gene 1) is a member of the nuclear receptor superfamily that can repress diverse nuclear receptors and has a key role in adreno-gonadal development. Our previous report has demonstrated that DAX-1 can inhibit hepatocyte nuclear factor 4alpha transactivity and negatively regulate gluconeogenic gene expression (Nedumaran, B., Hong, S., Xie, Y. B., Kim, Y. H., Seo, W. Y., Lee, M. W., Lee, C. H., Koo, S. H., and Choi, H. S. (2009) J. Biol. Chem. 284, 27511-27523). Here, we further expand the role of DAX-1 in hepatic energy metabolism. Transfection assays have demonstrated that DAX-1 can inhibit the transcriptional activity of nuclear receptor liver X receptor alpha (LXRalpha). Physical interaction between DAX-1 and LXRalpha was confirmed Immunofluorescent staining in mouse liver shows that LXRalpha and DAX-1 are colocalized in the nucleus. Domain mapping analysis shows that the entire region of DAX-1 is involved in the interaction with the ligand binding domain region of LXRalpha. Competition analyses demonstrate that DAX-1 competes with the coactivator SRC-1 for repressing LXRalpha transactivity. Chromatin immunoprecipitation assay showed that endogenous DAX-1 recruitment on the SREBP-1c gene promoter was decreased in the presence of LXRalpha agonist. Overexpression of DAX-1 inhibits T7-induced LXRalpha target gene expression, whereas knockdown of endogenous DAX-1 significantly increases T7-induced LXRalpha target gene expression in HepG2 cells. Finally, overexpression of DAX-1 in mouse liver decreases T7-induced LXRalpha target gene expression, liver triglyceride level, and lipid accumulation. Overall, this study suggests that DAX-1, a novel corepressor of LXRalpha, functions as a negative regulator of lipogenic enzyme gene expression in liver.
Journal of Biological Chemistry 03/2010; 285(12):9221-32. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Transport of dopamine (DA) by the dopamine transporter from the synaptic cleft into the presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. The binding of psychostimulants to their recognition sites on the DA transporter leads to an inhibition of DA transport and a subsequent rising of the dopamine contents in the synaptic cleft is ascribed to a mode of psychostimulation. Discovery of dopamine transporter inhibitors would be useful with regard to substituting for cocaine and minimizing its abuse. Recently, a number of fluoxetine analogues were synthesized, especially focusing on the substitution of N-methyl amine group through modifying the structure of the fluoxetine, N-methyl-3-[p-trifluoromethylphenoxy]-3-phenylpropylamine, widely used as an antidepressant. Among them, the pharmacological properties of FD-2, (R)-N-ethanol-3-(4-trifluorophenoxy)-3-phenyl propaneamine and FD-4, N-(R)-3-trifluorophenoxy-3-phenylpropane-imidazole with a higher affinity for the DA transporter were characterized in terms of dopamine transporter inhibition expecting for useful cocaine substitutes. Effects of the compounds on [H(3)]dopamine uptake, [I(125)]RTI-55 binding, and DA transporter-associated currents were examined with the ligand binding assays and voltage clamping technique in human embryonic kidney (HEK)-293 cells where the recombinant human DA transporter (hDAT) was stably expressed. Our results showed that (i) fluoxetine was potent in inhibiting both the uptake of [H(3)]DA (IC(50) = 0.21 +/- 0.032 mM, n = 3) and the [I(125)]RTI-55 binding (IC(50) = 0.23 +/- 0.012 mM, n = 10); (ii) N-methyl amine substituted fluoxetine analogues, FD-2 and FD-4 were equally or more potent than fluoxetine itself in terms of inhibition of [H(3)]DA uptake (IC50 FD-2: 0.077 +/- 0.0032 mM (n = 3); FD-4: 0.26 +/- 0.13 mM (n = 3), inhibition of [I(125)]RTI-55 binding, and reduction in DA transporter-associated currents, suggesting that these analogues could be a new class of dopamine transporter inhibitors.
Archives of Pharmacal Research 12/2009; 32(12):1663-71. · 1.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: Liver plays a major role in regulating energy homeostasis in mammals. During feeding conditions, excessive glucose is converted into a preferred storage form of energy sources as triacylglycerol in liver via a collective metabolic pathway termed lipogenesis. Sterol regulatory element-binding protein 1c is a master regulator for this process by activating number of enzyme genes, such as Fasn or Acaca, that are involved in this pathway at the transcriptional level. Here we show that the salt-inducible kinase (SIK) family of proteins regulates the hepatic lipogenesis by modulating SREBP-1c activity. Overexpression of SIK1 inhibits hepatic expression of lipogenic genes, such as Fasn, whereas knockdown of SIK1 in liver greatly enhances their expression. Regulation of the Fasn gene by SIK kinases is mediated at the level of transcription via phosphorylation and inactivation of nuclear SREBP-1c. Among candidate sites for SIK-dependent regulation of SREBP-1c, the serine 329 residue is shown to be a critical regulatory site for SIK-mediated repression of SREBP-1c activity by in vitro kinase assay and reverse transcription-PCR analysis in primary hepatocytes. Finally, reduced hepatic triacylglycerol levels and lipogenic gene expression by adenoviral SIK1 transgenic expression are restored to normal levels by co-infection of mutant SREBP-1c, suggesting that SIK-dependent regulation of hepatic lipogenesis is indeed mediated through the phosphorylation of SREBP-1c in vivo. The process for the development of nonalcoholic fatty liver involves de novo lipogenesis via the activation of SREBP-1c. Modulation of SREBP-1c activity by SIK proteins would provide an attractive means for the regulation of such diseases.
Journal of Biological Chemistry 03/2009; 284(16):10446-52. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Enlarged or giant mitochondria have often been documented in aged tissues although their role and underlying mechanism remain unclear. We report here how highly elongated giant mitochondria are formed in and related to the senescent arrest. The mitochondrial morphology was progressively changed to a highly elongated form during deferoxamine (DFO)-induced senescent arrest of Chang cells, accompanied by increase of intracellular ROS level and decrease of mtDNA content. Interestingly, under exposure to subcytotoxic doses of H2O2 (200 microM), about 65% of Chang cells harbored elongated mitochondria with senescent phenotypes whereas ethidium bromide (EtBr) (50 ng/ml) only reformed the cristae structure. Elongated giant mitochondria were also observed in TGF beta1- or H2O2-induced senescent Mv1Lu cells and in old human diploid fibroblasts (HDFs). In all senescent progresses employed in this study Fis1 protein, a mitochondrial fission modulator, was commonly downexpressed. Overexpression of YFP-Fis1 reversed both mitochondrial elongation and appearance of senescent phenotypes induced by DFO, implying its critical involvement in the arrest. Finally, we found that direct induction of mitochondrial elongation by blocking mitochondrial fission process with Fis1-DeltaTM or Drp1-K38A was sufficient to develop senescent phenotypes with increased ROS production. These data suggest that mitochondrial elongation may play an important role as a mediator in stress-induced premature senescence.
Journal of Cellular Physiology 12/2006; 209(2):468-80. · 4.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidation-reduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex.
[show abstract][hide abstract] ABSTRACT: Transforming growth factor beta1 (TGF beta1) is a well-characterized cytokine that suppresses epithelial cell growth. We report here that TGF beta1 arrested lung epithelial Mv1Lu cells at G1 phase of the cell cycle with acquisition of senescent phenotypes in the presence of 10% serum, whereas it gradually induced apoptosis with lower concentrations of serum. The senescent arrest was accompanied by prolonged generation of reactive oxygen species (ROS) and persistent disruption of mitochondrial membrane potential (DeltaPsim). We demonstrated that the sustained ROS overproduction was derived from mitochondrial respiratory defect via decreased complex IV activity and was involved in the arrest. Moreover, we verified that hepatocyte growth factor released Mv1Lu cells from the arrest by protecting mitochondrial respiration, thereby preventing both the DeltaPsim disruption and the ROS generation. Our present results suggest the TGF beta1-induced senescent arrest as another plausible mechanism to suppress cellular growth in vivo and provide a new biochemical association between the mitochondrial functional defects and the cytokine-induced senescent arrest, emphasizing the importance of maintenance of mitochondrial function in cellular protection from the arrest.
[show abstract][hide abstract] ABSTRACT: When cells are deprived of iron, their growth is invariably inhibited. However, the mechanism involved remains largely unclear. Recently, we have reported that subcytotoxic concentration of deferoxamine mesylate (DFO), an iron chelator, specifically inhibited transition of Chang cell, a normal hepatocyte cell line, from G1 to S phase, which was accompanied by irreversible appearance of senescent biomarkers. To investigate factors responsible for the irreversible arrest, we examined mitochondrial activities because they require several irons for their proper structure and function. After exposure to 1 M DFO, total cellular ATP level was irreversibly decreased with concurrent disruption of mitochondrial membrane potential (DeltaPsim), implying that it might be one of the crucial factors involved in the arrest. DFO did not directly inhibit the mitochondrial respiratory activities in vitro. Among the respiratory activities, complex II activity was specifically inhibited through a down-regulation of the expression of its iron-sulfur subunit. We also observed that mitochondrial morphology was drastically changed to highly elongated form. Our results suggest that mitochondrial function is sensitive to cellular iron level and iron deprivation might be involved in inducing the senescent arrest. In addition, complex II, which is a part of both oxidative phosphorylation and the Krebs cycle, could be one of the critical factors that regulate mitochondrial function by responding to iron levels.
Annals of the New York Academy of Sciences 05/2004; 1011:123-32. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mitochondria play a pivotal role as an ATP generator in aerobically growing cells, and their defects have long been implicated in the cellular aging process, although its detailed underlying mechanisms remain unclear. Recently, we found that, in the cellular senescent process of Chang cells induced by desferroxamine mesylate, an iron chelator, a significant decrease of intracellular ATP level was accompanied by decline in complex II activity, which preceded acquisition of the senescent phenotype. In the present study, we investigated the mechanism of how the mitochondrial ATP productivity was damaged by iron chelation and how complex II defect was involved in the senescent arrest. The ATP loss was irreversible and accompanied by sustained collapse of mitochondrial membrane potential (Delta psi m), but the ATP loss itself did not seem to be essential in progression to the senescent arrest. The Delta psi m disruption was due to decreased mitochondrial respiration, which was primarily associated with the defective complex II activity. Furthermore, we found that the declined activity of complex II was mainly due to down-regulation of protein expression of the iron-sulfur subunit, which was associated with the irreversibility of the arrest. Finally, we demonstrated that specific inhibition of complex II with 2-thenoyltrifluoroacetone induced overall delay of the cell cycle, suggesting that the delayed arrest by desferroxamine mesylate might be in part due to inhibition of complex II activity. Taken together, our results suggest that complex II might be considered as one of the primary factors to regulate mitochondrial respiratory function by responding to the cellular iron level, thereby influencing cellular growth.
Journal of Biological Chemistry 01/2004; 278(51):51577-86. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated beta-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor beta1 (TGF-beta1) mRNA. When neutralized with an anti-(TGF-beta1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-beta1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-beta1, but was independent of p53.