👤 Hyun Eun Kim

Obese, insulin-resistant states are characterized by a paradoxical pathogenic condition in which the liver appears to be selectively insulin resistant. Specifically, insulin fails to suppress glucose production, yet successfully stimulates de novo lipogenesis. The mechanisms underlying this dysregulation remain controversial. Here, we hypothesized that carbohydrate-responsive element-binding protein (ChREBP), a transcriptional activator of glycolytic and lipogenic genes, plays a central role in this paradox. Administration of fructose increased hepatic hexose-phosphate levels, activated ChREBP, and caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hepatic steatosis in mice. Activation of ChREBP was required for the increased expression of glycolytic and lipogenic genes as well as glucose-6-phosphatase (G6pc) that was associated with the effects of fructose administration. We found that fructose-induced G6PC activity is a major determinant of hepatic glucose production and reduces hepatic glucose-6-phosphate levels to complete a homeostatic loop. Moreover, fructose activated ChREBP and induced G6pc in the absence of Foxo1a, indicating that carbohydrate-induced activation of ChREBP and G6PC dominates over the suppressive effects of insulin to enhance glucose production. This ChREBP/G6PC signaling axis is conserved in humans. Together, these findings support a carbohydrate-mediated, ChREBP-driven mechanism that contributes to hepatic insulin resistance. Show less

Nonalcoholic fatty liver disease (NAFLD) is a rapidly emerging global health-problem. NAFLD encompasses a range of conditions associated with hepatic steatosis, aberrant accumulation of fat in hepatocytes. Although obesity and metabolic syndrome are considered to have a strong association with NAFLD, genetic factors that predispose liver to NAFLD and molecular mechanisms by which excess hepatic lipid develops remain largely unknown. We report that the transcription cofactor CRTC1 confers broad spectrum protection against hepatic steatosis development. CRTC1 directly interferes with the expression of genes regulated by lipogenic transcription factors, most prominently liver x receptor α (LXRα). Accordingly, Crtc1 deficient mice develop spontaneous hepatic steatosis in young age. As a cyclic AMP effector, CRTC1 mediates anti-steatotic effects of calorie restriction (CR). Notably, CRTC1 also mediates anti-lipogenic effects of bile acid signaling, whereas it is negatively regulated by miR-34a, a pathogenic microRNA upregulated in a broad spectrum of NAFLD. These patterns of gene function and regulation of CRTC1 are distinct from other CR-responsive proteins, highlighting critical protective roles that CRTC1 selectively plays against NAFLD development, which in turn provides novel opportunities for selectively targeting beneficial therapeutic effects of CR. Show less

Melanogenesis is a physiological process that results in the synthesis of melanin pigments, which play a crucial protective role against skin photocarcinogenesis. We investigated the effects of a Polygoni Multiflori Ramulus extract on melanogenesis and isolated emodin from Polygoni Multiflori as an active compound. In addition, the possible mechanisms of action were examined. We found that emodin inhibited both melanin content and tyrosinase activity concentration and time dependently. Tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 mRNA levels decreased following emodin treatment. However, while the mRNA levels of microphthalmia-associated transcription factor (MITF) were not affected by emodin, emodin reduced MITF protein levels. Furthermore, expression of the liver X-receptor (LXR) α gene, but not the LXR β gene was upregulated by emodin. Moreover, emodin regulated melanogenesis by promoting degradation of the MITF protein by upregulating the LXR α gene. The emodin effects on MITF was found to be mediated by phosphorylation of p42/44 MAPK. Taken together, these findings indicate that the inhibition of melanogenesis by emodin occurs through reduced MITF protein expression, which is mediated by upregulation of the LXR α gene and suggest that emodin may be useful as a hyperpigmentation inhibitor. Show less

This is the first study to identify common genetic factors associated with the basal metabolic rate (BMR) and body mass index (BMI) in obese Korean women including overweight. This will be a basic study for future research of obese gene-BMR interaction. The experimental design was 2 by 2 with variables of BMR and BMI. A genome-wide association study (GWAS) of single nucleotide polymorphisms (SNPs) was conducted in the overweight and obesity (BMI > 23 kg/m(2)) compared to the normality, and in women with low BMR (< 1426.3 kcal/day) compared to high BMR. A total of 140 SNPs reached formal genome-wide statistical significance in this study (P < 1 × 10(-4)). Surveys to estimate energy intake using 24-h recall method for three days and questionnaires for family history, a medical examination, and physical activities were conducted. We found that two NRG3 gene SNPs in the 10q23.1 chromosomal region were highly associated with BMR (rs10786764; P = 8.0 × 10(-7), rs1040675; 2.3 × 10(-6)) and BMI (rs10786764; P = 2.5 × 10(-5), rs10786764; 6.57 × 10(-5)). The other genes related to BMI (HSD52, TMA16, MARCH1, NRG1, NRXN3, and STK4) yielded P <10 × 10(-4). Five new loci associated with BMR and BMI, including NRG3, OR8U8, BCL2L2-PABPN1, PABPN1, and SLC22A17 were identified in obese Korean women (P < 1 × 10(-4)). In the questionnaire investigation, significant differences were found in the number of starvation periods per week, family history of stomach cancer, coffee intake, and trial of weight control in each group. We discovered several common BMR- and BMI-related genes using GWAS. Although most of these newly established loci were not previously associated with obesity, they may provide new insights into body weight regulation. Our findings of five common genes associated with BMR and BMI in Koreans will serve as a reference for replication and validation of future studies on the metabolic rate. Show less

Milk-related traits (milk yield, fat and protein) have been crucial to selection of Holstein. It is essential to find the current selection trends of Holstein. Despite this, uncovering the current trends of selection have been ignored in previous studies. We suggest a new formula to detect the current selection trends based on single nucleotide polymorphisms (SNP). This suggestion is based on the best linear unbiased prediction (BLUP) and the Fisher's fundamental theorem of natural selection both of which are trait-dependent. Fisher's theorem links the additive genetic variance to the selection coefficient. For Holstein milk production traits, we estimated the additive genetic variance using SNP effect from BLUP and selection coefficients based on genetic variance to search highly selective SNPs. Through these processes, we identified significantly selective SNPs. The number of genes containing highly selective SNPs with p-value <0.01 (nearly top 1% SNPs) in all traits and p-value <0.001 (nearly top 0.1%) in any traits was 14. They are phosphodiesterase 4B (PDE4B), serine/threonine kinase 40 (STK40), collagen, type XI, alpha 1 (COL11A1), ephrin-A1 (EFNA1), netrin 4 (NTN4), neuron specific gene family member 1 (NSG1), estrogen receptor 1 (ESR1), neurexin 3 (NRXN3), spectrin, beta, non-erythrocytic 1 (SPTBN1), ADP-ribosylation factor interacting protein 1 (ARFIP1), mutL homolog 1 (MLH1), transmembrane channel-like 7 (TMC7), carboxypeptidase X, member 2 (CPXM2) and ADAM metallopeptidase domain 12 (ADAM12). These genes may be important for future artificial selection trends. Also, we found that the SNP effect predicted from BLUP was the key factor to determine the expected current selection coefficient of SNP. Under Hardy-Weinberg equilibrium of SNP markers in current generation, the selection coefficient is equivalent to 2*SNP effect. Show less

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius. Show less

The class III phosphoinositide 3-kinase (PI3K) Vps34 (also known as PIK3C3 in mammals) produces phosphatidylinositol 3-phosphate [PI(3)P] on both early and late endosome membranes to control membrane dynamics. We used Vps34-deficient cells to delineate whether Vps34 has additional roles in endocytic trafficking. In Vps34 Show less

As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Show less

Autophagy is a lysosome-dependent degradation pathway that maintains cellular homeostasis in response to a variety of cellular stresses. Accumulating reports based on animal models have indicated the importance of this catabolic program in many human pathophysiological conditions, including diabetes, neurodegenerative diseases, aging, and cancers. Therefore, autophagy has been highlighted as a novel therapeutic target with a wide range of beneficial effects on human diseases. Here, we review the recent advances of our knowledge toward autophagy, as well as the efforts for developing autophagy modulators. Areas covered: The relevant patents (published at 2012-2015) and the research literature claiming the pharmacological modulation of autophagy are reviewed. Also, their molecular mechanisms and potential therapeutic utilities are discussed. Expert opinion: Considering the molecular machinery involved in autophagy induction, the targeting of autophagy-specific protein is very important to design the therapeutic interventions for specifically treating a variety of autophagy-associated disorders. Many patents and the research literature described in this review have shown promising applications of the relevant autophagy modulators for cancer or neurodegeneration treatments, a few of which are already being considered for clinical evaluation. However, most patents have claimed the modulators of autophagy with little information regarding their mechanisms of action. To design highly potent therapeutics, further work, such as developing compounds that specifically target the autophagy-specific machinery, are required. Show less

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation. Show less

Ultraviolet radiation resistance-associated gene product (UVRAG) was originally identified as a protein involved in cellular responses to UV irradiation. Subsequent studies have demonstrated that UVRAG plays as an important role in autophagy, a lysosome-dependent catabolic program, as a part of a pro-autophagy PIK3C3/VPS34 lipid kinase complex. Several recent studies have shown that UVRAG is also involved in autophagy-independent cellular functions, such as DNA repair/stability and vesicular trafficking/fusion. Here, we examined the UVRAG protein interactome to obtain information about its functional network. To this end, we screened UVRAG-interacting proteins using a tandem affinity purification method coupled with MALDI-TOF/MS analysis. Our results demonstrate that UVRAG interacts with various proteins involved in a wide spectrum of cellular functions, including genome stability, protein translational elongation, protein localization (trafficking), vacuole organization, transmembrane transport as well as autophagy. Notably, the interactome list of high-confidence UVRAG-interacting proteins is enriched for proteins involved in the regulation of genome stability. Our systematic UVRAG interactome analysis should provide important clues for understanding a variety of UVRAG functions. Show less

Skeletal muscle regeneration occurs continuously to repair muscle damage incurred during normal activity and in chronic disease or injury. Herein, we report that A-kinase anchoring protein 6 (AKAP6) is important for skeletal myoblast differentiation and muscle regeneration. Compared with unstimulated skeletal myoblasts that underwent proliferation, differentiated cells show significant stimulation of AKAP6 expression. AKAP6 knockdown with siRNA effectively halts the formation of myotubes and decreases the expression of the differentiation markers myogenin and myosin heavy chain. When shAKAP6-lentivirus is delivered to mice with cardiotoxin (CTX)-induced muscle injury, muscle regeneration is impaired compared with that of mice injected with control shMock-lentivirus. The motor functions of mice infected with shAKAP6-lentivirus (CTX+shAK6) are significantly worse than those of mice infected with shMock-lentivirus (CTX+shMock). Mechanistic analysis showed that AKAP6 promotes myogenin expression through myocyte enhancer factor 2A (MEF2A). Notably, myogenin increases AKAP6 expression as well. The results of chromatin immunoprecipitation and luciferase assays showed that myogenin binds to an E-box site on the AKAP6 promoter. Taken together, our findings demonstrate a novel interplay between AKAP6 and myogenin, and we suggest that AKAP6 is an important regulator of myoblast differentiation, myotube formation, and muscle regeneration. Show less

Genome-wide association studies have been used extensively to identify genetic variants linked to metabolic syndrome (MetS), but most of them have been conducted in non-Asian populations. This study aimed to evaluate the association between MetS and previously studied single nucleotide polymorphisms (SNPs), and their interaction with health-related behavior in Korean men. Seventeen SNPs were genotyped and their association with MetS and its components was tested in 1193 men who enrolled in the study at Seoul National University Hospital. We found that rs662799 near APOA5 and rs769450 in APOE had significant association with MetS and its components. The SNP rs662799 was associated with increased risk of MetS, elevated triglyceride (TG) and low levels of high-density lipoprotein, while rs769450 was associated with a decreased risk of TG. The SNPs showed interactions between alcohol drinking and physical activity, and TG levels in Korean men. We have identified the genetic association and environmental interaction for MetS in Korean men. These results suggest that a strategy of prevention and treatment should be tailored to personal genotype and the population. Show less

Previous studies have indicated that supplementation with probiotics might improve lipid metabolism. The objective of the study was to evaluate the effect of supplementation with probiotic strains Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on triglyceride (TG) and apolipoprotein A-V (apo A-V) levels. A randomized, double-blinded, placebo-controlled study was conducted with 128 non-diabetic subjects with hypertriglyceridemia. Over a 12-week test period, the probiotic group consumed 2 g/day of a powdered supplement containing L. curvatus HY7601 and L. plantarum KY1032, whereas the placebo group consumed a powder lacking probiotics. After the treatment, the probiotic group showed an 18.3% (P < 0.001) reduction in TGs and increases of 21.1% (P = 0.001) and 15.6% (P < 0.001) in the apo A-V and LDL particle size, respectively. The probiotic group had a significant reduction in TGs (P = 0.040) and increases in the plasma apo A-V (P = 0.003) and LDL particle size (P < 0.001) compared with the placebo group. In the probiotic group, the reduction in the TG levels was negatively correlated with changes in the apo A-V and baseline TGs, regardless of the APOA5 -1131T > C genotype. The consumption of two probiotic strains for 12 weeks reduced TGs and increased the apo A-V and LDL particle size in hypertriglyceridemic subjects. This effect was more pronounced in subjects with higher levels of fasting TGs regardless of their APOA5 -1131T > C genotype. Show less

This study evaluated the triglyceride (TG)-lowering effects of consuming dual probiotic strains of Lactobacillus curvatus (L. curvatus) HY7601 and Lactobacillus plantarum (L. plantarum) KY1032 on the fasting plasma metabolome. A randomized, double-blind, placebo-controlled study was conducted on 92 participants with hypertriglyceridemia but without diabetes. Over a 12-week testing period, the probiotic group consumed 2 g of powder containing 5 × 10(9) colony-forming units (cfu) of L. curvatus HY7601 and 5 × 10(9) cfu of L. plantarum KY1032 each day, whereas the placebo group consumed the same product without probiotics. Fasting plasma metabolomes were profiled using UPLC-LTQ-Orbitrap MS. After 12 weeks of treatment, the probiotic group displayed a 20% reduction (p = 0.001) in serum TGs and 25% increases (p=0.001) in apolipoprotein A-V (apoA-V). At the 12-week follow-up assessment, the following 11 plasma metabolites were significantly reduced in the probiotic group than the placebo group: palmitoleamide, palmitic amide, oleamide, and lysophosphatidyl choline (lysoPC) containing C14:0, C16:1, C16:0, C17:0, C18:3, C18:2, C18:1, and C20:3. In the probiotic group, changes (▵) in TG were negatively correlated with ▵ apoA-V, which was positively correlated with ▵ FFA. In addition, ▵ FFA was strongly and positively correlated with ▵ lysoPCs in the probiotic group but not the placebo group. The triglyceride-lowering effects of probiotic supplementation, partly through elevated apoA-V, in borderline to moderate hypertriglyceridemic subjects showed reductions in plasma metabolites, fatty acid primary amides and lysoPCs (NCT02215694; http://www.clinicaltrials.gov). Clinical trials: NCT02215694; http://www.clinicaltrials.gov. Show less

SUMO-modification of nuclear proteins has profound effects on gene expression. However, non-toxic chemical tools that modulate sumoylation in cells are lacking. Here, to identify small molecule sumoylation inhibitors we developed a cell-based screen that focused on the well-sumoylated substrate, human Liver Receptor Homolog-1 (hLRH-1, NR5A2). Our primary gene-expression screen assayed two SUMO-sensitive transcripts, APOC3 and MUC1, that are upregulated by SUMO-less hLRH-1 or by siUBC9 knockdown, respectively. A polyphenol, tannic acid (TA) emerged as a potent sumoylation inhibitor in vitro (IC50 = 12.8 µM) and in cells. TA also increased hLRH-1 occupancy on SUMO-sensitive transcripts. Most significantly, when tested in humanized mouse primary hepatocytes, TA inhibits hLRH-1 sumoylation and induces SUMO-sensitive genes, thereby recapitulating the effects of expressing SUMO-less hLRH-1 in mouse liver. Our findings underscore the benefits of phenotypic screening for targeting post-translational modifications, and illustrate the potential utility of TA for probing the cellular consequences of sumoylation. Show less

Cellular proteins are degraded in either proteasomes or lysosomes depending on the types of ubiquitin chains that covalently modify them. It is not known whether the choice between these two pathways is physiologically regulated. The Lys48-polyubiquitin chain is the major signal directing proteins for degradation in proteasomes. Here, we report the unexpected finding that canonical Wnt signaling translocates some K48-linked polyubiquitinated proteins to the endolysosomal pathway. Proteasomal target proteins, such as b-catenin, Smad1, and Smad4, were targeted into endolysosomes in a process dependent on GSK3 activity. Relocalization was also dependent on Axin1 and the multivesicular body (MVB) proteins HRS/Vps27 and Vps4. The Wnt-induced accumulation of K48-linked polyubiquitinated proteins in endolysosomal organelles was accompanied by a transient decrease in cellular levels of free mono-ubiquitin, which may contribute to Wnt-regulated stabilization of proteins (Wnt/ STOP). We conclude that Wnt redirects Lys48-polyubiquitinated proteins that are normally degraded in proteasomes to endolysosomes. Show less

Ends of human chromosomes consist of the six nucleotide repeat d[pTTAGGG]n known as telomeric DNA, which protects chromosomes. We have previously shown that the DHX36 gene product, G4 Resolvase 1 (G4R1), binds parallel G-quadruplex (G4) DNA with an unusually tight apparent Kd. Recent work associates G4R1 with the telomerase holoenzyme, which may allow it to access telomeric G4-DNA. Here we show that G4R1 can tightly bind telomeric G4-DNA, and in the context of the telomeric sequence, we determine length, sequence, and structural requirements sufficient for tight G4R1 telomeric binding. Specifically, G4R1 binds telomeric DNA in the K+-induced "3+1" G4-topology with an apparent Kd = 10 ± 1.9 pM, a value similar as previously found for binding to unimolecular parallel G4-DNA. G4R1 binds to the Na+-induced "2+2" basket G4-structure formed by the same DNA sequence with an apparent Kd = 71 ± 2.2 pM. While the minimal G4-structure is not sufficient for G4R1 binding, a 5' G4-structure with a 3' unstructured tail containing a guanine flanked by adenine(s) is sufficient for maximal binding. Mutations directed to disrupt G4-structure similarly disrupt G4R1 binding; secondary mutations that restore G4-structure also restore G4R1 binding. We present a model showing that a replication fork disrupting a T-loop could create a 5' quadruplex with an opened 3'tail structure that is recognized by G4R1. Show less

Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1/2 activity both in resting and LPS-stimulated microglia. In turn, DUSP6 suppressed the basal phosphorylation of ERK1/2, but exerted no suppressive effect on LPS-induced phosphorylation. Although DUSP6 is acknowledged as a negative regulator of the ERK1/2 pathway, such roles of DUSP6 need to be examined further in activated microglia. Show less

Brown fat has been highlight as a new therapeutic target for treatment of obesity and diabetes. However, molecular mechanism underlying brown adipogenesis are not fully understood. Here, we identified that MAP kinase phosphatase 3 (MKP3) has a novel role as regulator of brown adipocyte differentiation. The expression of MKP3 was significantly decreased during the early stage(s) of brown adipocyte differentiation in HIB-1B cells and primary cells. Ectopic expression of MKP3 led to reduced brown adipocyte differentiation, whereas depletion of MKP3 significantly enhanced the differentiation of primary brown preadipocytes. Consistently, we found an increased brown adipocyte differentiation in MKP3-null MEF cells. These inhibitory effects of MKP3 could be resulted via the temporal regulation of Erk activation. In recent, it was reported that MKP3 deficient mice are resistant to diet-induced obesity, and display enhanced energy expenditure. Taken together, we suggest that MKP3 could be an important factor in the regulation of brown adipocyte differentiation. Show less

In nonalcoholic fatty liver disease, hepatic gene expression and fatty acid (FA) composition have been reported independently, but a comprehensive gene expression profiling in relation to FA composition is lacking. The aim was to assess this relationship. In a cross-sectional study, hepatic gene expression (Illumina Microarray) was first compared among 20 patients with simple steatosis (SS), 19 with nonalcoholic steatohepatitis (NASH), and 24 healthy controls. The FA composition in hepatic total lipids was compared between SS and NASH, and associations between gene expression and FAs were examined. Gene expression differed mainly between healthy controls and patients (SS and NASH), including genes related to unsaturated FA metabolism. Twenty-two genes were differentially expressed between NASH and SS; most of them correlated with disease severity and related more to cancer progression than to lipid metabolism. Biologically active long-chain polyunsaturated FAs (PUFAs; eicosapentaenoic acid + docosahexaenoic acid, arachidonic acid) in hepatic total lipids were lower in NASH than in SS. This may be related to overexpression of FADS1, FADS2, and PNPLA3. The degree and direction of correlations between PUFAs and gene expression were different among SS and NASH, which may suggest that low PUFA content in NASH modulates gene expression in a different way compared with SS or, alternatively, that gene expression influences PUFA content differently depending on disease severity (SS versus NASH). Well-defined subjects with either healthy liver, SS, or NASH showed distinct hepatic gene expression profiles including genes involved in unsaturated FA metabolism. In patients with NASH, hepatic PUFAs were lower and associations with gene expression were different compared to SS. Show less

Human cord blood (CB) is enriched in circulating endothelial colony forming cells (ECFCs) that display high proliferative potential and in vivo vessel forming ability. Because Notch signaling is critical for embryonic blood vessel formation in utero, we hypothesized that Notch pathway activation may enhance cultured ECFC vasculogenic properties in vivo. In vitro ECFC stimulation with an immobilized chimeric Notch ligand (Delta-like1(ext-IgG)) led to significant increases in the mRNA and protein levels of Notch regulated Hey2 and EphrinB2 that were blocked by treatment with γ-secretase inhibitor addition. However, Notch stimulated preconditioning in vitro failed to enhance ECFC vasculogenesis in vivo. In contrast, in vivo co-implantation of ECFCs with OP9-Delta-like 1 stromal cells that constitutively expressed the Notch ligand delta-like 1 resulted in enhanced Notch activated ECFC-derived increased vessel density and enlarged vessel area in vivo, an effect not induced by OP9 control stromal implantation. This Notch activation was associated with diminished apoptosis in the exposed ECFC. We conclude that Notch pathway activation in ECFC in vivo via co-implanted stromal cells expressing delta-like 1 promotes vasculogenesis and augments blood vessel formation via diminishing apoptosis of the implanted ECFC. Show less

Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens. 468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens. An average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair). Whereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution. Show less

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance. Show less

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes. Show less

Liver X receptor (LXR) is a member of the nuclear receptor superfamily, and it regulates various biologic processes, including de novo lipogenesis, cholesterol metabolism, and inflammation. Selective inhibition of LXR may aid the treatment of nonalcoholic fatty liver diseases. In the present study, we evaluated the effects of three cinnamamide derivatives on ligand-induced LXRα activation and explored whether these derivatives could attenuate steatosis in mice. N-(4-trifluoromethylphenyl) 3,4-dimethoxycinnamamide (TFCA) decreased the luciferase activity in LXRE-tk-Luc-transfected cells and also suppressed ligand-induced lipid accumulation and expression of the lipogenic genes in murine hepatocytes. Furthermore, it significantly attenuated hepatic neutral lipid accumulation in a ligand-induced fatty liver mouse system. Modeling study indicated that TFCA inhibited activation of the LXRα ligand-binding domain by hydrogen bonding to Arg305 in the H5 region of that domain. It regulated the transcriptional control exerted by LXRα by influencing coregulator exchange; this process involves dissociation of the thyroid hormone receptor-associated proteins (TRAP)/DRIP coactivator and recruitment of the nuclear receptor corepressor. These results show that TFCA has the potential to attenuate ligand-induced lipogenesis and fatty liver by selectively inhibiting LXRα in the liver. Show less

20(S)-protopanaxatriol (PPT) is an aglycone of ginsenosides isolated from Panax ginseng and has several interesting activities, including anti-inflammatory and anti-oxidative stress effects. Herein, PPT was identified as an inhibitor against the ligand-dependent transactivation of liver X receptor α (LXRα) using a Gal4-TK-luciferase reporter system. LXRα is a transcription factor of nuclear hormone receptor family and stimulates the transcription of many metabolic genes, such as lipogenesis- or reverse cholesterol transport (RCT)-related genes. Quantitative RT-PCR analysis showed that PPT inhibited the LXRα-dependent transcription of lipogenic genes, such as sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase, and stearoyl CoA desaturase 1. These inhibitory effects of PPT are, at least in part, a consequence of the reduced recruitment of RNA polymerase II to the LXR response element (LXRE) of the SREBP-1c promoter. Furthermore, LXRα-dependent triglyceride accumulation in primary mouse hepatocytes was significantly reduced by PPT. Interestingly, PPT did not inhibit the LXRα-dependent transcription of ABCA1, a crucial LXRα target gene involved in RCT. Chromatin immunoprecipitation assays revealed that PPT repressed recruitment of the lipogenic coactivator TRAP80 to the SREBP-1c LXRE, but not the ABCA1 LXRE. Overall, these data suggest that PPT has selective inhibitory activity against LXRα-mediated lipogenesis, but not LXRα-stimulated RCT. Show less

The goals of this study were (1) to examine the effects of Cyperus rotundus (CR) rhizome on cellular lipogenesis and non-alcoholic/diet-induced fatty liver disease, and (2) to elucidate the molecular mechanism behind its actions. The present investigation showed that the hexane fraction of CR rhizome (CRHF) reduced the elevated transcription levels of sterol regulatory element binding protein-1c (SREBP-1c) in primary hepatocytes following exposure to the liver X receptor α (LXRα) agonist. The SREBP-1c gene is a master regulator of lipogenesis and a key target of LXRα. CRHF inhibited not only the LXRα-dependent activation of the synthetic LXR response element (LXRE) promoter, but also the activation of the natural SREBP-1c promoter. Moreover, CRHF decreased (a) the recruitment of RNA polymerase II to the LXRE of the SREBP-1c gene; (b) the LXRα-dependent up-regulation of various lipogenic genes; and (c) the LXRα-mediated accumulation of triglycerides in primary hepatocytes. Furthermore, CRHF ameliorated fatty liver disease and reduced the expression levels of hepatic lipogenic genes in high sucrose diet (HSD)-fed mice. Interestingly, CRHF did not affect the expression of ATP-binding cassette transporter A1, another important LXR target gene that is required for reverse cholesterol transport (RCT) and protects against atherosclerosis. Taken together, these results suggest that CRHF might be a novel therapeutic remedy for fatty liver disease through the selective inhibition of the lipogenic pathway. Show less

Irisin is a newly identified myokine related to exercise and the browning of white fat. Recently, it was reported that irisin serum levels are associated with intrahepatic triglyceride content, suggesting that it might have an important role in the liver. The aim of this study was to determine the role of irisin in hepatocytes. Specifically, the effect of recombinant irisin on palmitic acid (PA)-induced lipogenesis and its related signal pathways were examined in AML12 cells and mouse primary hepatocytes. In the present study, we observed the presence of irisin inside the cells in response to the treatment of recombinant irisin by flow cytometry and cell imaging technique. Recombinant irisin significantly inhibited the PA-induced increase in lipogenic markers ACC and FAS at the mRNA and protein levels, and prevented the PA-induced lipid accumulation in hepatocytes. Additionally, irisin inhibited the PA-induced increase in the expression, nuclear localization, and transcriptional activities of the master regulators of lipogenesis (LXRα and SREBP-1c). Moreover, irisin attenuated PA-induced oxidative stress, which was confirmed by measuring the expression of inflammatory markers (NFκB, COX-2, p38 MAPK, TNF, IL-6) and superoxide indicator (dihydroethidium). The preventive effects of irisin against lipogenesis and oxidative stress were mediated by the inhibition of protein arginine methyltransferase-3 (PRMT3). These findings suggested that irisin might have a beneficial role in the prevention of hepatic steatosis by altering the expression of lipogenic genes and attenuating oxidative stress in a PRMT3 dependent manner. Show less