Role of Carbohydrate response element-binding protein in mediating dexamethasone-induced glucose transporter 5 expression in Caco-2BBE cells and during the developmental phase in mice.

Glucose transporter 5 (GLUT5), the main fructose transporter in mammals, is primarily responsible for absorbing dietary fructose in the small intestine. The expression of this intestinal gene significantly increases in response to developmental and dietary cues that reach the glucocorticoid receptor and carbohydrate response element-binding protein (ChREBP), respectively. Our study demonstrates that ChREBP is involved in the dexamethasone (Dex)-induced expression of GLUT5 in Caco-2BBE cells and the small intestine of both wild-type and ChREBP-knockout mice. Dex, a glucocorticoid, demonstrated an increase in GLUT5 mRNA levels in a dose- and time-dependent manner. While the overexpression of ChREBP moderately increased GLUT5 expression, its synergistic increase in the presence of Dex was noteworthy, whereas the suppression of ChREBP significantly reduced Dex-induced GLUT5 expression. Dex did not increase ChREBP protein levels but facilitated its nuclear translocation, thereby increasing the activity of the GLUT5 promoter. In vivo experiments conducted on 14-day-old mice pups treated with Dex for three days revealed that only wild-type mice (not ChREBP-knockout mice) exhibited Dex-mediated Glut5 gene induction, which further supports the role of ChREBP in regulating GLUT5 expression. Collectively, our results provide insights into the molecular mechanisms involved in the regulation of GLUT5 expression in response to developmental and dietary signals mediated by glucocorticoids and ChREBP. General significance: The transcription factor ChREBP is important for Dex-mediated Glut5 gene expression in the small intestine.

Targeting E3 ubiquitin ligases and their adaptors as a therapeutic strategy for metabolic diseases.

Posttranslational modification of proteins via ubiquitination determines their activation, translocation, dysregulation, or degradation. This process targets a large number of cellular proteins, affecting all biological pathways involved in the cell cycle, development, growth, and differentiation. Thus, aberrant regulation of ubiquitination is likely associated with several diseases, including various types of metabolic diseases. Among the ubiquitin enzymes, E3 ubiquitin ligases are regarded as the most influential ubiquitin enzymes due to their ability to selectively bind and recruit target substrates for ubiquitination. Continued research on the regulatory mechanisms of E3 ligases and their adaptors in metabolic diseases will further stimulate the discovery of new targets and accelerate the development of therapeutic options for metabolic diseases. In this review, based on recent discoveries, we summarize new insights into the roles of E3 ubiquitin ligases and their adaptors in the pathogenesis of metabolic diseases by highlighting recent evidence obtained in both human and animal model studies.

Kctd17-mediated Chop degradation promotes adipogenic differentiation.

Obesity is commonly associated with excessive adipogenesis, a process by which preadipocytes undergo differentiation into mature adipocytes; however, the mechanisms underlying adipogenesis are not completely understood. Potassium channel tetramerization domain-containing 17 (Kctd17) belongs to the Kctd superfamily and act as a substrate adaptor of the Cullin 3-RING E3 ubiquitin ligase, which is involved in a wide variety of cell functions. However, its function in the adipose tissue remains largely unknown. Here, we found that Kctd17 expression levels were increased in white adipose tissue, especially in adipocytes, in obese mice compared to lean control mice. Gain or loss of function of Kctd17 in preadipocytes inhibited or promoted adipogenesis, respectively. Furthermore, we found that Kctd17 bound to C/EBP homologous protein (Chop) to target it for ubiquitin-mediated degradation, and this process was likely associated with increased adipogenesis. In conclusion, these data suggest that Kctd17 plays an important role in adipogenesis and can be a novel therapeutic target for obesity.

Fructose malabsorption in ChREBP-deficient mice disrupts the small intestine immune microenvironment and leads to diarrhea-dominant bowel habit changes.

BACKGROUND: The mechanism by which incompletely absorbed fructose causes gastrointestinal symptoms is not fully understood. In this study, we investigated the immunological mechanisms of bowel habit changes associated with fructose malabsorption by examining Chrebp-knockout mice exhibiting defective fructose absorption. METHODS: Mice were fed a high-fructose diet (HFrD), and stool parameters were monitored. The gene expression in the small intestine was analyzed by RNA sequencing. Intestinal immune responses were assessed. The microbiota composition was determined by 16S rRNA profiling. Antibiotics were used to assess the relevance of microbes for HFrD-induced bowel habit changes. RESULTS: Chrebp-knockout (KO) mice fed HFrD showed diarrhea. Small-intestine samples from HFrD-fed Chrebp-KO mice revealed differentially expressed genes involved in the immune pathways, including IgA production. The number of IgA-producing cells in the small intestine decreased in HFrD-fed Chrebp-KO mice. These mice showed signs of increased intestinal permeability. Chrebp-KO mice fed a control diet showed intestinal bacterial imbalance, which the HFrD exaggerated. Bacterial reduction improved diarrhea-associated stool parameters and restored the decreased IgA synthesis induced in HFrD-fed Chrebp-KO mice. CONCLUSIONS: The collective data indicate that gut microbiome imbalance and disrupting homeostatic intestinal immune responses account for the development of gastrointestinal symptoms induced by fructose malabsorption.

Hepatocyte Kctd17 Inhibition Ameliorates Glucose Intolerance and Hepatic Steatosis Caused by Obesity-induced Chrebp Stabilization.

BACKGROUND & AIMS: Obesity predisposes to type 2 diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD), but underlying mechanisms are incompletely understood. Potassium channel tetramerization domain-containing protein 17 (Kctd17) levels are increased in livers from obese mice and humans. In this study, we investigated the mechanism of increased Kctd17 and whether it is causal to obesity-induced metabolic complications. METHODS: We transduced Rosa26-LSL-Cas9 knockin mice with AAV8-TBG-Cre (Control), AAV8-U6-Kctd17 sgRNA-TBG-Cre (L-Kctd17), AAV8-U6-Oga sgRNA-TBG-Cre (L-Oga), or AAV8-U6-Kctd17/Oga sgRNA-TBG-Cre (DKO). We fed mice a high-fat diet (HFD) and assessed for hepatic glucose and lipid homeostasis. We generated Kctd17, O-GlcNAcase (Oga), or Kctd17/Oga-knockout hepatoma cells by CRISPR-Cas9, and Kctd17-directed antisense oligonucleotide to test therapeutic potential in vivo. We analyzed transcriptomic data from patients with NAFLD. RESULTS: Hepatocyte Kctd17 expression was increased in HFD-fed mice due to increased Srebp1c activity. HFD-fed L-Kctd17 or Kctd17 antisense oligonucleotide-treated mice show improved glucose tolerance and hepatic steatosis, whereas forced Kctd17 expression caused glucose intolerance and hepatic steatosis even in lean mice. Kctd17 induced Oga degradation, resulting in increasing carbohydrate response element-binding protein (Chrebp) protein, so concomitant Oga knockout negated metabolic benefits of hepatocyte Kctd17 deletion. In patients with NAFLD, KCTD17 messenger RNA was positively correlated with expression of Chrebp target and other lipogenic genes. CONCLUSIONS: Srebp1c-induced hepatocyte Kctd17 expression in obesity disrupted glucose and lipid metabolism by stabilizing Chrebp, and may represent a novel therapeutic target for obesity-induced T2D and NAFLD.

Emerging roles of PHLPP phosphatases in metabolism.

Over the last decades, research has focused on the role of pleckstrin homology (PH) domain leucine-rich repeat protein phosphatases (PHLPPs) in regulating cellular signaling via PI3K/Akt inhibition. The PKB/Akt signaling imbalances are associated with a variety of illnesses, including various types of cancer, inflammatory response, insulin resistance, and diabetes, demonstrating the relevance of PHLPPs in the prevention of diseases. Furthermore, identification of novel substrates of PHLPPs unveils their role as a critical mediator in various cellular processes. Recently, researchers have explored the increasing complexity of signaling networks involving PHLPPs whereby relevant information of PHLPPs in metabolic diseases was obtained. In this review, we discuss the current knowledge of PHLPPs on the well-known substrates and metabolic regulation, especially in liver, pancreatic beta cell, adipose tissue, and skeletal muscle in relation with the stated diseases. Understanding the context-dependent functions of PHLPPs can lead to a promising treatment strategy for several kinds of metabolic diseases. [BMB Reports 2021; 54(9): 451-457].

Deletion of KLF10 Leads to Stress-Induced Liver Fibrosis upon High Sucrose Feeding.

Liver fibrosis is a consequence of chronic liver injury associated with chronic viral infection, alcohol abuse, and nonalcoholic fatty liver. The evidence from clinical and animal studies indicates that transforming growth factor-ß (TGF-ß) signaling is associated with the development of liver fibrosis. Krüppel-like factor 10 (KLF10) is a transcription factor that plays a significant role in TGF-ß-mediated cell growth, apoptosis, and differentiation. In recent studies, it has been reported to be associated with glucose homeostasis and insulin resistance. In the present study, we investigated the role of KLF10 in the progression of liver disease upon a high-sucrose diet (HSD) in mice. Wild type (WT) and Klf10 knockout (KO) mice were fed either a control chow diet or HSD (50% sucrose) for eight weeks. Klf10 KO mice exhibited significant hepatic steatosis, inflammation, and liver injury upon HSD feeding, whereas the WT mice exhibited mild hepatic steatosis with no apparent liver injury. The livers of HSD-fed Klf10 KO mice demonstrated significantly increased endoplasmic reticulum stress, oxidative stress, and proinflammatory cytokines. Klf10 deletion led to the development of sucrose-induced hepatocyte cell death both in vivo and in vitro. Moreover, it significantly increased fibrogenic gene expression and collagen accumulation in the liver. Increased liver fibrosis was accompanied by increased phosphorylation and nuclear localization of Smad3. Here, we demonstrate that HSD-fed mice develop a severe liver injury in the absence of KLF10 due to the hyperactivation of the endoplasmic reticulum stress response and CCAAT/enhance-binding protein homologous protein (CHOP)-mediated apoptosis of hepatocytes. The current study suggests that KLF10 plays a protective role against the progression of hepatic steatosis into liver fibrosis in a lipogenic state.

Interaction of BRAF-induced ETS factors with mutant TERT promoter in papillary thyroid cancer.

Synergistic effects of BRAFV600E and TERT promoter mutations on the poor clinical outcomes in papillary thyroid cancer (PTC) have been demonstrated. The potential mechanism of this phenomenon has been proposed: MAPK pathway activation by the BRAFV600E mutation may upregulate E-twenty six (ETS) transcription factors, increasing TERT expression by binding to the ETS-binding site generated by the TERT promoter mutation; however, it has not yet been fully proven. This article provides transcriptomic insights into the interaction between BRAFV600E and TERT promoter mutations mediated by ETS factors in PTC. RNA sequencing data on 266 PTCs from The Cancer Genome Atlas and 65 PTCs from our institute were analyzed for gene expression changes and related molecular pathways, and the results of transcriptomic analyses were validated by in vitro experiments. TERT mRNA expression was increased by the coexistence of BRAFV600E and TERT promoter mutations (fold change, 16.17; q-value = 7.35 × 10-12 vs no mutation). In the ETS family of transcription factors, ETV1, ETV4 and ETV5 were upregulated by the BRAFV600E/MAPK pathway activation. These BRAFV600E-induced ETS factors selectively bound to the mutant TERT promoter. The molecular pathways activated by BRAFV600E were further augmented by adding the TERT promoter mutation, and the pathways related to immune responses or adhesion molecules were upregulated by TERT expression. The mechanism of the synergistic effect between BRAFV600E and TERT promoter mutations on cancer invasiveness and progression in PTC may be explained by increased TERT expression, which may result from the BRAF-induced upregulation of several ETS transcription factors.

ChREBP deficiency leads to diarrhea-predominant irritable bowel syndrome.

OBJECTIVE: Fructose malabsorption is a common digestive disorder in which absorption of fructose in the small intestine is impaired. An abnormality of the main intestinal fructose transporter proteins has been proposed as a cause for fructose malabsorption. However the underlying molecular mechanism for this remains unclear. In this study, we investigated whether carbohydrate response element-binding protein (ChREBP) plays a role in intestinal fructose absorption through the regulation of genes involved in fructose transport and metabolism and ion transport. METHODS: Wild type (WT) and Chrebp knockout (KO) mice (6 or 8 weeks old) were fed a control diet (55% starch, 15% maltodextrin 10) or high-fructose diet (HFrD, 60% fructose, 10% starch) for 3-12 days. Body weight and food intake were measured, signs of fructose malabsorption were monitored, and the expression of genes involved in fructose transport/metabolism and ion transport was evaluated. Furthermore, transient transfection and chromatin immunoprecipitation were performed to show the direct interaction between ChREBP and carbohydrate response elements in the promoter of Slc2A5, which encodes the fructose transporter GLUT5. RESULTS: Chrebp KO mice fed the control diet maintained a constant body weight, whereas those fed a HFrD showed significant weight loss within 3-5 days. In addition, Chrebp KO mice fed the HFrD exhibited a markedly distended cecum and proximal colon containing both fluid and gas, suggesting incomplete fructose absorption. Fructose-induced increases of genes involved in fructose transport (GLUT5), fructose metabolism (fructokinase, aldolase B, triokinase, and lactate dehydrogenase), and gluconeogenesis (glucose-6-phosphatase and fructose-1,6-bisphosphatase) were observed in the intestine of WT but not of Chrebp KO mice. Moreover the Na+/H+ exchanger NHE3, which is involved in Na+ and water absorption in the intestine, was significantly decreased in HFrD-fed Chrebp KO mice. Consistent with this finding, the high-fructose diet-fed Chrebp KO mice developed severe diarrhea. Results of chromatin immunoprecipitation assays showed a direct interaction of ChREBP with the Glut5 promoter, but not the Nhe3 promoter, in the small intestine. Ectopic co-expression of ChREBP and its heterodimer partner Max-like protein X activated the Glut5 promoter in Caco-2BBE cells. CONCLUSIONS: ChREBP plays a key role in the dietary fructose transport as well as conversion into lactate and glucose through direct transcriptional control of genes involved in fructose transport, fructolysis, and gluconeogenesis. Moreover, ablation of Chrebp results in a severe diarrhea in mice fed a high-fructose diet, which is associated with the insufficient induction of GLUT5 in the intestine.

CRISPR-Cas9-mediated generation of obese and diabetic mouse models.

Mouse models of obesity (ob/ob) and diabetes (db/db) in which the leptin (Lep) and leptin receptor (Lepr) genes have been mutated, respectively, have contributed to a better understanding of human obesity and type 2 diabetes and to the prevention, diagnosis, and treatment of these metabolic diseases. In this study, we report the first CRISPR-Cas9-induced Lep and Lepr knockout (KO) mouse models by co-microinjection of Cas9 mRNA and sgRNAs that specifically targeted Lep or Lepr in C57BL/6J embryos. Our newly established Lep and Lepr KO mouse models showed phenotypic disorders nearly identical to those found in ob/ob and db/db mice, such as an increase in body weight, hyperglycemia, and hepatic steatosis. Thus, Cas9-generated Lep and Lepr KO mouse lines will be easier for genotyping, to maintain the lines, and to use for future obesity and diabetes research.

Bach2 represses the AP-1-driven induction of interleukin-2 gene transcription in CD4＋ T cells.

The transcription repressor Bach2 has been proposed as a regulator of T cell quiescence, but the underlying mechanism is not fully understood. Given the importance of interleukin-2 in T cell activation, we investigated whether Bach2 is a component of the network of factors that regulates interleukin-2 expression. In primary and transformed CD4＋ T cells, Bach2 overexpression counteracted T cell receptor/CD28- or PMA/ionomycin-driven induction of interleukin-2 expression, and silencing of Bach2 had the opposite effect. Luciferase and chromatin immunoprecipitation assays revealed that Bach2 binds to multiple Maf-recognition element-like sites on the interleukin-2 proximal promoter in a manner competitive with AP-1, and thereby represses AP-1-driven induction of interleukin-2 transcription. Thus, this study demonstrates that Bach2 is a direct repressor of the interleukin-2 gene in CD4＋ T cells during the immediate early phase of AP-driven activation, thereby playing an important role in the maintenance of immune quiescence in the steady state. [BMB Reports 2017; 50(9): 472-477].

Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c.

Elongation of very long chain fatty acids protein 6 (ELOVL6), a rate-limiting enzyme for the elongation of saturated and monounsaturated fatty acids with 12, 14, and 16 carbons, plays a key role in energy metabolism and insulin sensitivity. Hepatic Elovl6 expression is upregulated in the fasting-refeeding response and in leptin-deficient ob/ob mice. Mouse Elovl6 has been shown to be a direct target of sterol regulatory element binding protein-1 (SREBP-1) in response to insulin. In the present study, we demonstrated that mouse and human Elovl6 expression is under the direct transcriptional control of carbohydrate response element binding protein (ChREBP), a mediator of glucose-induced gene expression. Serial deletion and site-directed mutagenesis studies revealed functional carbohydrate response elements (ChoREs) in the mouse and human Elovl6 promoters and gel shift assays and chromatin immunoprecipitation assays confirmed the binding of ChREBP to the Elovl6-ChoRE sites. In addition, the ectopic co-expression of ChREBP and SREBP-1c in HepG2 cells synergistically stimulated Elovl6 promoter activity and this synergistic activation was abolished by mutating the Elovl6 promoter ChoREs. Taken together, these results suggest that the synergistic action of ChREBP and SREBP-1c is necessary for the maximal induction of Elovl6 expression in the liver.

Ursodeoxycholic acid decreases age-related adiposity and inflammation in mice.

Ursodeoxycholic acid (UDCA), a natural, hydrophilic nontoxic bile acid, is clinically effective for treating cholestatic and chronic liver diseases. We investigated the chronic effects of UDCA on age-related lipid homeostasis and underlying molecular mechanisms. Twenty-week-old C57BL/6 male and female mice were fed a diet with or without 0.3% UDCA supplementation for 25 weeks. UDCA significantly reduced weight gain, adiposity, hepatic triglyceride, and hepatic cholesterol without incidental hepatic injury. UDCA-mediated hepatic triglyceride reduction was associated with downregulated hepatic expression of peroxisome proliferator-activated receptor-γ, and of other genes involved in lipogenesis (Chrebp, Acaca, Fasn, Scd1, and Me1) and fatty acid uptake (Ldlr, Cd36). The inflammatory cytokines Tnfa, Ccl2, and Il6 were significantly decreased in liver and/or white adipose tissues of UDCA-fed mice. These data suggest that UDCA exerts beneficial effects on age-related metabolic disorders by lowering the hepatic lipid accumulation, while concurrently reducing hepatocyte and adipocyte susceptibility to inflammatory stimuli. [BMB Reports 2016; 49(2): 105-110].

Frequent premature ventricular complex is associated with left atrial enlargement in patients with normal left ventricular ejection fraction.

BACKGROUND: Premature ventricular complex (PVC) has been regarded as benign; however, when frequent, the arrhythmia can induce left ventricular (LV) systolic dysfunction. Meanwhile, the influence of PVCs on cardiac structural remodeling and functional change before occurrence of overt systolic heart failure has not been fully described. In this study, we attempted to identify early cardiac structural/functional manifestations of frequent PVCs in patients with normal LV systolic function. METHODS: A total of 146 patients (age: 55 ± 15 years, 48 males) with frequent PVCs observed on 24-hour Holter monitoring (>10/h) and normal LV ejection fraction (LV EF ≥ 55% on echocardiography) were enrolled. Clinical characteristics and echocardiographic parameters of the patients were compared with those of an age-/sex-matched control group (n = 292, age: 55 ± 15 years, 96 males). RESULTS: Patients with frequent PVCs had significantly larger left atrial volume index (LAVI [28 ± 9 mL/m(2) vs. 24 ± 7 mL/m(2) ]), along with larger LV end-diastolic dimension (49.4 ± 4.4 mm vs. 48.5 ± 3.9 mm), lower LV EF (63 ± 7% vs. 66 ± 6%), and lower peak systolic mitral annular velocity (7 ± 2 cm/s vs. 8 ± 2 cm/s; P < 0.05 for all), whereas other clinical characteristics were similar. In particular, in patients with frequent PVCs, LAVI showed linear correlation with PVC burden (R = 0.30, P < 0.001), and, in a multiple regression model, PVC burden independently estimated LAVI, even after controlling for age, sex, comorbidities, and systolic function (ß = 0.309, P < 0.001). CONCLUSION: Frequent PVC is associated with LA enlargement in patients with normal LV EF. Atrial anatomical remodeling may precede LV geometry change and systolic dysfunction in patients with frequent PVCs.

The Unusual Suspect: Anemia-induced Systolic Anterior Motion of the Mitral Valve and Intraventricular Dynamic Obstruction in a Hyperdynamic Heart as Unexpected Causes of Exertional Dyspnea after Cardiac Surgery.

Dynamic left ventricular (LV) outflow tract obstruction is a characteristic feature of hypertrophic cardiomyopathy; however, it can also occur in association with hyperdynamic LV contraction and/or changes in the cardiac loading condition, even in a structurally normal or near-normal heart. Here, we report a case of anemia-induced systolic anterior motion of the mitral valve and the resultant intraventricular obstruction in a patient who underwent coronary artery bypass grafting and suffered from anemia associated with recurrent gastrointestinal bleeding.

Regulation of Cholesterol Metabolism in Liver: Link to NAFLD and Impact of n-3 PUFAs.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of chronic liver disease that affects one-third of adults in westernized countries. NAFLD represents a wide spectrum of hepatic alterations, ranging from simple triglyceride accumulation in the liver to steatohepatitis. Several pharmaceutical approaches to NAFLD management have been examined, but no particular treatment has been considered both safe and highly effective. Growing evidence reveal that supplemental fish oil, seal oil and purified n-3 fatty acids can reduce hepatic lipid content in NAFLD through extensive regulation by inhibiting lipogenesis, promoting fatty acid oxidation and suppressing inflammatory responses. Recently, the fat-1 transgenic mice capable of converting n-6 to n-3 polyunsaturated fatty acids (PUFAs) have been used to examine the effects of endogenous n-3 PUFAs on NAFLD. The increased n-3 PUFAs in fat-1 transgenic mice reduced diet-induced hyperlipidemia and fatty liver through induction of CYP7A1 expression and activation of cholesterol catabolism to bile acid. This article introduces the n-3 PUFAs, and addresses the evidence and mechanisms by which endogenously synthesized n-3 PUFAs or increased dietary n-3 PUFAs may ameliorate NAFLD.

The insect peptide CopA3 inhibits lipopolysaccharide-induced macrophage activation.

We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide-linked dimeric peptide, exerts antimicrobial and anti-inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL-6 and TNF-α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS-induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL-6 and TNF-α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL-6 and TNF-α and indicate that CopA3 may be useful as an immune-modulating agent.