Obesity-induced metabolic imbalance allosterically modulates CtBP2 to inhibit PPAR-alpha transcriptional activity.

Maintenance of metabolic homeostasis is secured by metabolite-sensing systems, which can be overwhelmed by constant macronutrient surplus in obesity. Not only the uptake processes but also the consumption of energy substrates determine the cellular metabolic burden. We herein describe a novel transcriptional system in this context comprised of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator for fatty acid oxidation, and C-terminal binding protein 2 (CtBP2), a metabolite-sensing transcriptional corepressor. CtBP2 interacts with PPARα to repress its activity, and the interaction is enhanced upon binding to malonyl-CoA, a metabolic intermediate increased in tissues in obesity and reported to suppress fatty acid oxidation through inhibition of carnitine palmitoyltransferase 1. In line with our preceding observations that CtBP2 adopts a monomeric configuration upon binding to acyl-CoAs, we determined that mutations in CtBP2 that shift the conformational equilibrium toward monomers increase the interaction between CtBP2 and PPARα. In contrast, metabolic manipulations that reduce malonyl-CoA decreased the formation of the CtBP2-PPARα complex. Consistent with these in vitro findings, we found that the CtBP2-PPARα interaction is accelerated in obese livers while genetic deletion of CtBP2 in the liver causes derepression of PPARα target genes. These findings support our model where CtBP2 exists primarily as a monomer in the metabolic milieu of obesity to repress PPARα, representing a liability in metabolic diseases that can be exploited to develop therapeutic approaches.

New balance capability index as a screening tool for mild cognitive impairment.

BACKGROUND: Mild cognitive impairment (MCI) is not just a prodrome to dementia, but a very important intervention point to prevent dementia caused by Alzheimer's disease (AD). It has long been known that people with AD have a higher frequency of falls with some gait instability. Recent evidence suggests that vestibular impairment is disproportionately prevalent among individuals with MCI and dementia due to AD. Therefore, we hypothesized that the measurement of balance capability is helpful to identify individuals with MCI. METHODS: First, we developed a useful method to evaluate balance capability as well as vestibular function using Nintendo Wii balance board as a stabilometer and foam rubber on it. Then, 49 healthy volunteers aged from 56 to 75 with no clinically apparent cognitive impairment were recruited and the association between their balance capability and cognitive function was examined. Cognitive functions were assessed by MoCA, MMSE, CDR, and TMT-A and -B tests. RESULTS: The new balance capability indicator, termed visual dependency index of postural stability (VPS), was highly associated with cognitive impairment assessed by MoCA, and the area under the receiver operating characteristic (ROC) curve was more than 0.8, demonstrating high sensitivity and specificity (app. 80% and 60%, respectively). CONCLUSIONS: Early evidence suggests that VPS measured using Nintendo Wii balance board as a stabilometer helps identify individuals with MCI at an early and preclinical stage with high sensitivity, establishing a useful method to screen MCI.

High protein diet-induced metabolic changes are transcriptionally regulated via KLF15-dependent and independent pathways.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

CtBP2 confers protection against oxidative stress through interactions with NRF1 and NRF2.

While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically.

Hepatocyte ELOVL Fatty Acid Elongase 6 Determines Ceramide Acyl-Chain Length and Hepatic Insulin Sensitivity in Mice.

BACKGROUND AND AIMS: Dysfunctional hepatic lipid metabolism is a cause of nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, and is closely associated with insulin resistance and type 2 diabetes. ELOVL fatty acid elongase 6 (Elovl6) is responsible for converting C16 saturated and monounsaturated fatty acids (FAs) into C18 species. We have previously shown that Elovl6 contributes to obesity-induced insulin resistance by modifying hepatic C16/C18-related FA composition. APPROACH AND RESULTS: To define the precise molecular mechanism by which hepatic Elovl6 affects energy homeostasis and metabolic disease, we generated liver-specific Elovl6 knockout (LKO) mice. Unexpectedly, LKO mice were not protected from high-fat diet-induced insulin resistance. Instead, LKO mice exhibited higher insulin sensitivity than controls when consuming a high-sucrose diet (HSD), which induces lipogenesis. Hepatic patatin-like phospholipase domain-containing protein 3 (Pnpla3) expression was down-regulated in LKO mice, and adenoviral Pnpla3 restoration reversed the enhancement in insulin sensitivity in HSD-fed LKO mice. Lipidomic analyses showed that the hepatic ceramide(d18:1/18:0) content was lower in LKO mice, which may explain the effect on insulin sensitivity. Ceramide(d18:1/18:0) enhances protein phosphatase 2A (PP2A) activity by interfering with the binding of PP2A to inhibitor 2 of PP2A, leading to Akt dephosphorylation. Its production involves the formation of an Elovl6-ceramide synthase 4 (CerS4) complex in the endoplasmic reticulum and a Pnpla3-CerS4 complex on lipid droplets. Consistent with this, liver-specific Elovl6 deletion in ob/ob mice reduced both hepatic ceramide(d18:1/18:0) and PP2A activity and ameliorated insulin resistance. CONCLUSIONS: Our study demonstrates the key role of hepatic Elovl6 in the regulation of the acyl-chain composition of ceramide and that C18:0-ceramide is a potent regulator of hepatic insulin signaling linked to Pnpla3-mediated NAFLD.

Transcriptional co-repressor CtBP2 orchestrates epithelial-mesenchymal transition through a novel transcriptional holocomplex with OCT1.

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-ß (TGF-ß) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.

Deciphering genetic signatures by whole exome sequencing in a case of co-prevalence of severe renal hypouricemia and diabetes with impaired insulin secretion.

BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic ß-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 µmol/l (226-416 µmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 µg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic ß-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic ß-cell functions that deserve further scrutiny.

Influence of Intermittent Cold Stimulations on CREB and Its Targeting Genes in Muscle: Investigations into Molecular Mechanisms of Local Cryotherapy.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

Effect of sodium-glucose cotransporter 2 (SGLT2) inhibition on weight loss is partly mediated by liver-brain-adipose neurocircuitry.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry.

[Fingertip Screen Using POCT HbAlc Analyzer at Community Pharmacies Is Effective for Early Recognition of Diabetes].

Type 2 diabetes and prediabetes are highly prevalent in Japan, affecting nearly 25% of the population. Recent advances in the development of POCT analyzers now enable us to screen for undiagnosed diabetes using easy fingertip self-testing of blood hemoglobin A1c (HbA1c) in such public places as community phar- macies. Using this method in 20 pharmacies in Adachi Ward, Tokyo and in Tokushima Prefecture, we screened more than 4,700 people who were not under treatment at hospital in our study, named "Diabetes Screening Revolution." As a result, we identified more than 500 (app. 11%) diabetic and 670 (app. 14%) prediabetic people who were not under treatment. This result demonstrates that fingertip screening using the POCT HbAlc analyzer at pharmacies is effective for the early recognition of diabetes and prediabetes. Moreover, in response to our research results, Japan's government has revised the legislation governing la- boratory medical technologists to allow fingertip self-testing in such public places as community pharmacies, with such a site being referred to as a "Specimen measurement office" in the new legal system. Collectively, we established a new scheme of self-screening for diabetes in Japan. [Review].

Skeletal muscle-specific HMG-CoA reductase knockout mice exhibit rhabdomyolysis: A model for statin-induced myopathy.

HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs.

Identification of human ELOVL5 enhancer regions controlled by SREBP.

Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.

Ablation of Elovl6 protects pancreatic islets from high-fat diet-induced impairment of insulin secretion.

ELOVL family member 6, elongation of very long-chain fatty acids (Elovl6) is a microsomal enzyme that regulates the elongation of C12-16 saturated and monounsaturated fatty acids and is related to the development of obesity-induced insulin resistance via the modification of the fatty acid composition. In this study, we investigated the role of systemic Elovl6 in the pancreatic islet and ß-cell function. Elovl6 is expressed in both islets and ß-cell lines. In mice fed with chow, islets of the Elovl6(-/-) mice displayed normal architecture and ß-cell mass compared with those of the wild-type mice. However, when fed a high-fat, high-sucrose (HFHS) diet, the islet hypertrophy in response to insulin resistance observed in normal mice was attenuated and glucose-stimulated insulin secretion (GSIS) increased in the islets of Elovl6(-/-) mice compared with those of the wild-type mice. Enhanced GSIS in the HFHS Elovl6(-/-) islets was associated with an increased ATP/ADP ratio and the suppression of ATF-3 expression. Our findings suggest that Elovl6 could be involved in insulin secretory capacity per ß-cell and diabetes.

Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance.

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.

Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

GR-KLF15 pathway controls hepatic lipogenesis during fasting.

During periods of fasting, the body undergoes a metabolic shift from carbohydrate utilization to the use of fats and ketones as an energy source, as well as the inhibition of de novo lipogenesis and the initiation of gluconeogenesis in the liver. The transcription factor sterol regulatory element-binding protein-1 (SREBP-1), which plays a critical role in the regulation of lipogenesis, is suppressed during fasting, resulting in the suppression of hepatic lipogenesis. We previously demonstrated that the interaction of fasting-induced Kruppel-like factor 15 (KLF15) with liver X receptor serves as the essential mechanism for the nutritional regulation of SREBP-1 expression. However, the underlying mechanisms of KLF15 induction during fasting remain unclear. In this study, we show that the glucocorticoid receptor (GR) regulates the hepatic expression of KLF15 and, subsequently, lipogenesis through the KLF15-SREBP-1 pathway during fasting. KLF15 is necessary for the suppression of SREBP-1 by GR, as demonstrated through experiments using KLF15 knockout mice. Additionally, we show that GR is involved in the fasting response, with heightened binding to the KLF15 enhancer. It has been widely known that the hypothalamic-pituitary-adrenal (HPA) axis regulates the secretion of glucocorticoids and plays a significant role in the metabolic response to undernutrition. These findings demonstrate the importance of the HPA-axis-regulated GR-KLF15 pathway in the regulation of lipid metabolism in the liver during fasting.

Single-Cell Transcriptome Profiling of Pancreatic Islets From Early Diabetic Mice Identifies Anxa10 for Ca2+ Allostasis Toward ß-Cell Failure.

Type 2 diabetes is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in ß-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here, we performed single-cell RNA sequencing of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of type 2 diabetes. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of ß- and α-cells. We recognized a new prediabetic gene, Anxa10, that was induced by and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed ß-cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassium-induced insulin secretion. Pseudotime analysis of ß-cells predicted that this Ca2+-surge responder cluster would proceed to mitochondria dysfunction and endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provide a new insight into Ca2+ allostasis and ß-cell failure processes. ARTICLE HIGHLIGHTS: The transcriptome of single-islet cells from healthy, prediabetic, and diabetic mice was studied. Distinct ß-cell heterogeneity and islet cell-cell network in prediabetes and diabetes were found. A new prediabetic ß-cell marker, Anxa10, regulates intracellular Ca2+ and insulin secretion. Diabetes triggers ß-cell to acinar cell transdifferentiation.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Genetic and Functional Analyses of Patients with Marked Hypo-High-Density Lipoprotein Cholesterolemia.

AIM: This study aimed to analyze two cases of marked hypo-high-density lipoprotein (HDL) cholesterolemia to identify mutations in ATP-binding cassette transporter A1 (ABCA1) and elucidate the molecular mechanism by which these novel pathological mutations contribute to hypo-HDL cholesterolemia in Tangier disease. METHODS: Wild type and mutant expression plasmids containing a FLAG tag inserted at the C-terminus of the human ABCA1 gene were generated and transfected into HEK293T cells. ABCA1 protein expression and cholesterol efflux were evaluated via Western blotting and efflux assay. The difference in the rate of change in protein expression was evaluated when proteolytic and protein-producing systems were inhibited. RESULTS: In case 1, a 20-year-old woman presented with a chief complaint of gait disturbance. Her HDL-C level was only 6.2 mg/dL. Tangier disease was suspected because of muscle weakness, decreased nerve conduction velocity, and splenomegaly. Whole-exome analysis showed compound heterozygosity for a W484* nonsense mutation and S1343I missense mutation, which confirmed Tangier disease. Cholesterol efflux decreased by a mixture of W484* and S1343I mutations. The S1343I mutation decreased the protein production rate but increased the degradation rate, decreasing the protein levels. This patient also had Krabbe disease. The endogenous ABCA1 protein level of macrophage cell decreased by knocking down its internal galactocerebrosidase.Case 2, a 51-year-old woman who underwent tonsillectomy presented with peripheral neuropathy, corneal opacity, and HDL-C of 3.4 mg/dL. Whole-exome analysis revealed compound heterozygosity for R579* and R1572* nonsense mutations, which confirmed Tangier disease. CONCLUSION: Case 1 is a new ABCA1 mutation with complex pathogenicity, namely, a W484*/S1343I compound heterozygote with marked hypo-HDL cholesterolemia. Analyses of the compound heterozygous mutations indicated that decreases in ABCA1 protein levels and cholesterol efflux activity caused by the novel S1343I mutation combined with loss of W484* protein activity could lead to marked hypo-HDL cholesterolemia. Galactocerebrosidase dysfunction could also be a potential confounding factor for ABCA1 protein function.

TFE3 inhibits myoblast differentiation in C2C12 cells via down-regulating gene expression of myogenin.

Transcription factor E3 (TFE3) belongs to a basic helix-loop-helix family, and is involved in the biology of osteoclasts, melanocytes and their malignancies. We previously reported the metabolic effects of TFE3 on insulin in the liver and skeletal muscles in animal models. In the present study, we explored a novel role for TFE3 in a skeletal muscle cell line. When TFE3 was overexpressed in C2C12 myoblasts by adenovirus before induction of differentiation, myogenic differentiation of C2C12 cells was significantly inhibited. Adenovirus-mediated TFE3 overexpression also suppressed the gene expression of muscle regulatory factors (MRFs), such as MyoD and myogenin, during C2C12 differentiation. In contrast, knockdown of TFE3 using adenovirus encoding short-hairpin RNAi specific for TFE3 dramatically promoted myoblast differentiation associated with significantly increased expression of MRFs. Consistent with these findings, promoter analyses via luciferase reporter assay and electrophoretic mobility shift assay suggested that TFE3 negatively regulated myogenin promoter activity by direct binding to an E-box, E2, in the myogenin promoter. These findings indicated that TFE3 has a regulatory role in myoblast differentiation, and that transcriptional suppression of myogenin expression may be part of the mechanism of action.