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1.
J Endod ; 50(7): 1011-1016, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38642733

ABSTRACT

INTRODUCTION: The purpose of this study was to evaluate the effect of side flattening of cutting flutes on the cyclic resistance and torsional resistance of nickel-titanium files. METHODS: Both novel flattened Platinum V.EU (PL) and standard nonflattened CC Premium V.EU (CC) rotaries were tested. For cyclic fatigue tests, all the files were rotated in an artificial root canal with a curvature of 45° and a radius of 6.06 mm at 300 rpm (n = 15 in each group). The number of cycles to failure (NCF) was calculated. For torsional tests, the files were rotated at 2 rpm clockwise until fracture occurred. The maximum torque value at fracture was measured and the toughness and distortion angle were computed. Subsequently, 5 fragments were randomly selected in each experiment, the cross-section and longitudinal direction of the fragments were photographed using a scanning electron microscope. An unpaired t-test was performed at a significance level of 95%. RESULTS: There was a statistically significant difference in NCF between CC and PL (P < .05). CC showed higher NCF than PL. There was no statistically significant difference between CC and PL with regards to the parameters related to torsional resistance (distortion angle, ultimate strength, and toughness) (P > .05). CONCLUSION: Within the limitations of this study, side flattening of the file did not improve cyclic resistance or torsional resistance of the files. As side flattening may reduce a file's cyclic resistance, such files should be used with caution in clinical practice.


Subject(s)
Equipment Failure , Nickel , Root Canal Preparation , Titanium , Torsion, Mechanical , Root Canal Preparation/instrumentation , Materials Testing , Equipment Design , Torque , Microscopy, Electron, Scanning , Dental Instruments , Dental Alloys/chemistry
2.
Cell ; 175(4): 947-961.e17, 2018 11 01.
Article in English | MEDLINE | ID: mdl-30401435

ABSTRACT

Interactions between the gut microbiota, diet, and the host potentially contribute to the development of metabolic diseases. Here, we identify imidazole propionate as a microbially produced histidine-derived metabolite that is present at higher concentrations in subjects with versus without type 2 diabetes. We show that imidazole propionate is produced from histidine in a gut simulator at higher concentrations when using fecal microbiota from subjects with versus without type 2 diabetes and that it impairs glucose tolerance when administered to mice. We further show that imidazole propionate impairs insulin signaling at the level of insulin receptor substrate through the activation of p38γ MAPK, which promotes p62 phosphorylation and, subsequently, activation of mechanistic target of rapamycin complex 1 (mTORC1). We also demonstrate increased activation of p62 and mTORC1 in liver from subjects with type 2 diabetes. Our findings indicate that the microbial metabolite imidazole propionate may contribute to the pathogenesis of type 2 diabetes.


Subject(s)
Diabetes Mellitus, Type 2/metabolism , Gastrointestinal Microbiome , Imidazoles/metabolism , Insulin/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Signal Transduction , Animals , Cells, Cultured , Diabetes Mellitus, Type 2/microbiology , HEK293 Cells , Histidine/metabolism , Humans , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Sequestosome-1 Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
3.
Cell Signal ; 51: 130-138, 2018 11.
Article in English | MEDLINE | ID: mdl-30092354

ABSTRACT

Regulation of tyrosine phosphorylation on insulin receptor substrate-1 (IRS-1) is essential for insulin signaling. The protein tyrosine phosphatase (PTP) C1-Ten/Tensin2 has been implicated in the regulation of IRS-1, but the molecular basis of this dephosphorylation is not fully understood. Here, we demonstrate that the cellular phosphatase activity of C1-Ten/Tensin2 on IRS-1 is mediated by the binding of the C1-Ten/Tensin2 Src-homology 2 (SH2) domain to phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). We show that the role of C1-Ten/Tensin2 is dependent on insulin-induced phosphoinositide 3-kinase activity. The C1-Ten/Tensin2 SH2 domain showed strong preference and high affinity for PtdIns(3,4,5)P3. Using site-directed mutagenesis, we identified three basic residues in the C1-Ten/Tensin2 SH2 domain that were critical for PtdIns(3,4,5)P3 binding but were not involved in phosphotyrosine binding and PTP activity. Using a PtdIns(3,4,5)P3 binding-deficient mutant, we showed that the specific binding of the C1-Ten/Tensin2 SH2 domain to PtdIns(3,4,5)P3 allowed C1-Ten/Tensin2 to function as a PTP in cells. Collectively, our findings suggest that the interaction between the C1-Ten/Tensin2 SH2 domain and PtdIns(3,4,5)P3 produces a negative feedback loop of insulin signaling through IRS-1.


Subject(s)
Insulin Receptor Substrate Proteins/metabolism , Phosphatidylinositol Phosphates/metabolism , Tensins/chemistry , Tensins/metabolism , src Homology Domains , Animals , Escherichia coli , HEK293 Cells , Humans , L Cells , Mice , Mutagenesis, Site-Directed , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Tensins/genetics
4.
Sci Rep ; 7(1): 17777, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29259227

ABSTRACT

Insulin resistance causes type 2 diabetes; therefore, increasing insulin sensitivity is a therapeutic approach against type 2 diabetes. Activating AMP-activated protein kinase (AMPK) is an effective approach for treating diabetes, and reduced insulin receptor substrate-1 (IRS-1) protein levels have been suggested as a molecular mechanism causing insulin resistance. Thus, dual targeting of AMPK and IRS-1 might provide an ideal way to treat diabetes. We found that 15,16-dihydrotanshinone I (DHTS), as a C1-Ten protein tyrosine phosphatase inhibitor, increased IRS-1 stability, improved glucose tolerance and reduced muscle atrophy. Identification of DHTS as a C1-Ten inhibitor revealed a new function of C1-Ten in AMPK inhibition, possibly through regulation of IRS-1. These findings suggest that C1-Ten inhibition by DHTS could provide a novel therapeutic strategy for insulin resistance-associated metabolic syndrome through dual targeting of IRS-1 and AMPK.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Insulin Receptor Substrate Proteins/metabolism , Insulin Resistance/physiology , Phenanthrenes/pharmacology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Signal Transduction/drug effects , Animals , Cell Line , Enzyme Activation/drug effects , Furans , Glucose/metabolism , Glucose Tolerance Test/methods , Humans , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Male , Metabolic Syndrome/drug therapy , Metabolic Syndrome/metabolism , Mice , Mice, Inbred C57BL , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Atrophy/drug therapy , Muscular Atrophy/metabolism , Quinones
5.
Sci Rep ; 7(1): 12346, 2017 09 27.
Article in English | MEDLINE | ID: mdl-28955049

ABSTRACT

Hypertrophy is a prominent feature of damaged podocytes in diabetic kidney disease (DKD). mTORC1 hyperactivation leads to podocyte hypertrophy, but the detailed mechanism of how mTORC1 activation occurs under pathological conditions is not completely known. Moreover, reduced nephrin tyrosine phosphorylation has been observed in podocytes under pathological conditions, but the molecular mechanism linking nephrin phosphorylation and pathology is unclear so far. In this study, we observed a significant increase in C1-Ten level in diabetic kidney and in high glucose-induced damaged podocytes. C1-Ten acts as a protein tyrosine phosphatase (PTPase) at the nephrin-PI3K binding site and renders PI3K for IRS-1, thereby activating mTORC1. Furthermore, C1-Ten causes podocyte hypertrophy and proteinuria by increasing mTORC1 activity in vitro and in vivo. These findings demonstrate the relationship between nephrin dephosphorylation and the mTORC1 pathway, mediated by C1-Ten PTPase activity. We suggest that C1-Ten contributes to the pathogenesis of DKD by inducing podocyte hypertrophy under high glucose conditions.


Subject(s)
Diabetic Nephropathies/pathology , Mechanistic Target of Rapamycin Complex 1/metabolism , Membrane Proteins/metabolism , Podocytes/pathology , Protein Tyrosine Phosphatases/metabolism , Tensins/metabolism , Animals , Glucose/metabolism , HEK293 Cells , Humans , Hypertrophy/pathology , Insulin Receptor Substrate Proteins/metabolism , Male , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteinuria/etiology , Proteinuria/pathology , Signal Transduction
6.
Sci Rep ; 6: 21772, 2016 Feb 23.
Article in English | MEDLINE | ID: mdl-26902888

ABSTRACT

Resveratrol (RSV) is a natural polyphenol that has a beneficial effect on health, and resveratrol-induced autophagy has been suggested to be a key process in mediating many beneficial effects of resveratrol, such as reduction of inflammation and induction of cancer cell death. Although various resveratrol targets have been suggested, the molecule that mediates resveratrol-induced autophagy remains unknown. Here, we demonstrate that resveratrol induces autophagy by directly inhibiting the mTOR-ULK1 pathway. We found that inhibition of mTOR activity and presence of ULK1 are required for autophagy induction by resveratrol. In line with this mTOR dependency, we found that resveratrol suppresses the viability of MCF7 cells but not of SW620 cells, which are mTOR inhibitor sensitive and insensitive cancer cells, respectively. We also found that resveratrol-induced cancer cell suppression occurred ULK1 dependently. For the mechanism of action of resveratrol on mTOR inhibition, we demonstrate that resveratrol directly inhibits mTOR. We found that resveratrol inhibits mTOR by docking onto the ATP-binding pocket of mTOR (i.e., it competes with ATP). We propose mTOR as a novel direct target of resveratrol, and inhibition of mTOR is necessary for autophagy induction.


Subject(s)
Adenosine Triphosphate/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Autophagy/drug effects , Gene Expression Regulation, Neoplastic , Stilbenes/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Antineoplastic Agents, Phytogenic/chemistry , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Binding, Competitive , Cell Line, Tumor , Genes, Reporter , HEK293 Cells , HeLa Cells , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Luciferases/genetics , Luciferases/metabolism , MCF-7 Cells , Molecular Docking Simulation , Protein Binding , Protein Domains , Protein Structure, Secondary , Resveratrol , Signal Transduction , Stilbenes/chemistry , TOR Serine-Threonine Kinases/chemistry , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism
7.
Cell Signal ; 26(11): 2470-80, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25101860

ABSTRACT

C1-Ten is a member of the tensin family of focal adhesion molecules but recent studies suggest it plays a more active role in many biological processes because of its potential association with diabetes and cancers. However, relatively little is known about the regulation of C1-Ten, such as changes in its protein level or cellular localization. The cellular localization of C1-Ten is unique because it is expressed in cytoplasmic puncta but nothing is known about these puncta. Here, we show that p62 sequestrates C1-Ten into puncta, making C1-Ten diffuse into the cytoplasm upon p62 depletion. More importantly, p62-mediated C1-Ten sequestration promoted C1-Ten ubiquitination and proteasomal degradation. p62-mediated protein reduction was specific to C1-Ten, and not other tensins such as tensin1 and tensin3. Thus, our results link cellular localization of C1-Ten to an off-switch site for C1-Ten. Additionally, p62 expression increased but C1-Ten protein decreased during muscle differentiation, supporting a role for p62 as a physiological regulator of C1-Ten.


Subject(s)
Adaptor Proteins, Signal Transducing/biosynthesis , Cytoplasm/metabolism , Microfilament Proteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Ubiquitination/physiology , Adaptor Proteins, Signal Transducing/genetics , Cytoplasm/genetics , Gene Expression Regulation/physiology , HEK293 Cells , HeLa Cells , Humans , Microfilament Proteins/genetics , Phosphoric Monoester Hydrolases/genetics , Proteasome Endopeptidase Complex/genetics , Sequestosome-1 Protein , Tensins
8.
Cell Signal ; 26(10): 2122-30, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25007995

ABSTRACT

mTORC1, a kinase complex that is considered a master regulator of cellular growth and proliferation, is regulated by many extra- and intracellular signals. Among these signals, mitochondrial status is known to have an impact on the effects of mTORC1 on cell growth and survival. However, how mitochondrial status affects mTORC1 activity, notably the molecular link, is not fully elucidated. Here, we found that Parkin can interact with and ubiquitinate mTOR. We also identified K2066 and K2306 as Parkin-dependent and mitochondrial stress-induced mTOR ubiquitination residues. This ubiquitination by Parkin is required for maintenance of mTORC1 activity under mitochondrial stress. With regard to the physiological meaning of mTORC1 activity under mitochondrial stress, we suggest that mTORC1 plays a pro-survival role.


Subject(s)
Mitochondria/metabolism , Multiprotein Complexes/metabolism , TOR Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis/drug effects , Carbonyl Cyanide m-Chlorophenyl Hydrazone/analogs & derivatives , Carbonyl Cyanide m-Chlorophenyl Hydrazone/toxicity , HEK293 Cells , Humans , Mechanistic Target of Rapamycin Complex 1 , Mitochondria/drug effects , Multiprotein Complexes/antagonists & inhibitors , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Regulatory-Associated Protein of mTOR , Sirolimus/toxicity , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/genetics , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitination
9.
Mol Cell Biol ; 33(8): 1608-20, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23401856

ABSTRACT

Muscle atrophy occurs under various catabolic conditions, including insulin deficiency, insulin resistance, or increased levels of glucocorticoids. This results from reduced levels of insulin receptor substrate 1 (IRS-1), leading to decreased phosphatidylinositol 3-kinase activity and thereby activation of FoxO transcription factors. However, the precise mechanism of reduced IRS-1 under a catabolic condition is unknown. Here, we report that C1-Ten is a novel protein tyrosine phosphatase (PTPase) of IRS-1 that acts as a mediator to reduce IRS-1 under a catabolic condition, resulting in muscle atrophy. C1-Ten preferentially dephosphorylated Y612 of IRS-1, which accelerated IRS-1 degradation. These findings suggest a novel type of IRS-1 degradation mechanism which is dependent on C1-Ten and extends our understanding of the molecular mechanism of muscle atrophy under catabolic conditions. C1-Ten expression is increased by catabolic glucocorticoid and decreased by anabolic insulin. Reflecting these hormonal regulations, the muscle C1-Ten is upregulated in atrophy but downregulated in hypertrophy. This reveals a previously unidentified role of C1-Ten as a relevant PTPase contributing to skeletal muscle atrophy.


Subject(s)
Insulin Receptor Substrate Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscular Atrophy/metabolism , Phosphoprotein Phosphatases/metabolism , Animals , Cell Line , Dexamethasone/pharmacology , Down-Regulation , Glucocorticoids/pharmacology , HEK293 Cells , Humans , Insulin/metabolism , Male , Mice , Mice, Obese , Muscle Fibers, Skeletal/pathology , Phosphatidylinositol 3-Kinase/metabolism , Phosphoprotein Phosphatases/genetics , Phosphorylation , Protein Stability , RNA Interference , RNA, Small Interfering , Signal Transduction , Tensins
10.
Cell Signal ; 25(2): 539-51, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23178303

ABSTRACT

Ras homolog enriched in brain (Rheb) regulates diverse cellular functions by modulating its nucleotide-bound status. Although Rheb contains a high basal GTP level, the regulatory mechanism of Rheb is not well understood. In this study, we propose soluble αß-tubulin acts as a constitutively active Rheb activator, which may explain the reason why Rheb has a high basal GTP levels. We found that soluble αß-tubulin is a direct Rheb-binding protein and that its deacetylated form has a high binding affinity for Rheb. Modulation of both soluble and acetylated αß-tubulin levels affects the level of GTP-bound Rheb. This occurs in the mitotic phase in which the level of acetylated αß-tubulin is increased but that of GTP-bound Rheb is decreased. Constitutively active Rheb-overexpressing cells showed an abnormal mitotic progression, suggesting the deacetylated αß-tubulin-mediated regulation of Rheb status may be important for proper mitotic progression. Taken together, we propose that deacetylated soluble αß-tubulin is a novel type of positive regulator of Rheb and may play a role as a temporal regulator for Rheb during the cell cycle.


Subject(s)
Guanosine Triphosphate/metabolism , Monomeric GTP-Binding Proteins/metabolism , Neuropeptides/metabolism , Tubulin/metabolism , Acetylation , Cell Line, Tumor , HEK293 Cells , HeLa Cells , Histidine/genetics , Histidine/metabolism , Humans , MCF-7 Cells , Microtubules/metabolism , Mitosis , Monomeric GTP-Binding Proteins/chemistry , Monomeric GTP-Binding Proteins/genetics , Neuropeptides/chemistry , Neuropeptides/genetics , Oligopeptides/genetics , Oligopeptides/metabolism , Protein Binding , Ras Homolog Enriched in Brain Protein , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Transfection
11.
J Biol Chem ; 287(22): 18398-407, 2012 May 25.
Article in English | MEDLINE | ID: mdl-22493283

ABSTRACT

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Osmotic Pressure , TOR Serine-Threonine Kinases/metabolism , Base Sequence , Cell Line , Chromatin Immunoprecipitation , Humans , JNK Mitogen-Activated Protein Kinases/genetics , Phosphorylation , RNA, Small Interfering , Regulatory-Associated Protein of mTOR , Tandem Mass Spectrometry
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