All-trans retinoic acid induces lipophagy through the activation of the AMPK-Beclin1 signaling pathway and reduces Rubicon expression in adipocytes.

Lipophagy is defined as a lipolysis pathway that degrades lipid droplet (LD) via autophagy. All-trans retinoic acid (atRA), a metabolite of vitamin A, stimulates lipolysis through hormone-sensitive lipase and ß-oxidation. However, the regulation of lipolysis by atRA-induced autophagy in adipocytes remains unclear. In this study, we investigated the effect of atRA on autophagy in epididymal fat of mice and the molecular mechanisms of autophagy in 3T3-L1 adipocytes. Western blotting showed that atRA decreased the expression of p62, a cargo receptor for autophagic degradation, and increased the expression of the lipidated LC3B (LC3B-II), an autophagy marker, in epididymal fat. Next, we confirmed that atRA increased autophagic flux in differentiated 3T3-L1 cells using the GFP-LC3-RFP-LC3ΔG probe. Immunofluorescent staining revealed that the colocalization of LC3B with perilipin increased in differentiated 3T3-L1 cells treated with atRA. The knockdown of Atg5, an essential gene in autophagy induction, partly suppressed the atRA-induced release of non-esterified fatty acid (NEFA) from LDs in differentiated 3T3-L1 cells. atRA time-dependently elicited the phosphorylation of AMPK and Beclin1, autophagy-inducing factors, in mature 3T3-L1 adipocytes. Inversely, atRA decreased the protein expression of Rubicon, an autophagy repressor, in differentiated 3T3-L1 cells and epididymal fat. Interestingly, the expression of ALDH1A1, atRA-synthesizing enzymes, increased in epididymal fat with decreased protein expression of Rubicon in aged mice. These results suggest that atRA may partially induce lipolysis through lipophagy by activating the AMPK-Beclin1 signaling pathway in the adipocytes and increased atRA levels may contribute to decreased Rubicon expression in the epididymal fat of aged mice. (248/250 words).

Humoral and cellular factors inhibit phosphate-induced vascular calcification during the growth period.

Hyperphosphatemia is an independent and non-classical risk factor of cardiovascular disease and mortality in patients with chronic kidney disease (CKD). Increased levels of extracellular inorganic phosphate (Pi) are known to directly induce vascular calcification, but the detailed underlying mechanism has not been clarified. Although serum Pi levels during the growth period are as high as those observed in hyperphosphatemia in adult CKD, vascular calcification does not usually occur during growth. Here, we have examined whether the defence system against Pi-induced vascular calcification can exist during the growth period using mice model. We found that calcification propensity of young serum (aged 3 weeks) was significantly lower than that of adult serum (10 months), possibly due to high fetuin-A levels. In addition, when the aorta was cultured in high Pi medium in vitro, obvious calcification was observed in the adult aorta but not in the young aorta. Furthermore, culture in high Pi medium increased the mRNA level of tissue-nonspecific alkaline phosphatase (TNAP), which degrades pyrophosphate, only in the adult aorta. Collectively, our findings indicate that the aorta in growing mouse may be resistant to Pi-induced vascular calcification via a mechanism in which high serum fetuin-A levels and suppressed TNAP expression.

Integration of bioassay and non-target metabolite analysis of tomato reveals that ß-carotene and lycopene activate the adiponectin signaling pathway, including AMPK phosphorylation.

Adiponectin, an adipokine, regulates glucose metabolism and insulin sensitivity through the adiponectin receptor (AdipoR). In this study, we searched for metabolites that activate the adiponectin signaling pathway from tomato (Solanum lycopersicu). Metabolites of mature tomato were separated into 55 fractions by liquid chromatography, and then each fraction was examined using the phosphorylation assay of AMP-protein kinase (AMPK) in C2C12 myotubes and in AdipoR-knockdown cells by small interfering RNA (siRNA). Several fractions showed AMPK phosphorylation in C2C12 myotubes and siRNA-mediated abrogation of the effect. Non-targeted metabolite analysis revealed the presence of 721 diverse metabolites in tomato. By integrating the activity of fractions on AMPK phosphorylation and the 721 metabolites based on their retention times of liquid chromatography, we performed a comprehensive screen for metabolites that possess adiponectin-like activity. As the screening suggested that the active fractions contained four carotenoids, we further analyzed ß-carotene and lycopene, the major carotenoids of food. They induced AMPK phosphorylation via the AdipoR, Ca2+/calmodulin-dependent protein kinase kinase and Ca2+ influx, in addition to activating glucose uptake via AdipoR in C2C12 myotubes. All these events were characteristic adiponectin actions. These results indicated that the food-derived carotenoids, ß-carotene and lycopene, activate the adiponectin signaling pathway, including AMPK phosphorylation.

Sulforaphane induces lipophagy through the activation of AMPK-mTOR-ULK1 pathway signaling in adipocytes.

Lipophagy, a form of selective autophagy, degrades lipid droplet (LD) in adipose tissue and the liver. The chemotherapeutic isothiocyanate sulforaphane (SFN) contributes to lipolysis through the activation of hormone-sensitive lipase and the browning of white adipocytes. However, the details concerning the regulation of lipolysis in adipocytes by SFN-mediated autophagy remain unclear. In this study, we investigated the effects of SFN on autophagy in the epididymal fat of mice fed a high-fat diet (HFD) or control-fat diet and on the molecular mechanisms of autophagy in differentiated 3T3-L1 cells. Western blotting revealed that the protein expression of lipidated LC3 (LC3-II), an autophagic substrate, was induced after 3T3-L1 adipocytes treatment with SFN. In addition, SFN increased the LC3-II protein expression in the epididymal fat of mice fed an HFD. Immunofluorescence showed that the SFN-induced LC3 expression was co-localized with LDs in 3T3-L1 adipocytes and with perilipin, the most abundant adipocyte-specific protein, in adipocytes of mice fed an HFD. Next, we confirmed that SFN activates autophagy flux in differentiated 3T3-L1 cells using the mCherry-EGFP-LC3 and GFP-LC3-RFP-LC3ΔG probe. Furthermore, we examined the induction mechanisms of autophagy by SFN in 3T3-L1 adipocytes using western blotting. ATG5 knockdown partially blocked the SFN-induced release of fatty acids from LDs in mature 3T3-L1 adipocytes. SFN time-dependently elicited the phosphorylation of AMPK, the dephosphorylation of mTOR, and the phosphorylation of ULK1 in differentiated 3T3-L1 cells. Taken together, these results suggest that SFN may provoke lipophagy through AMPK-mTOR-ULK1 pathway signaling, resulting in partial lipolysis of adipocytes.

Identification of Dietary Phytochemicals Capable of Enhancing the Autophagy Flux in HeLa and Caco-2 Human Cell Lines.

Autophagy is a major degradation system for intracellular macromolecules. Its decline with age or obesity is related to the onset and development of various intractable diseases. Although dietary phytochemicals are expected to enhance autophagy for preventive medicine, few studies have addressed their effects on the autophagy flux, which is the focus of the current study. Herein, 67 dietary phytochemicals were screened using a green fluorescent protein (GFP)-microtubule-associated protein light chain 3 (LC3)-red fluorescent protein (RFP)-LC3ΔG probe for the quantitative assessment of autophagic degradation. Among them, isorhamnetin, chrysoeriol, 2,2',4'-trihydroxychalcone, and zerumbone enhanced the autophagy flux in HeLa cells. Meanwhile, analysis of the structure-activity relationships indicated that the 3'-methoxy-4'-hydroxy group on the B-ring in the flavone skeleton and an ortho-phenolic group on the chalcone B-ring were crucial for phytochemicals activities. These active compounds were also effective in colon carcinoma Caco-2 cells, and some of them increased the expression of p62 protein, a typical substrate of autophagic proteolysis, indicating that phytochemicals impact p62 levels in autophagy-dependent and/or -independent manners. In addition, these compounds were characterized by distinct modes of action. While isorhamnetin and chrysoeriol enhanced autophagy in an mTOR signaling-dependent manner, the actions of 2,2',4'-trihydroxychalcone and zerumbone were independent of mTOR signaling. Hence, these dietary phytochemicals may prove effective as potential preventive or therapeutic strategies for lifestyle-related diseases.

Isorhamnetin, a 3'-methoxylated flavonol, enhances the lysosomal proteolysis in J774.1 murine macrophages in a TFEB-independent manner.

Lysosome is the principal organelle for the ultimate degradation of cellular macromolecules, which are delivered through endocytosis, phagocytosis, and autophagy. The lysosomal functions have been found to be impaired by fatty foods and aging, and more importantly, the lysosomal dysfunction in macrophages has been reported as a risk of atherosclerosis development. In this study, we searched for dietary polyphenols which possess the activity for enhancing the lysosomal degradation in J774.1, a murine macrophage-like cell line. Screening test utilizing DQ-BSA digestion identified isorhamnetin (3'-O-methylquercetin) as an active compound. Interestingly, structural comparison to inactive flavonols revealed that the chemical structure of the B-ring moiety in isorhamnetin is the primary determinant of its lysosome-enhancing activity. Unexpectedly isorhamnetin failed to inhibit mTORC1-TFEB signaling, a master regulator of lysosomal biogenesis and function. Our data suggested that the other molecular mechanism might be critical for the regulation of lysosomes in macrophages.Abbreviations: ANOVA: analysis of variance; ApoE: apolipoprotein E; ATP6V0D2: ATPase H+ transporting V0 subunit d2; BAF: bafilomycin A1; BODIPY: boron dipyrromethene; BSA: bovine serum albumin; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified eagle medium; DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; FBS: fetal bovine serum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LC-MS/MS: liquid chromatography tandem mass spectrometry; MITF: microphthalmia-associated transcription factor; MRM: multiple reaction monitoring; mTORC1: mechanistic target of rapamycin complex 1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor γ; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SDS: sodium dodecyl sulfate; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TBS: Tris-buffered saline; TFA: trifluoroacetic acid; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcriptional factor EB; TFEC: transcription factor EC; V-ATPase: vacuolar-type proton ATPase.

Wide-range screening of anti-inflammatory compounds in tomato using LC-MS and elucidating the mechanism of their functions.

Obesity-induced chronic inflammation is a key factor in type 2 diabetes. A vicious cycle involving pro-inflammatory mediators between adipocytes and macrophages is a common cause of chronic inflammation in the adipose tissue. Tomato is one of the most popular vegetables and is associated with a reduced risk of diabetes. However, the molecular mechanism underlying the effect of tomato on diabetes is unclear. In this study, we focused on anti-inflammatory compounds in tomato. We found that the extract of tomato reduced plasma glucose and inflammatory markers in mice. We screened anti-inflammatory fractions in tomato using lipopolysaccharide-stimulated RAW264.7 macrophages, and active compounds were estimated by liquid chromatography-mass spectrometry over a wide range. Surprisingly, a large number of compounds including oxylipin and coumarin derivatives were estimated as anti-inflammatory compounds. Especially, 9-oxo-octadecadienoic acid and daphnetin suppressed pro-inflammatory cytokines in RAW264.7 macrophages inhibiting mitogen-activated protein kinase phosphorylation and inhibitor of kappa B α protein degradation. These findings suggest that tomato containing diverse anti-inflammatory compounds ameliorates chronic inflammation in obese adipose tissue.

A Survey of Dental Implant Instruction in Predoctoral Dental Curricula in North America.

The aim of this study was to investigate the extent and forms of predoctoral implant dentistry instruction in North American dental schools and to identify future directions and challenges. The survey collected data on curriculum content, departmental oversight, techniques, and materials, as well as current problems to be solved. The 30-question survey was sent in 2012 to the dean or administrator in charge of the predoctoral curriculum of all 73 dental schools in the U.S. and Canada at the time; four reminders were sent. Forty-seven schools responded, for a response rate of 64%. Of the 47 responding schools, 46 (98%) offered didactic instruction (mean of 17 hours); 87% had a laboratory component (mean of 14.46 hours); and 57% had a clinical requirement. In the responding schools, students had an average of 1.85 implant restorative cases and 0.61 surgical cases. Forty-two of the schools (89%) had implemented observation of implant surgery and/or assisting with implant surgery in their curricula. Major challenges reported in implementing a comprehensive predoctoral implant curriculum included expense of implant systems to the schools and to patients, shortage of predoctoral cases, and lack of curriculum time and trained faculty. These results show that implant education for predoctoral dental students continues to expand, with a trend towards more preclinical exercises and clinical experiences and fewer didactic courses.

Wearing complete dentures is associated with changes in the three-dimensional shape of the oropharynx in edentulous older people that affect swallowing.

OBJECTIVE: To investigate the effects of wearing complete dentures on pharyngeal shape for swallowing in edentulous older people. BACKGROUND: In the absence of complete dentures, edentulous older people often lose the occlusal support necessary to position the mandible, which leads to an anterosuperior shift of the mandible during swallowing. This may result in pharyngeal shape changes effecting swallowing function in older people. However, the details of this phenomenon are currently unclear. MATERIALS AND METHODS: Participants were 17 older edentulous volunteers. Cone-beam computed tomography imaging was performed with the participant in the seated position and wearing (i) both maxillary and mandibular dentures, (ii) maxillary dentures only and (iii) no dentures. During imaging, participants were instructed to keep their mouth closed to the mandibular position determined in advance during swallowing for each denture-wearing condition. The volume, height and average cross-sectional area of the velopharynx and oropharynx were measured, and the positions of the epiglottis and mandible were recorded. RESULTS: While the vertical height of the oral cavity and pharynx significantly decreased, the volume and average cross-sectional area of the oropharynx significantly increased when dentures were not worn (p < 0.01). The absence of dentures caused an anterosuperior shift of the mandible when swallowing and drew the epiglottis forward, resulting in expansion of the oropharynx where the tongue base forms the anterior wall. CONCLUSION: The absence of dentures results in anatomical changes in oropharyngeal shape that may exacerbate the pharyngeal expansion caused by ageing and reduce the swallowing reserve.

Color difference thresholds in dentistry.

PURPOSE: The aim of this prospective multicenter study was to determine 50:50% perceptibility threshold (PT) and 50:50% acceptability threshold (AT) of dental ceramic under simulated clinical settings. MATERIALS AND METHODS: The spectral radiance of 63 monochromatic ceramic specimens was determined using a non-contact spectroradiometer. A total of 60 specimen pairs, divided into 3 sets of 20 specimen pairs (medium to light shades, medium to dark shades, and dark shades), were selected for psychophysical experiment. The coordinating center and seven research sites obtained the Institutional Review Board (IRB) approvals prior the beginning of the experiment. Each research site had 25 observers, divided into five groups of five observers: dentists-D, dental students-S, dental auxiliaries-A, dental technicians-T, and lay persons-L. There were 35 observers per group (five observers per group at each site ×7 sites), for a total of 175 observers. Visual color comparisons were performed using a viewing booth. Takagi-Sugeno-Kang (TSK) fuzzy approximation was used for fitting the data points. The 50:50% PT and 50:50% AT were determined in CIELAB and CIEDE2000. The t-test was used to evaluate the statistical significance in thresholds differences. RESULTS: The CIELAB 50:50% PT was ΔEab = 1.2, whereas 50:50% AT was ΔEab = 2.7. Corresponding CIEDE2000 (ΔE00 ) values were 0.8 and 1.8, respectively. 50:50% PT by the observer group revealed differences among groups D, A, T, and L as compared with 50:50% PT for all observers. The 50:50% AT for all observers was statistically different than 50:50% AT in groups T and L. CONCLUSION: A 50:50% perceptibility and ATs were significantly different. The same is true for differences between two color difference formulas ΔE00 /ΔEab . Observer groups and sites showed high level of statistical difference in all thresholds. CLINICAL SIGNIFICANCE: Visual color difference thresholds can serve as a quality control tool to guide the selection of esthetic dental materials, evaluate clinical performance, and interpret visual and instrumental findings in clinical dentistry, dental research, and subsequent standardization. The importance of quality control in dentistry is reinforced by increased esthetic demands of patients and dental professionals.

Assessment of a prototype computer colour matching system to reproduce natural tooth colour on ceramic restorations.

OBJECTIVES: The aim of this study was to assess the accuracy of a prototype computer colour matching (CCM) system for dental ceramics targeting the colour of natural maxillary central incisors employing a dental spectrophotometer and the Kubelka-Munk theory. METHODS: Seventeen human volunteers with natural intact maxillary central incisors were selected to participate in this study. One central incisor from each subject was measured in the body region by a spectrophotometer and the reflectance values were used by the CCM system in order to generate a prescription for a ceramic mixture to reproduce the target tooth's colour. Ceramic discs were fabricated based on these prescriptions and layered on a zirconia ceramic core material of a specified colour. The colour match of each two-layered specimen to the target natural tooth was assessed by CIELAB colour coordinates (ΔE(*), ΔL(*), Δa(*) and Δb(*)). RESULTS: The average colour difference ΔE(*) value was 2.58±84 for the ceramic specimen-natural tooth (CS-NT) pairs. ΔL(*) values ranged from 0.17 to 2.71, Δa(*) values ranged from -1.70 to 0.61, and Δb(*) values ranged from -1.48 to 3.81. There was a moderate inverse correlation (R=-0.44, p-value=0.0721) between L(*) values for natural target teeth and ΔE(*) values; no such correlation was found for a(*) and b(*) values. CONCLUSION: The newly developed prototype CCM system has the potential to be used as an efficient tool in the reproduction of natural tooth colour.

The optical effect of composite luting cement on all ceramic crowns.

PURPOSE: To investigate the optical properties of resin-based composite cements and assess their effects on the color of all ceramic crowns. MATERIAL AND METHODS: Optical properties of three cements (Variolink II, Esthetic, Nexus II) were analyzed using disk samples (100 microm and 1.0 mm diameter) based on CIELAB color coordinates L*a*b*C* and opacity index. Empress (Ivoclar) and Katana (Noritake) crowns were filled with four different shades of die material (IPS die material ST3, ST5, ST9 and Tetric Evo Ceram Bleach XL). The shade of the crowns was measured with each of three shades of composite cement (Chroma, Bleach and Opaque, Esthetic cement). ST3 and glycerin served as the control. Color differences were calculated in incisal, body and cervical regions. RESULTS: In the cervical and body regions, the color change caused by the bleach abutment could not be altered by using different colored cements for the Empress and Katana crowns. In the cervical region, the color changes effected by the dark brown abutment could be reversed with the bleach luting cement; in the body region this was true only for the Empress crown. Neither different abutment colors nor different luting cement shades resulted in perceptible color changes in the incisal regions. Mean DeltaE* values in the three areas (incisal, body, cervical) were significantly different (p-value<0.01); in the cervical and body regions Empress mean DeltaE* was significantly larger (p<0.01). CONCLUSION: The composite cements evaluated in this study created perceptible color differences with particular combinations of die material, cement and ceramic crown.

Clinical evaluation of perceptibility of color differences between natural teeth and all-ceramic crowns.

OBJECTIVES: Color matching still remains as one of the most challenging tasks in clinical dentistry. With the advancement of intraoral color matching instruments the importance of the development of color thresholds becomes increasingly important. The objective of this study was to set a gold standard for the color difference (DeltaE) at which all-ceramic crowns cannot be distinguished from natural teeth. METHOD: All-ceramic crowns with a zirconia coping (LAVA, 3M ESPE, St. Paul, MN, USA) and layered porcelain (Cerabien CZR, Noritake, Nagoya, Japan) fabricated to match contralateral maxillary central incisors were assessed by experienced observers. Eleven all-ceramic crowns (AC) were assessed as "excellent/perfect matches;" these crowns were then measured using a spectrophotometer and compared to their natural counterparts. Unrestored natural maxillary teeth (NT) were compared to unrestored contralateral teeth in 22 subjects. Six areas were measured in both groups to determine color differences (DeltaE*). RESULTS: Mean DeltaE* value between the restorations and natural teeth was 1.6. Mean DeltaE* between contralateral natural teeth was 0.9. Incisal areas of natural teeth exhibited greater differences in color than other compared areas. CONCLUSION: A DeltaE*=1.6 represented the color difference that could not be detected by the human eye.

Spontaneous hepatic rupture due to metastatic tumor of lung adenocarcinoma.

A 64-year-old man diagnosed as lung adenocarcinoma with hepatic tumor was admitted to our hospital. He carried the hepatitis B virus but was negative for PIVKA-II and alpha-fetoprotein, and hence we diagnosed a case of stage IV lung adenocarcinoma. We planned to administer systemic chemotherapy, but he experienced sudden-onset abdominal discomfort accompanied with decreased blood pressure. We diagnosed hemorrhagic ascites due to spontaneous rupture of the liver tumor. Emergency angiography and therapeutic embolization stabilized his clinical condition. Hemorrhagic ascites due to metastatic liver tumor is rare and the sudden onset of abdominal symptoms is an indicator of rupture.