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.

Vascular endothelial growth factor-A (VEGF-A) is a master regulator of angiogenesis that controls several angiogenic processes in endothelial cells. However, the detailed mechanisms of VEGF-A responsible for pleiotropic functions and crosstalk with other signaling pathways have not been fully understood. Here, we found that VEGF-A utilizes the connective tissue growth factor (CTGF)/formyl peptide receptor-like 1 (FPRL1) axis as one of its mediators in angiogenesis. Using a proteomic approach, we found increased secretion of a matricellular protein, CTGF, from VEGF-A-treated human umbilical vein endothelial cells (HUVECs). Then, we studied the effect of CTGF binding to FPRL1 in VEGF-A-induced angiogenesis. CTGF directly binds to FPRL1 through a linker region and activates the downstream signals of FPRL1, such as increase in extracellular signal-regulated kinase (ERK) phosphorylation and intracellular Ca(2+) concentration. We found that linker region-induced FPRL1 activation promotes the migration and network formation of HUVECs, while disruption of FPRL1 inhibits VEGF-A-induced HUVEC migration and network formation. In addition, similar results were observed by the chorioallantoic membrane (CAM) assay based evaluation of angiogenesis in vivo. To summarize, our data reveal a novel working model for VEGF-A-induced angiogenesis via the VEGF-A/CTGF/FPRL1 axis that might prolong and enhance the signals initiated from VEGF-A.

This study proposes a tactile estimation method of molded plastic plates based on human tactile perception characteristics. Plastic plates are often used in consumer products. The tactile evaluation plays an important role in product development. However, physical quantities not taking into account human tactile perception have been employed in previous tactile estimation procedures. Hence, in this study, we adopted the vibrational thresholds of the mechanoreceptive units-FA I, FA II, SA I and SA II-for stimuli detection and developed a tactile estimation method for plastic plates that clarified the mechanoreceptive units related to tactile sensation. The developed tactile sensor consists of a base and a silicone rubber pad that contains strain gauges in it. We detected vibration during touch by the sensor and calculated the estimation of the firing values of the cutaneous mechanoreceptors, which are the essential data obtained by humans during tactile perception, in comparison to the amplitude spectrum of the vibration with the threshold amplitude of each mechanoreceptive unit. Simultaneously, we calculated the relationship between the normal and tangential forces recorded while the sensor ran over the samples. As a result of stepwise linear regression analysis using these values as explanatory variables, the evaluation scores for Soft were successfully estimated using the firing value of FA II and the relationship between normal/tangential forces, and the evaluation scores for Rough were estimated using the SA I firing value.

Increased fatty acid (FA) is often observed in highly proliferative tumors. FAs have been shown to modulate the secretion of proteins from tumor cells, contributing to tumor survival. However, the secreted factors affected by FA have not been systematically explored. Here, we found that treatment of oleate, a monounsaturated omega-9 FA, promoted the proliferation of HepG2 cells. To examine the secreted factors associated with oleate-induced cell proliferation, we performed a comprehensive secretome profiling of oleate-treated and untreated HepG2 cells. A comparison of the secretomes identified 349 differentially secreted proteins (DSPs; 145 upregulated and 192 downregulated) in oleate-treated samples, compared to untreated samples. The functional enrichment and network analyses of the DSPs revealed that the 145 upregulated secreted proteins by oleate treatment were mainly associated with cell proliferation-related processes, such as lipid metabolism, inflammatory response, and ER stress. Based on the network models of the DSPs, we selected six DSPs (MIF, THBS1, PDIA3, APOA1, FASN, and EEF2) that can represent such processes related to cell proliferation. Thus, our results provided a secretome profile indicative of an oleate-induced proliferation of HepG2 cells.

Voltage-gated Ca2+ channels (Cav) are essential for pancreatic beta cell function as they mediate Ca2+ influx, which leads to insulin exocytosis. The β3 subunit of Cav (Cavβ3) has been suggested to regulate cytosolic Ca2+ ([Ca2+]i) oscillation frequency and insulin secretion under physiological conditions, but its role in diabetes is unclear. Here, we report that islets from diabetic mice show Cavβ3 overexpression, altered [Ca2+]i dynamics, and impaired insulin secretion upon glucose stimulation. Consequently, in high-fat diet (HFD)-induced diabetes, Cavβ3-deficient (Cavβ3-/-) mice showed improved islet function and enhanced glucose tolerance. Normalization of Cavβ3 expression in ob/ob islets by an antisense oligonucleotide rescued the altered [Ca2+]i dynamics and impaired insulin secretion. Importantly, transplantation of Cavβ3-/- islets into the anterior chamber of the eye improved glucose tolerance in HFD-fed mice. Cavβ3 overexpression in human islets also impaired insulin secretion. We thus suggest that Cavβ3 may serve as a druggable target for diabetes treatment.

Robust mitochondrial respiration provides energy to support physical performance and physiological well-being, whereas mitochondrial malfunction is associated with various pathologies and reduced longevity. In the current study, we tested whether myricetin, a natural flavonol with diverse biological activities, may impact mitochondrial function and longevity. The mice were orally administered myricetin (50 mg/kg/day) for 3 weeks. Myricetin significantly potentiated aerobic capacity in mice, as evidenced by their increased running time and distance. The elevated mitochondrial function was associated with induction of genes for oxidative phosphorylation and mitochondrial biogenesis in metabolically active tissues. Importantly, myricetin treatment led to decreased PGC-1α acetylation through SIRT1 activation. Furthermore, myricetin significantly improved the healthspan and lifespan of wild-type, but not Sir-2.1-deficient, C. elegans. These results demonstrate that myricetin enhances mitochondrial activity, possibly by activating PGC-1α and SIRT1, to improve physical endurance, strongly suggesting myricetin as a mitochondria-activating agent.

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.

Interleukin 6 (IL6) is a pleiotropic cytokine that not only affects the immune system, but also plays an active role in many physiological events in various organs. Notably, 35% of systemic IL6 originates from adipose tissues under noninflammatory conditions. Here, we describe a previously unknown function of melanocortins in regulating Il6 gene expression and production in 3T3-L1 adipocytes through membrane receptors which are called melanocortin receptors (MCRs). Of the five MCRs that have been cloned, MC2R and MC5R are expressed during adipocyte differentiation. alpha-Melanocyte-stimulating hormone (alpha-MSH) or ACTH treatment of 3T3-L1 adipocytes induces Il6 gene expression and production in a time- and concentration-dependent manner via various signaling pathways including the protein kinase A, p38 mitogen-activated protein kinase, cJun N-terminal kinase, and IkappaB kinase pathways. Specific inhibition of MC2R and MC5R expression with short interfering Mc2r and Mc5r RNAs significantly attenuated the alpha-MSH-induced increase of intracellular cAMP and both the level of Il6 mRNA and secretion of IL6 in 3T3-L1 adipocytes. Finally, when injected into mouse tail vein, alpha-MSH dramatically increased the Il6 transcript levels in epididymal fat pads. These results suggest that alpha-MSH in addition to ACTH may function as a regulator of inflammation by regulating cytokine production.

F2L, a peptide derived from heme-binding protein, was originally identified as an endogenous ligand for formyl peptide receptor-like (FPRL)2. Previously, we reported that F2L inhibits FPR and FPRL1-mediated signaling in neutrophils. Since endothelial cells express functional FPRL1, we examined the effect of F2L on LL-37 (an FPRL1 agonist)-induced signaling in human umbilical vein endothelial cells (HUVECs). F2L stimulated the chemotactic migration in HUVECs. However, F2L inhibited FPRL1 activity, resulting in the inhibition of cell proliferation and tube formation induced by LL-37 in HUVECs. We suggest that F2L will potentially be useful in the study of FPRL1 signaling and the development of drugs to treat diseases involving the FPRL1 in the vascular system.

Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A) regulates the level of the inositol polyphosphates, inositol trisphosphate (IP3) and inositol tetrakisphosphate to modulate cellular signaling and intracellular calcium homeostasis in the central nervous system. IP3K-A binds to F-actin in an activity-dependent manner and accumulates in dendritic spines, where it is involved in the regulation of synaptic plasticity. IP3K-A knockout mice exhibit deficits in some forms of hippocampus-dependent learning and synaptic plasticity, such as long-term potentiation in the dentate gyrus synapses of the hippocampus. In the present study, to further elucidate the role of IP3K-A in the brain, we developed a transgenic (Tg) mouse line in which IP3K-A is conditionally overexpressed approximately 3-fold in the excitatory neurons of forebrain regions, including the hippocampus. The Tg mice showed an increase in both presynaptic release probability of evoked responses, along with bigger synaptic vesicle pools, and miniature excitatory postsynaptic current amplitude, although the spine density or the expression levels of the postsynaptic density-related proteins NR2B, synaptotagmin 1, and PSD-95 were not affected. Hippocampal-dependent learning and memory tasks, including novel object recognition and radial arm maze tasks, were partially impaired in Tg mice. Furthermore, (R,S)-3,5-dihydroxyphenylglycine-induced metabotropic glutamate receptor long-term depression was inhibited in Tg mice and this inhibition was dependent on protein kinase C but not on the IP3 receptor. Long-term potentiation and depression dependent on N-methyl-d-aspartate receptor were marginally affected in Tg mice. In summary, this study shows that overexpressed IP3K-A plays a role in some forms of hippocampus-dependent learning and memory tasks as well as in synaptic transmission and plasticity by regulating both presynaptic and postsynaptic functions.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasma membrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaVα2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.

Interstitial fibrosis is a common feature of chronic kidney disease, and platelet-derived growth factor receptor-β (PDGFR-β)-positive mesenchymal cells are reportedly the major source of scar-producing myofibroblasts. We had previously demonstrated that albumin and its derivative R-III (a retinol-binding protein-albumin domain III fusion protein) inhibited the transdifferentiation/activation of hepatic stellate cells (HSCs) to myofibroblasts and that R-III administration reduced liver fibrosis. In this study, we isolated cells (referred to as renal stellate cells, RSCs) from rat kidney tissues using the HSC isolation protocol and compared their morphological and biochemical characteristics with those of HSCs. RSCs shared many characteristics with HSCs, such as storage of vitamin A-containing lipid droplets and expression of HSC markers as well as pericyte markers. RSCs underwent spontaneous transdifferentiation into myofibroblasts in in vitro culture, which was inhibited by albumin expression or R-III treatment. We also evaluated the therapeutic effects of R-III in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Injected R-III localized predominantly in cytoglobin/stellate cell activation-associated protein (Cygb/STAP)-positive cells in the kidney and reduced renal fibrosis. These findings suggest that RSCs can be recognized as the renal counterparts of HSCs and that RSCs represent an attractive therapeutic target for anti-fibrotic therapy.

Anterior gradient protein 2 homolog (AGR2) belongs to the disulfide isomerase family of endoplasmic reticulum proteins. Itis overexpressed in several types of solid tumors, including tumors of the prostate, lung, and pancreas. However, the role of AGR2 in breast cancer and the regulatory mechanisms underlying AGR2 protein expressionare not fullyunderstood. We demonstrated that AGR2 levels are increased under hypoxic conditions and in breast cancer tumors. Mechanistically, Twist1 binds to, and activates the AGR2 promoter via an E-box sequence. Under hypoxic conditions, the increased expression of ARG2 is attenuated when Twist1 levels are reduced by shRNA. Conversely, Twist1 overexpression fully reverses decreased AGR2 levels upon HIF-1α knockdown. Notably, AGR2 is required for Twist1-induced proliferation, migration, and invasion of breast cancer cells. Collectively, these findings extend our understanding of AGR2 regulation in breast cancer and may contribute to development of Twist1-AGR2 targeting therapeutics for breast cancer.

Phospholipase D (PLD)2 via its enzymatic activity regulates cell proliferation and migration and thus is implicated in cancer. However, the role of PLD2 in obesity and type 2 diabetes has not previously been investigated. Here, we show that during diet-induced thermogenesis and obesity, levels of PLD2 but not PLD1 in adipose tissue are inversely related with uncoupling protein 1, a key thermogenic protein. We demonstrate that the thermogenic program in adipose tissue is significantly augmented in mice with adipocyte-specific Pld2 deletion or treated with a PLD2-specific inhibitor and that these mice are resistant to high fat diet-induced obesity, glucose intolerance, and insulin resistance. Mechanistically, we show that Pld2 deletion in adipose tissue or PLD2 pharmacoinhibition acts via p62 to improve mitochondrial quality and quantity in adipocytes. Thus, PLD2 inhibition is an attractive therapeutic approach for obesity and type 2 diabetes by resolving defects in diet-induced thermogenesis.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Adipose tissue functions as an endocrine organ, and the development of systemic inflammation in adipose tissue is closely associated with metabolic diseases, such as obesity and insulin resistance. Accordingly, the fine regulation of the inflammatory response caused by obesity has therapeutic potential for the treatment of metabolic syndrome. In this study, we analyzed the role of DJ-1 (PARK7) in adipogenesis and inflammation related to obesity in vitro and in vivo. Many intracellular functions of DJ-1, including oxidative stress regulation, are known. However, the possibility of DJ-1 involvement in metabolic disease is largely unknown. Our results suggest that DJ-1 deficiency results in reduced adipogenesis and the down-regulation of pro-inflammatory cytokines in vitro. Furthermore, DJ-1-deficient mice show a low-level inflammatory response in the high-fat diet-induced obesity model. These results indicate previously unknown functions of DJ-1 in metabolism and therefore suggest that precise regulation of DJ-1 in adipose tissue might have a therapeutic advantage for metabolic disease treatment.

Gut microbes might influence host metabolic homeostasis and contribute to the pathogenesis of type 2 diabetes (T2D), which is characterized by insulin resistance. Bacteria-derived extracellular vesicles (EVs) have been suggested to be important in the pathogenesis of diseases once believed to be non-infectious. Here, we hypothesize that gut microbe-derived EVs are important in the pathogenesis of T2D. In vivo administration of stool EVs from high fat diet (HFD)-fed mice induced insulin resistance and glucose intolerance compared to regular diet (RD)-fed mice. Metagenomic profiling of stool EVs by 16S ribosomal DNA sequencing revealed an increased amount of EVs derived from Pseudomonas panacis (phylum Proteobacteria) in HFD mice compared to RD mice. Interestingly, P. panacis EVs blocked the insulin signaling pathway in both skeletal muscle and adipose tissue. Moreover, isolated P. panacis EVs induced typical diabetic phenotypes, such as glucose intolerance after glucose administration or systemic insulin injection. Thus, gut microbe-derived EVs might be key players in the development of insulin resistance and impairment of glucose metabolism promoted by HFD.

Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and critical for normal embryonic development and repair of pathophysiological conditions in adults. Although phospholipase D (PLD) activity has been implicated in angiogenic processes, its role in VEGF signaling during angiogenesis in mammals is unclear. Here, we found that silencing of PLD2 by siRNA blocked VEGF-mediated signaling in immortalized human umbilical vein endothelial cells (iHUVECs). Also, VEGF-induced endothelial cell survival, proliferation, migration, and tube formation were inhibited by PLD2 silencing. Furthermore, while Pld2-knockout mice exhibited normal development, loss of PLD2 inhibited VEGF-mediated ex vivo angiogenesis. These findings suggest that PLD2 functions as a key mediator in the VEGF-mediated angiogenic functions of endothelial cells. [BMB Reports 2016; 49(3): 191-196].

Periostin is an extracellular matrix (ECM) protein that is overexpressed in a variety of human cancers, and its functions appear to be linked to tumor growth, metastasis, and angiogenesis. Recent clinical evidence suggests that aberrant periostin expression is correlated with poor outcome in patients with breast cancer. To identify novel tools to regulate the functional role of periostin, we generated benzyl-d(U)TP-modified DNA aptamers that were directed against human periostin (PNDAs) and characterized their functional roles in breast cancer progression. PNDA-3 selectively bound to the FAS-1 domain of periostin with nanomolar affinity and disrupted the interaction between periostin and its cell surface receptors, αvβ3 and αvβ5 integrins. PNDA-3 markedly antagonized the periostin-induced adhesion, migration, and invasion of breast cancer cells and blocked the activation of various components of the αvβ3 and αvβ5 integrin signal transduction pathways. In a 4T1 orthotopic mouse model, PNDA-3 administration significantly reduced primary tumor growth and distant metastasis. Thus, our results demonstrated that periostin-integrin signaling regulates breast cancer progression at multiple levels in tumor cells and the tumor microenvironment. DNA aptamers targeting periostin may potentially be used to inhibit breast cancer progression.