Ginsenosides Rh1, Rg2, and Rg3 ameliorate dexamethasone-induced muscle atrophy in C2C12 myotubes.

High doses or prolonged use of the exogenous synthetic glucocorticoid dexamethasone (Dex) can lead to muscle atrophy. In this study, the anti-atrophic effects of ginsenosides Rh1, Rg2, and Rg3 on Dex-induced C2C12 myotube atrophy were assessed by XTT, myotube diameter, fusion index, and western blot analysis. The XTT assay results showed that treatment with Rh1, Rg2, and Rg3 enhanced cell viability in Dex-injured C2C12 myotubes. Compared with the control group, the myotube diameter and fusion index were both reduced in Dex-treated cells, but treatment with Rh1, Rg2, and Rg3 increased these parameters. Furthermore, Rh1, Rg2, and Rg3 significantly downregulated the protein expression of FoxO3a, MuRF1, and Fbx32, while also upregulating mitochondrial biogenesis through the SIRT1/PGC-1α pathway. It also prevents myotube atrophy by regulating the IGF-1/Akt/ mTOR signaling pathway. These findings indicate that Rh1, Rg2, and Rg3 have great potential as useful agents for the prevention and treatment of muscle atrophy.

Improvement of analytical method for three azo dyes in processed milk and cheese using HPLC-PDA.

This study aims to develop and validate a method for simultaneously measuring three azo dyes (azorubine, brilliant black BN, lithol rubine BK) not designated in Korea. The HPLC-PDA analysis method was validated based on the ICH guidelines, and the color stability was evaluated. The milk and cheese samples were spiked with azo dyes, the correlation coefficient of calibration curve ranged from 0.999 to 1.000 and the recovery rates of azo dyes were 98.81 âˆ¼ 115.94%, with RSD of 0.08 âˆ¼ 3.71%. The LOD and the LOQ in milk and cheese ranged from 1.14 to 1.73 µg/mL and 3.46 to 5.25 µg/mL, respectively. In addition, the expanded uncertainties of the measurements ranged from 3.3421 to 3.8146%. The azo dyes appeared to be color stable for more than 14 days. The results indicate that this analytical method is suitable for extracting and analyzing azo dyes in milk and cheese samples, which are not permitted in Korea.

Radical Scavenging-Linked Anti-Obesity Effect of Standardized Ecklonia stolonifera Extract on 3T3-L1 Preadipocytes and High-Fat Diet-Fed ICR Mice.

Ecklonia stolonifera, belonging to the Laminariaceae family, is an edible widely distributed perennial brown marine alga that is rich in polyphenols. Dieckol, a bioactive component of the E. stolonifera extract (ESE), is a major phlorotannin compound found only in brown algae. This study aimed to evaluate the ability of ESE to inhibit lipid accumulation caused by oxidative stress in 3T3-L1 adipocytes and high-fat diet-fed obese ICR mice. We report that ESE-treated obese ICR mice, which were fed a high-fat diet, showed reduced whole-body and adipose tissue weights with improved plasma lipid profiles. In vitro and in vivo studies have indicated that ESE inhibited the expression of adipogenesis-related genes associated with fat accumulation through AMP-activated protein kinase activity and increased the expression of lipolysis-related genes. In addition, ESE reduced the expression of enzymes involved in reactive oxygen species (ROS) production and increased the expression of antioxidant enzymes, thereby reducing ROS levels. These findings suggest that ESE possesses strong antioxidant properties and inhibits oxidative stress-induced lipid accumulation by reducing ROS production during adipocyte generation.

Anti-adipogenic Effects of Sulforaphane-rich Ingredient with Broccoli Sprout and Mustard Seed in 3T3-L1 Preadipocytes.

Glucoraphanin (GRA) is a precursor of sulforaphane (SFN), which can be synthesized by the enzyme myrosinase. In this study, we developed and validated HPLC analytical methods for the determination of GRA and SFN in mustard seed powder (MSP), broccoli sprout powder (BSP), and the MSP-BSP mixture powder (MBP), and evaluated their anti-adipogenic effects in 3T3-L1 adipocytes. We found that the analysis methods were suitable for the determination of GRA and SFN in MSP, BSP, and MBP. The content of GRA in BSP was 131.11 ± 1.84 µmol/g, and the content of SFN in MBP was 162.29 ± 1.24 µmol/g. In addition, BSP and MBP effectively decreased lipid accumulation content without any cytotoxicity. Both BSP and MBP significantly inhibited the expression of adipogenic proteins and increased the expression of proteins related to lipolysis and lipid metabolism. BSP and MBP inhibited the expression of adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein-α (C/EBP-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) in 3T3-L1 adipocytes, and inhibited the expression of fatty acid synthase (FAS) through AMP-activated protein kinase (AMPK). Meanwhile, BSP and MBP also increased the expression of the lipolysis-related proteins, uncoupling protein-1 (UCP-1) and carnitine palmitoyltransferase-1 (CPT-1). Moreover, MBP exerted anti-adipogenic to a greater extent than BSP in 3T3-L1 preadipocytes.

In-Depth Understanding of Ecklonia stolonifera Okamura: A Review of Its Bioactivities and Bioactive Compounds.

Ecklonia stolonifera Okamura (ES) is mainly distributed in the coastal areas of the middle Pacific, around Korea and Japan, and has a long-standing edible value. It is rich in various compounds, such as polysaccharides, fatty acids, alginic acid, fucoxanthin, and phlorotannins, among which the polyphenol compound phlorotannins are the main active ingredients. Studies have shown that the extracts and active components of ES exhibit anti-cancer, antioxidant, anti-obesity, anti-diabetic, antibacterial, cardioprotective, immunomodulatory, and other pharmacological properties in vivo and in vitro. Although ES contains a variety of bioactive compounds, it is not widely known and has not been extensively studied. Based on its potential health benefits, it is expected to play an important role in improving the nutritional value of food both economically and medically. Therefore, ES needs to be better understood and developed so that it can be utilized in the development and application of marine medicines, functional foods, bioactive substances, and in many other fields. This review provides a comprehensive overview of the bioactivities and bioactive compounds of ES to promote in-depth research and a reference for the comprehensive utilization of ES in the future.

Whole structural reconstruction and quantification of epidermal innervation through the suction blister method and skin-clearing technique.

Three-dimensional (3-D) analysis of intraepidermal nerve fibers (IENFs) is conducted to advance assessment methods for peripheral neuropathies and pruritic skin disorders. The skin-clearing technique was proven to be a reliable method for 3-D imaging of IENFs. Nonetheless, it still requires further improvement in the imaging process. The aim of this study was to standardize the 3-D evaluation method of IENFs and to suggest promising 3-D biomarkers for clinical application. A total of nine healthy individuals were prospectively enrolled. The newly adopted suction blister method was combined with the tissue-clearing technique. The detailed structure of the IENFs was reconstructed and quantified using the neuron tracing software. The suction blister method showed improved linear integrity of IENFs compared with those obtained from the previously used salt-split skin test. The 3-D parameters most significantly related to natural aging were the convex hull two-dimensional perimeter and the total length (both p = 0.020). The meaningful correlations were followed by total volume (p = 0.025), ends (p = 0.026), convex hull 3-D surface, and complexity (both p = 0.030). Thus, the 3-D parameters could be utilized as possible biomarkers to identify ambiguous pathologies of peripheral neuropathies and pruritic skin disorders.

Anti-Obesogenic Effects of Sulforaphane-Rich Broccoli (Brassica oleracea var. italica) Sprouts and Myrosinase-Rich Mustard (Sinapis alba L.) Seeds In Vitro and In Vivo.

Glucoraphanin (GRA), a glucosinolate particularly abundant in broccoli (Brassica oleracea var. italica) sprouts, can be converted to sulforaphane (SFN) by the enzyme myrosinase. Herein, we investigated the anti-obesogenic effects of broccoli sprout powder (BSP), mustard (Sinapis alba L.) seed powder (MSP), and sulforaphane-rich MSP-BSP mixture powder (MBP) in bisphenol A (BPA)-induced 3T3-L1 cells and obese C57BL/6J mice. In vitro experiments showed that MBP, BSP, and MSP have no cytotoxic effects. Moreover, MBP and BSP inhibited the lipid accumulation in BPA-induced 3T3-L1 cells. In BPA-induced obese mice, BSP and MBP treatment inhibited body weight gain and ameliorated dyslipidemia. Furthermore, our results showed that BSP and MBP could activate AMPK, which increases ACC phosphorylation, accompanied by the upregulation of lipolysis-associated proteins (UCP-1 and CPT-1) and downregulation of adipogenesis-related proteins (C/EBP-α, FAS, aP2, PPAR-γ, and SREBP-1c), both in vitro and in vivo. Interestingly, MBP exerted a greater anti-obesogenic effect than BSP. Taken together, these findings indicate that BSP and MBP could inhibit BPA-induced adipocyte differentiation and adipogenesis by increasing the expression of the proteins related to lipid metabolism and lipolysis, effectively treating BPA-induced obesity. Thus, BSP and MBP can be developed as effective anti-obesogenic drugs.

Improvement of methods for analysing sucrose acetate isobutyrate (SAIB) in soft drinks using GC-FID and evaluating antioxidant effects in the SAIB emulsion system.

Sucrose acetate isobutyrate SAIB (E444) is a mixture produced by the esterification of sucrose with acetic anhydride and isobutyric anhydride. It is a food additive that is used as an emulsifier in soft drinks. It is difficult to analyse SAIB quantitatively because there are 256 synthesisable structures in the mixture. This study developed an analytical method for SAIB using gas chromatography-flame ionization detection (GC-FID). The pre-treatment of SAIB in soft drinks was performed using a liquid-liquid extraction method, which demonstrated a recovery rate of 107.8 ± 7.2%. In the GC-FID analysis of SAIB, numerous peaks were observed in the chromatogram, and the content of SAIB was calculated as the sum of these peak areas. A series of analytical methods were validated according to International Conference for Harmonization (ICH) guidelines. Accordingly, the applicability of the developed analytical method was confirmed for both domestic and imported soft drinks distributed in Korea. Additionally, in the linoleic acid emulsion, SAIB exhibited better lipid oxidation stability than the natural antioxidant α-tocopherol and had similar efficacy to the synthetic antioxidant butylated hydroxytoluene (BHT). Although SAIB has excellent lipid oxidation stability, it must be used within legal standards according to consumer demand to reduce the use of synthetic materials in processed foods. The validated GC-FID analytical method will enable subsequent monitoring of the distributed products.

Immunomodulatory Effect of Polysaccharide from Fermented Morinda citrifolia L. (Noni) on RAW 264.7 Macrophage and Balb/c Mice.

This study aims to determine the immunomodulatory effects of a polysaccharide fraction from fermented M. citrifolia L. (FMP) in RAW 264.7 macrophages and Balb/c mice. M. citrifolia was fermented for 72 h using Lactobacillus brevis; polysaccharides were extracted using ethanol precipitation. The RAW 264.7 cells exposed to FMP (50, 100, and 200 µg/mL) for 24 h showed increased NO production, proinflammatory cytokine (IL-1ß, IL-6, and TNF-α) release, and COX-2 and iNOS protein expression. FMP (100, 200 mg/kg) and deacetylasperulosidic acid (DAA) (20 mg/kg) administered orally to Balb/c mice for 14 days upregulated NO production and NK cytotoxicity in abdominal cavity and spleen, respectively. Th1 and Th2 cytokines production and immune cell numbers increased in spleen, mesenteric lymph nodes (MLN), peritoneal exudate cells (PEC), Peyer's patches (PP), and peripheral blood mononuclear cells (PBMC). Therefore, FMP containing DAA can be used as materials for health functional foods to enhance immune responses.

Three-dimensional imaging for the analysis of human epidermal melanocytes.

Three-dimensional (3-D) analysis of human epidermal melanocytes is required for deeper understanding of melanocytic disorders. The purpose of this study was to standardize 3-D imaging and quantification for the evaluation of epidermal melanocytes. The epidermal specimen was obtained using the suction blister method from a patient with melanocytic nevus on the forearm skin. Cutaneous ACT-PRESTO, the tissue-clearing and labeling technique, was subsequently performed. With the 3-D image analysis program, morphological reconstruction and quantification of selected perilesional and melanocytic nevus areas were possible. The region of melanocytic nevus showed higher numbers of total melanocytic dendrites and similar numbers of cell bodies compared with perilesional area. In addition, the mean area and volume of cell bodies increased in the melanocytic nevus area compared with the results in the perilesional area. The 3-D evaluation method of human epidermal melanocytes can be applied to investigate novel pathologies related to hyper- or hypo-pigmentary disorders.

Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma.

PTEN is one of the most frequently altered tumor suppressor genes in malignant tumors. The dominant-negative effect of PTEN alteration suggests that the aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hotspot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that overexpress these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes ('edge mutations') localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties regarding their subcellular localization. Further, some missense mutations ('edge mutations') in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

Down-regulation of habenular calcium-dependent secretion activator 2 induces despair-like behavior.

Calcium-dependent secretion activator 2 (CAPS2) regulates the trafficking and exocytosis of neuropeptide-containing dense-core vesicles (DCVs). CAPS2 is prominently expressed in the medial habenula (MHb), which is related to depressive behavior; however, how MHb neurons cause depressive symptoms and the role of CAPS2 remains unclear. We hypothesized that dysfunction of MHb CAPS neurons might cause defects in neuropeptide secretion and the activity of monoaminergic centers, resulting in depressive-like behaviors. In this study, we examined (1) CAPS2 expression in the habenula of depression animal models and major depressive disorder patients and (2) the effects of down-regulation of MHb CAPS2 on the animal behaviors, synaptic transmission in the interpeduncular nucleus (IPN), and neuronal activity of monoamine centers. Habenular CAPS2 expression was decreased in the rat chronic restraint stress model, mouse learned helplessness model, and showed tendency to decrease in depression patients who died by suicide. Knockdown of CAPS2 in the mouse habenula evoked despair-like behavior and a reduction of the release of DCVs in the IPN. Neuronal activity of IPN and monoaminergic centers was also reduced. These results implicate MHb CAPS2 as playing a pivotal role in depressive behavior through the regulation of neuropeptide secretion of the MHb-IPN pathway and the activity of monoaminergic centers.

Extracorporeal shockwave therapy enhances peripheral nerve remyelination and gait function in a crush model.

BACKGROUND: Conservative treatment, such as electrical stimulation and steroid injection, have been employed in an attempt to improve symptoms after peripheral nerve injury, without significant success. Although non-invasive and safe extracorporeal shockwave therapy (ESWT) can be a practical alternative, the therapeutic effects of ESWT on peripheral nerve remyelination has not been established. OBJECTIVES: To investigate the effects of ESWT on peripheral nerve remyelination and gait function for 5 weeks in a sciatic nerve crush model. MATERIAL AND METHODS: In total, we divided 97 rats into 5 groups: group 1 - a healthy negative control group; group 2 - 3 weeks after sciatic nerve crush and 3 sessions of ESWT; group 3 - 5 weeks after crush injury with 3 sessions of ESWT; group 4 - 3 weeks after crush injury with no ESWT; and group 5 - 5 weeks after crush injury with no ESWT. The focused ESWT was applied to the unilateral sciatic nerve injury site. One session consisted of 1,500 stimuli, and the session were performed at intervals of 1 week. RESULTS: The degree of myelination and expression of myelin basic protein at the distal part of the injured sciatic nerve tended to increase in the ESWT groups compared with the no-ESWT groups 3 and 5 weeks after crush injury. Regarding the functional gait recovery, the print width and area of the injured leg in the ESWT groups was significantly larger than that in the no-ESWT groups 3 and 5 weeks after crush injury. CONCLUSIONS: The ESWT may enhance peripheral nerve remyelination and gait function in a nerve crush model. Long-term follow-up after ESWT and investigation of molecular mechanisms will be needed to confirm these therapeutic effects.

Differential synapse density between Purkinje cell dendritic spine and parallel fiber varicosity in the rat cerebellum among the phylogenic lobules.

The cerebellum is a region of the brain that plays an important role in motor control. It is classified phylogenetically into archicerebellum, paleocerebellum and neocerebellum. The Purkinje cells are lined in a row called Purkinje cell layer and it has a unique dendritic branches with many spines.The previous study reported that there is a difference of synapse density according to the lobules based on large two-dimensional data. However, recent study with high voltage electron microscopy showed there was no differences in dendritic spine density of the Purkinje cell according to its phylogenetic lobule. We analyzed Purkinje cell density in the II, VI and X lobules by stereological modules and synaptic density was estimated by double disector based on Purkinje cell density in the molecular layer of each lobule.The results showed that there was significant difference in the Purkinje cell density and synapse number according to their phylogenetic lobules. The number of Purkinje cell in a given volume was larger in the archicerebellum, but synapse density was higher in the neocerebellum.These data suggest that cellular and synaptic organization of the Purkinje cell is different according to their phylogenetic background.

Tissue-Clearing Technique and Cutaneous Nerve Biopsies: Quantification of the Intraepidermal Nerve-Fiber Density Using Active Clarity Technique-Pressure Related Efficient and Stable Transfer of Macromolecules Into Organs.

BACKGROUND AND PURPOSE: Cutaneous nerve biopsies based on two-dimensional analysis have been regarded as a creditable assessment tool for diagnosing peripheral neuropathies. However, advancements in methodological imaging are required for the analysis of intact structures of peripheral nerve fibers. A tissue-clearing and labeling technique facilitates three-dimensional imaging of internal structures in unsectioned, whole biological tissues without excessive time or labor costs. We sought to establish whether a tissue-clearing and labeling technique could be used for the diagnostic evaluation of peripheral neuropathies. METHODS: Five healthy individuals and four patients with small-fiber neuropathy (SFN) and postherpetic neuralgia (PHN) were prospectively enrolled. The conventional methods of indirect immunofluorescence (IF) and bright-field immunohistochemistry (IHC) were adopted in addition to the tissue-clearing and labeling method called active clarity technique-pressure related efficient and stable transfer of macromolecules into organs (ACT-PRESTO) to quantify the intraepidermal nerve-fiber density (IENFD). RESULTS: The mean IENFD values obtained by IF, bright-field IHC, and ACT-PRESTO in the healthy control group were 6.54, 6.44, and 90.19 fibers/mm², respectively; the corresponding values in the patients with SFN were 1.99, 2.32, and 48.12 fibers/mm², respectively, and 3.06, 2.87, and 47.21 fibers/mm², respectively, in the patients with PHN. CONCLUSIONS: This study has shown that a tissue-clearing method provided not only rapid and highly reproducible three-dimensional images of cutaneous nerve fibers but also yielded reliable quantitative IENFD data. Quantification of the IENFD using a tissue-clearing and labeling technique is a promising way to improve conventional cutaneous nerve biopsies.

Impacts of GFP-FoxP3+ regulatory T cells on lupus hallmarks differ by genetic background and type of GFP knock-in.

FoxP3 reporter mice expressing green fluorescence protein (GFP) have been used as a very convenient tool to investigate the impact of regulatory T (Treg) cells on pathogenesis in autoimmune diseases. Here, we found that GFP-FoxP3+ knock-in (KI) mice showed alterations in the production of anti-nuclear autoantibodies (ANAs) and nephritis with different extent, depending on the presence or absence of lupus susceptibility gene locus 1 (Sle1) and KI method: contrasting with B6.Sle1.fGFP-FoxP3 mice, expressing GFP via N-terminal insertion, B6.Sle1.iGFP-FoxP3, expressing GFP via bicistronic internal ribosome entry site-driven promotion, exhibited significantly lower penetrance of serum ANA, comparing to control B6.Sle1 mice. Moreover, B6.Sle1.GFP-FoxP3+ mice reduced the Sle1-induced splenomegaly and B-cell expansion independently of the KI method employed, mainly by reducing the numbers of transitional 1 (T1) B cells and CD21-CD23- B cells, including plasmablasts and plasma cells. The absolute numbers of both splenic CD4+ T cells and Treg cells from B6.Sle1.GFP-FoxP3 KI mice were significantly reduced but their proportion was not changed, compared to B6.Sle1 mice. Although the glomerular basement membranes were thickened in both B6.Sle1 and B6.Sle1.iGFP-FoxP3 mice, they were thinner in B6.Sle1.fGFP-FoxP3 mice. The latter mice expressed more nephrophilic autoantibodies and deposited more complement component 3 in glomeruli compared to B6.iGFP-FoxP3 mice. FoxP3+ Treg cells may modulate B-cell tolerance in lupus-prone B6.Sle1 mice, presumably by modulating pathogenic, nephrophilic autoantibody production and nephritis.

Quantification of intraepidermal nerve fiber density using three-dimensional microscopy.

Three-dimensional microscopy provides more extended depth of penetration compared with conventional light microscopy and is known to be useful in clinical evaluation of thick biological specimens. Skin nerve biopsy together with the quantification of intraepidermal nerve fibers in multiple thick sections has been widely adopted for evaluating peripheral neuropathies. The aim of the present study was to evaluate the effectivity of three-dimensional microscopy in reducing the required time and inter-rater discrepancies, especially in the case of personnel not familiar with the quantification methods. A total of six cryo-sectioned specimens were analyzed for the study and the skin samples were collected from one patient with postherpetic neuralgia who voluntarily participated in the study. Two investigators, a physician and non-physician assessed the intraepidermal nerve fiber densities and required analysis time using three different methods including direct visualization of tissue slides, and analysis with two- and three-dimensional images. Three-dimensional microscopy could produce images that enabled reliable evaluation of intraepidermal nerve fibers; the accuracy of analysis was statistically comparable between the physician and non-physician (p > .05). Three-dimensional microscopy also enabled the non-physician to proceed meaningfully faster evaluation compared with the direct visualization method (p = .03). Three-dimensional microscopy could be one of the useful methods to improve accuracy and convenience of the analysis of intraepidermal nerve fibers especially appropriate for unaccustomed physician or non-physician. RESEARCH HIGHLIGHTS: Three-dimensional microscopy is capable of producing images with more extended depth of penetration compared with conventional light microscopy and has been known to be suitable for clinical evaluation of thick biological specimens. Cutaneous nerve biopsy and the quantification of nerve fibers in thick sections has been widely adopted for evaluating peripheral neuropathies. Three-dimensional microscopy could be especially appropriate for unaccustomed physician or non-physician to improve accuracy and convenience of the analysis of intraepidermal nerve fibers.

Atmospheric scanning electron microscopy and its applications for biological specimens.

Scanning electron microscopy in ambient conditions (Air-SEM) was developed recently and has been used mainly for industrial applications. We assessed the potential application of Air-SEM for the analysis of biological tissues by using rat brain, kidney, human tooth, and bone. Hard tissues prepared by grinding and frozen sections were observed. Basic cytoarchitecture of bone and tooth was identified in the without heavy metal staining. Kidney tissue prepared using routine SEM methodology yielded images comparable to those of field emission (FE)-SEM. Sharpness was lower than that of FE-SEM, but foot process of podocytes was observed at high magnification. Air-SEM observation of semithin sections of kidney samples revealed glomerular basement membrane and podocyte processes, as seen using conventional SEM. Neuronal structures of soma, dendrites, axons, and synapses were clearly observed by Air-SEM with STEM detector and were comparable to conventional transmission electron microscopy images. Correlative light and electron microscopy observation of zebrafish embryos based on fluorescence microscopy and Air-SEM indicated the potential for a correlative approach. However, the image quality should be improved before becoming routine use in biomedical research.

Novel therapeutic roles of MC-4 in combination with everolimus against advanced renal cell carcinoma by dual targeting of Akt/pyruvate kinase muscle isozyme M2 and mechanistic target of rapamycin complex 1 pathways.

Current clinical trials of new anticancer therapies against metastatic renal cell carcinoma (RCC), including molecular-targeted therapies, have not shown promise. The purpose of this study was to preclinically assess the antitumor effects of MC-4, a partially purified material of Artemisia annua L., as a monotherapy or in combination with the known mechanistic target of rapamycin complex 1 (mTORC1) inhibitor, everolimus, against Caki-1 (Von Hippel-Lindau (VHL)+/+) and 786-O (VHL-/-) human RCC cells. MC-4 monotherapy significantly increased tumor growth inhibition and autophagic cell death in RCC cells in vitro and in vivo. Everolimus led to compensatory Akt activation by inhibiting only mTORC1 signaling pathway. In contrast to everolimus, MC-4 enhanced phosphatase and tensin homolog expression and reduced its downstream effector, Akt/pyruvate kinase muscle isozyme M2 (PKM2), leading to decreased expression of glucose transporter 1, which is associated with cancer cell metabolism. The synergistic antitumor and anti-metastatic effects induced by co-administration of MC-4 and everolimus involve cell growth inhibition and autophagic cell death via dual targeting of phosphatidylinositol 3-kinase (PI3K)/Akt/PKM2 and mTORC1. These findings suggest that MC-4 is a novel Akt/PKM2 inhibitor that can overcome the limitation of existing mTOR inhibitors and can be considered a novel strategy to treat patients with rapidly progressing advanced RCC.

Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue.

We investigated the role of connexin 43 (Cx43) in maintaining the integrity of mitochondria in brown adipose tissue (BAT). The functional effects of Cx43 were evaluated using inducible, adipocyte-specific Cx43 knockout in mice (Gja1 adipoq KO) and by overexpression and knockdown of Cx43 in cultured adipocytes. Mitochondrial morphology was evaluated by electron microscopy and mitochondrial function and autophagy were assessed by immunoblotting, immunohistochemistry, and qPCR. The metabolic effects of adipocyte-specific knockout of Cx43 were assessed during cold stress and following high fat diet feeding. Cx43 expression was higher in BAT compared to white adipose tissue. Treatment with the ß3-adrenergic receptor agonist CL316,243 increased Cx43 expression and mitochondrial localization. Gja1 adipoq KO mice reduced mitochondrial density and increased the presence of damaged mitochondria in BAT. Moreover, metabolic activation with CL316,243 further reduced mitochondrial integrity and upregulated autophagy in the BAT of Gja1 adipoq KO mice. Inhibition of Cx43 in cultured adipocytes increased the generation of reactive oxygen species and induction of autophagy during ß-adrenergic stimulation. Gja1 adipoq KO mice were cold intolerant, expended less energy in response to ß3-adrenergic receptor activation, and were more insulin resistant after a high-fat diet challenge. Collectively, our data demonstrate that Cx43 is required for maintaining the mitochondrial integrity and metabolic activity of BAT.