Dihydromyrcenol Modulates Involucrin Expression through the Akt Signaling Pathway.

The epidermis serves as a protective barrier against external threats and is primarily composed of keratinocytes, which ultimately form corneocytes. Involucrin, a protein integral to the cornified envelope, plays a pivotal role in preserving the functional integrity of the skin barrier. Previous studies have shown that Akt plays an important role in keratinocyte differentiation and skin barrier development. This study investigated whether dihydromyrcenol (DHM), a plant-derived terpene, could increase involucrin production in keratinocytes and sought to elucidate the possible underlying mechanisms. To accomplish this objective, we assessed the alterations in involucrin by DHM through quantitative PCR and Western blot on the HaCaT cell line. The changes in the promoter levels were investigated using luciferase assays. Furthermore, upstream mechanisms were explored through the use of siRNA and inhibitors. To strengthen our findings, the results were subsequently validated in primary cells and 3D skin equivalents. DHM significantly increased involucrin mRNA and protein levels in a concentration-dependent manner. In addition, the Fyn-Akt signaling pathway was found to be required for DHM-induced involucrin expression, as inhibition of Fyn or Akt blocked the increase in involucrin mRNA induced by DHM. The transcription factor Sp1, which is recognized as one of the transcription factors for involucrin, was observed to be activated in response to DHM treatment. Moreover, DHM increased epidermal thickness in a 3D human skin model. These findings suggest that the modulation of involucrin expression with DHM could improve skin barrier function and highlight the importance of manipulating the Akt pathway to achieve this improvement.

The Role of Cyclic Adenosine Monophosphate (cAMP) in Modulating Glucocorticoid Receptor Signaling and Its Implications on Glucocorticoid-Related Collagen Loss.

Glucocorticoid receptors (GRs) play a pivotal role in the stress response of the body, but overactivation can disrupt normal physiological functions. This study explores the role of cyclic adenosine monophosphate (cAMP) in GR activation and the associated mechanisms. We initially used the human embryonic kidney 293 cell line (HEK293) and found that cAMP enhancement, using forskolin and 3-isobutyl-1-methylxanthine (IBMX), did not alter glucocorticoid signaling under normal conditions, as evidenced by glucocorticoid response element (GRE) activity and the translocation of GR. However, in stressful conditions induced by dexamethasone, a synthetic glucocorticoid, cAMP was found to lessen glucocorticoid signaling within a short time frame but amplify it over an extended period in HEK293 cells. Bioinformatic analysis revealed that cAMP upregulation triggers the extracellular signal-regulated kinase (ERK) pathway, which influences GR translocation and ultimately regulates its activity. This stress-modulating function of cAMP was also investigated in the Hs68 dermal fibroblast line, known for its susceptibility to glucocorticoids. We found that cAMP enhancement via forskolin reduces GRE activity and reverses collagen loss in Hs68 cells exposed to dexamethasone. These findings underline the context-specific role of cAMP signaling in managing glucocorticoid signaling and its potential therapeutic application in treating stress-related pathological conditions like skin aging characterized by collagen reduction.

Activation of cAMP Signaling in Response to α-Phellandrene Promotes Vascular Endothelial Growth Factor Levels and Proliferation in Human Dermal Papilla Cells.

Dermal papilla cells (DPCs) are growth factor reservoirs that are specialized for hair morphogenesis and regeneration. Due to their essential role in hair growth, DPCs are commonly used as an in vitro model to investigate the effects of hair growth-regulating compounds and their molecular mechanisms of action. Cyclic adenosine monophosphate (cAMP), an intracellular second messenger, is currently employed as a growth-promoting target molecule. In a pilot test, we found that α-phellandrene, a naturally occurring phytochemical, increased cAMP levels in DPCs. Therefore, we sought to determine whether α-phellandrene increases growth factors and proliferation in human DPCs and to identify the underlying mechanisms. We demonstrated that α-phellandrene promotes cell proliferation concentration-dependently. In addition, it increases the cAMP downstream effectors, such as protein kinase A catalytic subunit (PKA Cα) and phosphorylated cAMP-responsive element-binding protein (CREB). Also, among the CREB-dependent growth factor candidates, we identified that α-phellandrene selectively upregulated vascular endothelial growth factor (VEGF) mRNA expression in DPCs. Notably, the beneficial effects of α-phellandrene were nullified by a cAMP inhibitor. This study demonstrated the cAMP-mediated growth effects in DPCs and the therapeutic potential of α-phellandrene for preventing hair loss.

UV-Irradiation- and Inflammation-Induced Skin Barrier Dysfunction Is Associated with the Expression of Olfactory Receptor Genes in Human Keratinocytes.

Olfactory receptors (ORs) have diverse physiological roles in various cell types, beyond their function as odorant sensors in the olfactory epithelium. These previous findings have suggested that ORs could be diagnostic markers and promising therapeutic targets in several pathological conditions. In the current study, we sought to characterize the changes in the expression of ORs in the HaCaT human keratinocytes cell line exposed to ultraviolet (UV) light or inflammation, well-recognized stimulus for skin barrier disruption. We confirmed that major olfactory signaling components, including ORs, GNAL, Ric8b, and adenylate cyclase type 3, are highly expressed in HaCaT cells. We have also demonstrated that the 12 ectopic ORs detectable in HaCaT cells are more highly expressed in UV-irradiated or inflamed conditions than in normal conditions. We further assessed the specific OR-mediated biological responses of HaCaT cells in the presence of known odorant ligands of ORs and observed that specific ligand-activated ORs downregulate skin barrier genes in HaCaT cells. This study shows the potential of OR as a marker for skin barrier abnormalities. Further research is needed to explore how OR is implicated in the development and progression of barrier dysfunction.

Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells).

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

Nonanal Stimulates Growth Factors via Cyclic Adenosine Monophosphate (cAMP) Signaling in Human Hair Follicle Dermal Papilla Cells.

Human hair follicle dermal papilla cells (DPCs) are a specialized population of cells located in the hair follicles and regulate hair growth and development, particularly by releasing numerous growth factors in response to various physiological conditions. In the present study, we aimed to test whether nonanal, a scent compound from plants, stimulated growth factors in DPCs and to delineate the underlying mechanisms involved. We found that nonanal promoted DPC proliferation in a dose-dependent manner. Meanwhile, it also increased the intracellular cyclic adenosine monophosphate (cAMP) levels and the expression of various growth factor genes such as vascular endothelial growth factor, keratinocyte growth factor, and insulin-like growth factor 1. Furthermore, nonanal treatment stimulated DPC migration. Notably, the benefits of nonanal use were abrogated by cAMP inhibition. Our results reveal the potential of nonanal in preventing hair loss and suggest that its effects are cAMP-mediated in DPCs.

Anti-Allergic and Anti-Inflammatory Effects of Undecane on Mast Cells and Keratinocytes.

The critical roles of keratinocytes and resident mast cells in skin allergy and inflammation have been highlighted in many studies. Cyclic adenosine monophosphate (cAMP), the intracellular second messenger, has also recently emerged as a target molecule in the immune reaction underlying inflammatory skin conditions. Here, we investigated whether undecane, a naturally occurring plant compound, has anti-allergic and anti-inflammatory activities on sensitized rat basophilic leukemia (RBL-2H3) mast cells and HaCaT keratinocytes and we further explored the potential involvement of the cAMP as a molecular target for undecane. We confirmed that undecane increased intracellular cAMP levels in mast cells and keratinocytes. In sensitized mast cells, undecane inhibited degranulation and the secretion of histamine and tumor necrosis factor α (TNF-α). In addition, in sensitized keratinocytes, undecane reversed the increased levels of p38 phosphorylation, nuclear factor kappaB (NF-κB) transcriptional activity and target cytokine/chemokine genes, including thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC) and interleukin-8 (IL-8). These results suggest that undecane may be useful for the prevention or treatment of skin inflammatory disorders, such as atopic dermatitis, and other allergic diseases.

Erratum: Tong, T., et al. α-Ionone Protects Against UVB-Induced Photoaging in Human Dermal Fibroblasts. Molecules 2019, 24, 1804.

The authors wish to make the following change to their paper [...].

Carvone Decreases Melanin Content by Inhibiting Melanoma Cell Proliferation via the Cyclic Adenosine Monophosphate (cAMP) Pathway.

Melanin, which determines the color of the skin and hair, is initially synthesized to protect the skin from ultraviolet light; however, excessive melanin pigmentation caused by abnormal cell proliferation can result in various melanocytic lesions. Cyclic adenosine monophosphate (cAMP) is known to regulate cell cycle progression and consequently to inhibit the division of abnormally proliferating cells. In this work, we aimed to test whether carvone, a scent compound from plants, inhibits proliferation and subsequently reduces melanin content of melanoma cells and to determine whether its beneficial effects are mediated by the cAMP pathway. We found that carvone decreases melanin content and inhibits melanoma cell proliferation in a concentration-dependent manner. Meanwhile, it inhibited the activation of cell cycle-associated proteins such as cyclin-dependent kinase 1 (CDK1). Of note, the beneficial effects of carvone were abrogated by cAMP inhibition. Our findings indicate potential benefits of carvone for the treatment of melanomas and presumably other hyperpigmentation-related dermatological disorders such as melasmas, lentigines, and excessive freckles.

α-Cedrene protects rodents from high-fat diet-induced adiposity via adenylyl cyclase 3.

OBJECTIVE: The increasing global prevalence of obesity and its associated disorders points to an urgent need for the development of novel and effective strategies for the prevention of weight gain. Here, we investigated the potential of α-cedrene, a volatile sesquiterpene compound derived from cedarwood oil, in regulation of obesity and delineated the mechanisms involved. METHODS: For the prevention of obesity, C57BL/6 N mice were fed a high-fat diet (HFD) and were orally administered either with vehicle or α-cedrene for 8 weeks. For the therapy of obesity, obese Sprague Dawley rats, induced by a HFD for 8 weeks, were orally treated either with vehicle or α-cedrene for 12 weeks. To determine whether the action of α-cedrene was Adcy3 dependent, Adcy3 heterozygous null mice (Adcy3+/-) and wild-type controls were fed either HFD or α-cedrene supplemented HFD for 17 weeks. RESULTS: Oral α-cedrene administration prevented or reversed HFD-induced obesity and abnormal metabolic aberrations in rodents, without affecting their food intake. Downregulation of Adcy3 expression by small interfering RNA abrogated the beneficial effects of α-cedrene on the oxygen consumption rate and intracellular lipid accumulation in 3T3-L1 adipocytes. Similarly, in Adcy3+/- mice, the α-cedrene-driven suppression of body weight gain observed in wild-type mice was substantially (~50%) attenuated. Expression of thermogenic and lipid oxidation genes was increased in adipose tissues of α-cedrene-treated mice, with concomitant downregulation of adipogenic gene expression. These beneficial molecular changes elicited by α-cedrene were blunted in adipose tissues of Adcy3+/- mice. CONCLUSIONS: Our results highlight the potential of α-cedrene for antiobesity interventions and suggest that the antiobesity effect of α-cedrene is mediated by Adcy3 in adipose tissues.

Transcriptome profiles of tomato plants after neutron irradiation and infection with TYLCV.

Ionizing radiation is ubiquitous in the environment and can cause mutagenesis in living organisms. In this study, we examined the effects of neutron irradiation on tomato plants. Neutron irradiation decreased tomato germination rates, but most irradiated tomato plants did not show any significant phenotype. However, tomato mutants infected by Tomato yellow leaf curl virus (TYLCV) displayed resistance against TYLCV compared to the wild type (WT), which showed disease symptoms. RNA-Seq data demonstrated that the expression profiles of eight tomato mutants were significantly different from that of the WT. The transcriptomes obtained from presoaked seeds were highly altered compared to those of dry seeds. Increased irradiation time resulted in severe changes in the tomato transcriptome; however, different neutron irradiation intensities affected the expressions of different sets of genes. A high number of single-nucleotide polymorphisms in tomato transcriptomes suggest that neutron irradiation strongly impacts plant transcriptomes. The transition/transversion values among mutants were almost constant and were lower than that of the non-irradiated sample (WT), suggesting that neutron irradiation caused an effect. Taken together, this is the first report showing the effects of neutron irradiation on tomato plants by transcriptome analyses.

α-Ionone Protects Against UVB-Induced Photoaging in Human Dermal Fibroblasts.

Ultraviolet (UV) light-induced wrinkle formation is a major dermatological problem and is associated with alteration in collagen. Here, we investigated the potential of α-ionone, a naturally occurring aromatic compound, in regulation of UVB-induced photoaging in human Hs68 dermal fibroblasts and identified the mechanisms involved. We found that in human dermal fibroblasts, α-ionone inhibited UVB-induced loss of collagen. α-Ionone upregulated the molecules participating in the TGF-ß-SMAD pathway (TGF-ß1, phospho-SMAD2/3, Col1A1, and Col1A2), but downregulated the molecules involved in the MAPK-AP-1 signaling pathway (phospho-p38, phospho-JNK, phospho-ERK, phospho-c-Fos, phospho-c-Jun, MMP1, MMP3, and MMP9), in human dermal fibroblasts. α-Ionone treatment also increased hyaluronic acid contents, and this effect was accompanied by an upregulation of mRNA expression of genes (HAS1 and HAS2) involved in hyaluronic acid synthesis. Thus, α-ionone is effective in the prevention of UVB-induced decrease of collagen and hyaluronic acid in human dermal fibroblasts. We propose that α-ionone may prove beneficial for the prevention of UV-induced wrinkle formation and skin damage.

Artemisia Iwayomogi Extract Attenuates High-Fat Diet-Induced Hypertriglyceridemia in Mice: Potential Involvement of the Adiponectin-AMPK Pathway and Very Low Density Lipoprotein Assembly in the Liver.

This study aimed to examine the protective effect of Artemisia iwayomogi extract (AI) against hypertriglyceridemia induced by a high-fat diet (HFD) in mice and to uncover the underlying molecular mechanisms. C57BL/6N mice were fed chow, HFD, HFD + 0.1% AI, HFD + 0.25% AI, or HFD + 0.5% AI for 10 weeks. The addition of 0.25% and 0.5% AI resulted in dose-dependent improvements in the major parameters of hypertriglyceridemia, including plasma triglyceride, free fatty acids, apolipoprotein B, and lipoprotein lipase, with parallel reductions in body weight gain, hepatic lipid accumulation, and insulin resistance. These beneficial effects were accompanied by the activation of adiponectin-adenosine monophosphate-activated protein kinase (AMPK) mediated signaling cascades in the liver, which downregulated molecules involved in lipogenesis and concurrently upregulated molecules related to fatty acid oxidation. The downregulation of molecules involved in very low density lipoprotein assembly, which was associated with improved hepatic insulin signaling, also appeared to contribute to the AI-induced attenuation of hypertriglyceridemia.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

IL-7 receptor deletion ameliorates diet-induced obesity and insulin resistance in mice.

AIM/HYPOTHESIS: Obesity-induced inflammation plays an important role in the development of insulin resistance and type 2 diabetes. Recent studies have demonstrated that adiposity can be improved by ablating certain inflammatory signalling pathways. Although the IL-7 receptor (IL-7R) is mostly known as a key regulator of T lymphocyte development and homeostasis, its role in obesity and metabolic diseases is unknown. Because IL-7 is markedly increased in the serum of obese individuals and IL-7R (also known as IL7R) is overexpressed in white adipose tissue (WAT) in obesity, we studied the metabolic consequences of genetic Il-7r ablation in mice. METHODS: Age-matched Il-7r-deficient (Il-7r KO) and wild-type (WT) littermates were fed a standard chow or high-fat diet (HFD) for 14 weeks. Their serum metabolic variables were measured. The expression of genes and proteins related to insulin resistance and inflammation was evaluated in WAT. RESULTS: We demonstrated that Il-7r KO mice exhibited significantly reduced body weight gain and visceral adiposity compared with WT controls on both chow and HFD. The expression of signalling molecules involved in adipogenesis was reduced in the WAT of Il-7r KO mice. We also found that Il-7r KO mice had significantly enhanced glucose homeostasis and insulin sensitivity. Consistent with an improved metabolic phenotype, proinflammatory cytokine production and macrophage infiltration was attenuated in the WAT of Il-7r KO mice. CONCLUSIONS/INTERPRETATION: The IL-7R plays an important role in the induction of HFD-induced adipogenesis and insulin resistance in mice.

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter.

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4(+) T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.

Quercetin protects against obesity-induced skeletal muscle inflammation and atrophy.

Skeletal muscle inflammation and atrophy are closely associated with metabolic impairment such as insulin resistance. Quercetin, a natural polyphenol flavonoid, is known to elicit anti-inflammatory and antioxidant activities. In this study, we investigated its effect on obesity-induced skeletal muscle inflammation and atrophy in mice. Male C57BL/6 mice were fed a regular diet, a high-fat diet (HFD), and an HFD supplemented with quercetin for nine weeks. Quercetin reduced levels of inflammatory cytokines and macrophage accumulation in the skeletal muscle of the HFD-fed obese mice. It also reduced transcript and protein levels of the specific atrophic factors, Atrogin-1 and MuRF1, in the skeletal muscle of the HFD-fed obese mice, and protected against the reduction of muscle mass and muscle fiber size. In vitro, quercetin markedly diminished transcript levels of inflammatory receptors and activation of their signaling molecules (ERK, p38 MAPK, and NF-κB) in cocultured myotubes/macrophages, and this was accompanied by reduced expression of the atrophic factors. Together, these findings suggest that quercetin reduces obesity-induced skeletal muscle atrophy by inhibiting inflammatory receptors and their signaling pathway. Quercetin may be useful for preventing obesity-induced muscle inflammation and sarcopenia.

Postprandial glucose-lowering effects of fermented red ginseng in subjects with impaired fasting glucose or type 2 diabetes: a randomized, double-blind, placebo-controlled clinical trial.

BACKGROUND: Red ginseng is prepared by steaming raw ginseng, a process believed to increase the pharmacological efficacy. Further bioconversion of red ginseng through fermentation is known to increase its intestinal absorption and bioactivity, and bioconversion diminishes the toxicity of red ginseng's metabolite. This study was conducted to investigate the effects of daily supplementation with fermented red ginseng (FRG) on glycemic status in subjects with impaired fasting glucose or type 2 diabetes. METHODS: This study was a four-week long, randomized, double-blind, placebo-controlled trial. Forty-two subjects with impaired fasting glucose or type 2 diabetes were randomly allocated to two groups assigned to consume either the placebo or fermented red ginseng (FRG) three times per day for four weeks. Fasting and postprandial glucose profiles during meal tolerance tests were assessed before and after the intervention. RESULTS: FRG supplementation led to a significant reduction in postprandial glucose levels and led to an increase in postprandial insulin levels compared to the placebo group. There was a consistently significant improvement in the glucose area under the curve (AUC) in the FRG group. However, fasting glucose, insulin, and lipid profiles were not different from the placebo group. CONCLUSION: Daily supplementation with FRG lowered postprandial glucose levels in subjects with impaired fasting glucose or type 2 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01826409.

Blockade of 4-1BB and 4-1BBL interaction reduces obesity-induced skeletal muscle inflammation.

Obesity-induced skeletal muscle inflammation is characterized by increased macrophage infiltration and inflammatory cytokine production. In this study, we investigated whether 4-1BB, a member of the TNF receptor superfamily (TNFRSF9) that provides inflammatory signals, participates in obesity-induced skeletal muscle inflammation. Expression of the 4-1BB gene, accompanied by increased levels of inflammatory cytokines, was markedly upregulated in the skeletal muscle of obese mice fed a high-fat diet, in muscle cells treated with obesity factors, and in cocultured muscle cells/macrophages. In vitro stimulation of 4-1BB with agonistic antibody increased inflammatory cytokine levels in TNFα-pretreated muscle cells, and this effect was absent in cells derived from 4-1BB-deficient mice. Conversely, disruption of the interaction between 4-1BB and its ligand (4-1BBL) with blocking antibody decreased the release of inflammatory cytokines from cocultured muscle cells/macrophages. Moreover, deficiency of 4-1BB markedly reduced macrophage infiltration and inflammatory cytokine production in the skeletal muscle of mice fed a high-fat diet. These findings indicate that 4-1BB mediates the inflammatory responses in obese skeletal muscle by interacting with its ligand 4-1BBL on macrophages. Therefore, 4-1BB and 4-1BBL may be useful targets for prevention of obesity-induced inflammation in skeletal muscle.

The influence of the Korean traditional Chungkookjang on variables of metabolic syndrome in overweight/obese subjects: study protocol.

BACKGROUND: Metabolic syndrome is a set of disorders that increases the risk of developing cardiovascular disease. The primary target of treatment of patients with metabolic syndrome is therapeutic lifestyle change. Numerous preclinical study have reported positive effects of chungkookjang in in vivo models of diabetes and obesity, but there is a paucity of controlled clinical trials on variables of metabolic syndrome in obese subjects. Thus, the objective of this trial is to examine the effect of chungkookjang compared to placebo on variables of metabolic syndrome in overweight/obese subjects. METHODS: This double-blind randomized controlled crossover trial will be conducted on 120 overweight/obese subjects; aged 19-29 years. Subjects will be recruited from the Chonbuk National University, Jeonju, South Korea. Enrolled subjects will be randomly assigned to two groups of equal number; one group received 35 g of chungkookjang (n = 60) and the other group received placebo (n = 60) on a regular daily basis for 12 weeks. After a 12-week washout period, the groups will be crossed over. In addition to anthropometric measures and blood pressure, glucose parameter, lipid profile, adipocytokine, and carnitine assay will be determined at baseline and 12 week. Also, safety will be assessing by measuring total bilirubin, alkaline phosphatase, alanine transaminase, aspartate aminotransferase, total protein, albumin, blood urea nitrogen, creatinine, and creatine kinase at baseline and 12 weeks. 24-hour dietary recalls were collected at the baseline and at the end of the trial. DISCUSSION: This trial will evaluate the effects of chungkookjang on variables of metabolic syndrome in overweight/obese subjects. The results of this study may contribute to the reduction of risk factor for metabolic syndrome caused by obesity. TRIAL REGISTRATION: Clinical trials NCT01811511.