GPR41 and GPR43: From development to metabolic regulation.

G-protein-coupled receptors are a diverse class of cell surface receptors that orchestrate numerous physiological functions. The G-protein-coupled receptors, GPR41 and GPR43, sense short-chain fatty acids (SCFAs), which are metabolites of dietary fermentation by the host's intestinal bacteria. These receptors have gained attention as potential therapeutic targets against various diseases because of their SCFA-mediated beneficial effects on the host's intestinal health. Mounting evidence has associated the activity of these receptors with chronic metabolic diseases, including obesity, diabetes, inflammation, and cardiovascular disease. However, despite intensive research using various strategies, including gene knockout (KO) mouse models, evidence about the precise roles of GPR41 and GPR43 in disease treatment remains inconsistent. Here, we comprehensively review the latest findings from functional studies of the signaling mechanisms that underlie the activities of GPR41 and GPR43, as well as highlight their multifaceted roles in health and disease. We anticipate that this knowledge will guide future research priorities and the development of effective therapeutic interventions.

Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression.

BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

NINJ1 mediates inflammatory cell death, PANoptosis, and lethality during infection conditions and heat stress.

Innate immunity provides the first line of defense through multiple mechanisms, including pyrogen production and cell death. While elevated body temperature during infection is beneficial to clear pathogens, heat stress (HS) can lead to inflammation and pathology. Links between pathogen exposure, HS, cytokine release, and inflammation have been observed, but fundamental innate immune mechanisms driving pathology during pathogen exposure and HS remain unclear. Here, we use multiple genetic approaches to elucidate innate immune pathways in infection or LPS and HS models. Our results show that bacteria and LPS robustly increase inflammatory cell death during HS that is dependent on caspase-1, caspase-11, caspase-8, and RIPK3 through the PANoptosis pathway. Caspase-7 also contributes to PANoptosis in this context. Furthermore, NINJ1 is an important executioner of this cell death to release inflammatory molecules, independent of other pore-forming executioner proteins, gasdermin D, gasdermin E, and MLKL. In an in vivo HS model, mortality is reduced by deleting NINJ1 and fully rescued by deleting key PANoptosis molecules. Our findings suggest that therapeutic strategies blocking NINJ1 or its upstream regulators to prevent PANoptosis may reduce the release of inflammatory mediators and benefit patients.

Evaluation of Caspase Activation to Assess Innate Immune Cell Death.

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum. This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including apoptosis, pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases, from other cell death pathways. Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

The anti-obesity effect of mulberry leaf (Mori Folium) extracts was increased by bioconversion with Pectinex.

Mulberry leaf (Mori Folium) extract (MLE) is known to have anti-obesity effects. In this study, the enhanced effects of MLE after bioconversion treatment using Pectinex (BMLE) on obesity were explored, and the underlying mechanisms were investigated using the active components, neochlorogenic acid (5-CQA) and cryptochlorogenic acid (4-CQA), whose amounts were increased by bioconversion of MLE. Both MLE and BMLE inhibited lipid accumulation in 3T3-L1 adipocytes without cytotoxicity and suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBPα). In addition, MLE and BMLE decreased high-fat diet-induced adipose tissue mass expansion. Notably, BMLE significantly increased antiadipogenic and anti-obesity effects compared to MLE in vitro and in vivo. The active ingredients increased by bioconversion, 5-CQA and 4-CQA, inhibited the protein levels of C/EBPα and the mRNA levels of stearoyl-CoA desaturase 1 (Scd1). These findings provide new insights into the therapeutic possibility of using bioconversion of MLE, by which upregulation of 5-CQA and 4-CQA potently inhibits adipogenesis.

Single cell analysis of PANoptosome cell death complexes through an expansion microscopy method.

In response to infection or sterile insults, inflammatory programmed cell death is an essential component of the innate immune response to remove infected or damaged cells. PANoptosis is a unique innate immune inflammatory cell death pathway regulated by multifaceted macromolecular complexes called PANoptosomes, which integrate components from other cell death pathways. Growing evidence shows that PANoptosis can be triggered in many physiological conditions, including viral and bacterial infections, cytokine storms, and cancers. However, PANoptosomes at the single cell level have not yet been fully characterized. Initial investigations have suggested that key pyroptotic, apoptotic, and necroptotic molecules including the inflammasome adaptor protein ASC, apoptotic caspase-8 (CASP8), and necroptotic RIPK3 are conserved components of PANoptosomes. Here, we optimized an immunofluorescence procedure to probe the highly dynamic multiprotein PANoptosome complexes across various innate immune cell death-inducing conditions. We first identified and validated antibodies to stain endogenous mouse ASC, CASP8, and RIPK3, without residual staining in the respective knockout cells. We then assessed the formation of PANoptosomes across innate immune cell death-inducing conditions by monitoring the colocalization of ASC with CASP8 and/or RIPK3. Finally, we established an expansion microscopy procedure using these validated antibodies to image the organization of ASC, CASP8, and RIPK3 within the PANoptosome. This optimized protocol, which can be easily adapted to study other multiprotein complexes and other cell death triggers, provides confirmation of PANoptosome assembly in individual cells and forms the foundation for a deeper molecular understanding of the PANoptosome complex and PANoptosis to facilitate therapeutic targeting.

Garcinia Cambogia Improves High-Fat Diet-Induced Glucose Imbalance by Enhancing Calcium/CaMKII/AMPK/GLUT4-Mediated Glucose Uptake in Skeletal Muscle.

SCOPE: Garcinia cambogia (G. cambogia) is known to have antiobesity effects. In this study, the therapeutic effects of G. cambogia on glucose homeostasis in obesity-induced diabetes are explored and the underlying mechanisms are investigated. METHODS AND RESULTS: C2C12 myotubes are treated with G. cambogia; glucose uptake, intracellular Ca2+ levels, and related alterations in signaling pathways are examined. High-fat diet (HFD)-fed mice are administered G. cambogia for 8 weeks; oral glucose tolerance is evaluated, and the regulation of identified targets of signaling pathways in quadriceps skeletal muscle are examined in vivo. G. cambogia increases glucose uptake in C2C12 myotubes and induces the upregulation of AMPK, ACC, and p38 MAPK phosphorylation. Notably, G. cambogia markedly elevates both intracellular Ca2+ levels, activating CaMKII, a Ca2+ -sensing protein, and TBC1D4-mediated GLUT4 translocation, to facilitate glucose uptake. Furthermore, high-glucose-induced inhibition of glucose uptake and signal transduction is reverted by G. cambogia. In an HFD-induced diabetes mouse model, G. cambogia administration results in significant blood glucose-lowering effects, which are attributed to the regulation of targets that have been identified in vitro, in quadricep skeletal muscle. CONCLUSION: These findings provide new insights into the mechanism by which G. cambogia regulates glucose homeostasis in obesity-induced diabetes.

Garcinia cambogia attenuates adipogenesis by affecting CEBPB and SQSTM1/p62-mediated selective autophagic degradation of KLF3 through RPS6KA1 and STAT3 suppression.

The overexpansion of adipose tissues leads to obesity and eventually results in metabolic disorders. Garcinia cambogia (G. cambogia) has been used as an antiobesity supplement. However, the molecular mechanisms underlying the effects of G. cambogia on cellular processes have yet to be fully understood. Here, we discovered that G. cambogia attenuated the expression of CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), an important adipogenic factor, suppressing its transcription in differentiated cells. In addition, G. cambogia inhibited macroautophagic/autophagic flux by decreasing autophagy-related gene expression and autophagosome formation. Notably, G. cambogia markedly elevated the expression of KLF3 (Kruppel-like factor 3 (basic)), a negative regulator of adipogenesis, by reducing SQSTM1/p62-mediated selective autophagic degradation. Furthermore, increased KLF3 induced by G. cambogia interacted with CTBP2 (C-terminal binding protein 2) to form a transcriptional repressor complex and inhibited Cebpa and Pparg transcription. Importantly, we found that RPS6KA1 and STAT3 were involved in the G. cambogia-mediated regulation of CEBPB and autophagic flux. In an obese animal model, G. cambogia reduced high-fat diet (HFD)-induced obesity by suppressing epididymal and inguinal subcutaneous white adipose tissue mass and adipocyte size, which were attributed to the regulation of targets that had been consistently identified in vitro. These findings provide new insight into the mechanism of G. cambogia-mediated regulation of adipogenesis and suggest molecular links to therapeutic targets for the treatment of obesity.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG: autophagy-related; Baf: bafilomycin A1; BECN1: beclin 1; CEBP: CCAAT/enhancer binding protein (C/EBP); CHX: cycloheximide; CREB: cAMP response element binding protein; CTBP: C-terminal binding protein; EGCG: (-)-epigallocatechin gallate; eWAT: epididymal white; G. cambogia: Garcinia cambogia; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HFD: high-fat diet; iWAT: inguinal subcutaneous white; KLF: Kruppel-like factor; LAP: liver-enriched transcriptional activating proteins; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; PPARG: peroxisome proliferator activated receptor gamma; qPCR: quantitative real-time PCR; RFP: red fluorescent protein; RPS6KA1: ribosomal protein S6 kinase A1; siRNA: small-interfering RNA; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TEM: transmission electron microscopy.

Garcinia cambogia Ameliorates Non-Alcoholic Fatty Liver Disease by Inhibiting Oxidative Stress-Mediated Steatosis and Apoptosis through NRF2-ARE Activation.

Excessive free fatty acids (FFAs) causes reactive oxygen species (ROS) generation and non-alcoholic fatty liver disease (NAFLD) development. Garcinia cambogia (G. cambogia) is used as an anti-obesity supplement, and its protective potential against NAFLD has been investigated. This study aims to present the therapeutic effects of G. cambogia on NAFLD and reveal underlying mechanisms. High-fat diet (HFD)-fed mice were administered G. cambogia for eight weeks, and steatosis, apoptosis, and biochemical parameters were examined in vivo. FFA-induced HepG2 cells were treated with G. cambogia, and lipid accumulation, apoptosis, ROS level, and signal alterations were examined. The results showed that G. cambogia inhibited HFD-induced steatosis and apoptosis and abrogated abnormalities in serum chemistry. G. cambogia increased in NRF2 nuclear expression and activated antioxidant responsive element (ARE), causing induction of antioxidant gene expression. NRF2 activation inhibited FFA-induced ROS production, which suppressed lipogenic transcription factors, C/EBPα and PPARγ. Moreover, the ability of G. cambogia to inhibit ROS production suppressed apoptosis by normalizing the Bcl-2/BAX ratio and PARP cleavage. Lastly, these therapeutic effects of G. cambogia were due to hydroxycitric acid (HCA). These findings provide new insight into the mechanism by which G. cambogia regulates NAFLD progression.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) accelerates vascular remodelling via p53 and JAK2-STAT3 regulation in vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Abnormal vascular smooth muscle cell (VSMC) proliferation and migration lead to neointima formation, which eventually results in cardiovascular hyperplastic diseases. The molecular mechanisms underlying these cellular processes have not been fully understood. Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) has been identified as an anti-apoptotic molecule, but little is known about its target genes and related pathways in VSMC dysfunction or its clinical implication in neointima formation following vascular injury. EXPERIMENTAL APPROACH: Determination, using loss/gain-of-function approaches by gene delivery, of whether CIAPIN1 modulates VSMC proliferation, migration and neointima formation and the underlying mechanisms was carried out. Balloon injury or ligation and local delivery of lentivirus were performed on rat or mouse carotid arteries. Rat aortic smooth muscle cells, the primary cell, was used as the model to evaluate the effect of CIAPIN1 on proliferation and migration. KEY RESULTS: CIAPIN1 was overexpressed in the neointimal region of rat arteries. CIAPIN1 deficiency markedly inhibited injury-induced or ligation-induced intimal hyperplasia and suppressed PDGF-BB-induced VSMC proliferation, migration and cell cycle progression, while overexpression promoted proliferation, migration and neointima formation. CIAPIN1 negatively regulated Tp53 transcription, which promoted cell cycle progression and migration via cyclin E1-CDK2/pRb/PCNA and the MMP2 pathway. CIAPIN1 also increased JAK2 expression, enhancing JAK2 and STAT3 phosphorylation by vascular injury, which forced phenotypic switching from contractile to synthetic state in injured arteries. CONCLUSIONS AND IMPLICATIONS: These findings provide new insights into the mechanism by which CIAPIN1 regulates vascular remodelling and suggest a novel therapeutic target for treating vascular proliferative diseases.

Rosuvastatin Inhibits the Apoptosis of Platelet-Derived Growth Factor-Stimulated Vascular Smooth Muscle Cells by Inhibiting p38 via Autophagy.

The secretion of platelet-derived growth factors (PDGFs) into vascular smooth muscle cells (VSMCs) induced by specific stimuli, such as oxidized low-density lipoprotein (LDL) cholesterol, initially increases the proliferation and migration of VSMCs, and continuous stimulation leads to VSMC apoptosis, resulting in the formation of atheroma. Autophagy suppresses VSMC apoptosis, and statins can activate autophagy. Thus, this study aimed to investigate the mechanism of the autophagy-mediated vasoprotective activity of rosuvastatin, one of the most potent statins, in VSMCs continuously stimulated with PDGF-BB, a PDGF isoform, at a high concentration (100 ng/ml) to induce phenotypic switching of VSMC. Rosuvastatin inhibited apoptosis in a concentration-dependent manner by reducing cleaved caspase-3 and interleukin-1ß (IL-1ß) levels and reduced intracellular reactive oxygen species (ROS) levels in PDGF-stimulated VSMCs. It also inhibited PDGF-induced p38 phosphorylation and increased the expression of microtubule-associated protein light chain 3 (LC3) and the conversion of LC3-I to LC3-II in PDGF-stimulated VSMCs. The ability of rosuvastatin to inhibit apoptosis and p38 phosphorylation was suppressed by treatment with 3-methyladenine (an autophagy inhibitor) but promoted by rapamycin (an autophagy activator) treatment. SB203580, a p38 inhibitor, reduced the PDGF-induced increase in intracellular ROS levels and inhibited the formation of cleaved caspase-3, indicating the suppression of apoptosis. In carotid ligation model mice, rosuvastatin decreased the thickness and area of the intima and increased the area of the lumen. In conclusion, our observations suggest that rosuvastatin inhibits p38 phosphorylation through autophagy and subsequently reduces intracellular ROS levels, leading to its vasoprotective activity. SIGNIFICANCE STATEMENT: This study shows the mechanism responsible for the vasoprotective activity of rosuvastatin in vascular smooth muscle cells under prolonged platelet-derived growth factor stimulation. Rosuvastatin inhibits p38 activation through autophagy, thereby suppressing intracellular reactive oxygen species levels, leading to the inhibition of apoptosis and reductions in the intima thickness and area. Overall, these results suggest that rosuvastatin can be used as a novel treatment to manage chronic vascular diseases such as atherosclerosis.

Regulation of autophagy by controlling Erk1/2 and mTOR for platelet-derived growth factor-BB-mediated vascular smooth muscle cell phenotype shift.

AIMS: Vascular smooth muscle cell (VSMC) phenotype shift is involved in the pathophysiology of vascular injury or platelet-derived growth factor (PDGF)-induced abnormal proliferation and migration of VSMCs. We aimed to investigate the underlying mechanism involved in PDGF-mediated signaling pathways and autophagy regulation followed by VSMC phenotype shift. MAIN METHODS: The proliferation, migration and apoptosis of cultured rat aortic VSMCs were measured, and cells undergoing phenotype shift and autophagy were examined. Specific inhibitors for target proteins in signaling pathways were applied to clarify their roles in regulating cell functions. KEY FINDINGS: PDGF-BB stimulation initiated autophagy activation and synthetic phenotype transition by decreasing α-smooth muscle-actin (SMA), calponin and myosin heavy chain (MHC) and increasing osteopontin (OPN) expression. However, U0126, a potent extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, decreased PDGF-BB-induced LC3 expression, while rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), increased it. Furthermore, U0126 decreased the expresseion of autophagy-related genes (Atgs) such as beclin-1, Atg7, Atg5, and Atg12-Atg5 complex, indicating that Erk1/2 is a regulator of PDGF-BB-induced VSMC autophagy. Regardless of autophagy inhibition by U0126 or activation by rapamycin, the PDGF-BB-induced decrease in SMA, calponin and MHC and increase in OPN expression were inhibited. Furthermore, PDGF-BB-stimulated VSMC proliferation, migration and proliferating cell nuclear antigen (PCNA) expression were inhibited by U0126 and rapamycin. SIGNIFICANCE: These findings suggest that PDGF-BB-induced autophagy is strongly regulated by Erk1/2, an mTOR-independent pathway, and any approach for targeting autophagy modulation is a potential therapeutic strategy for addressing abnormal VSMC proliferation and migration.

Garcinia cambogia suppresses adipogenesis in 3T3-L1 cells by inhibiting p90RSK and Stat3 activation during mitotic clonal expansion.

Obesity is associated with an increase in adipose tissue, which is mediated by hyperplasia and hypertrophy. Therefore, inhibiting cell proliferation during mitotic clonal expansion (MCE) is one of the major strategies for preventing obesity. The antagonistic effects of Garcinia cambogia (G. cambogia) on obesity have been studied in animal experimental models. However, the effects of G. cambogia extract on MCE, and the underlying molecular mechanisms, are poorly understood. In this study, 3T3-L1 cells were used to investigate whether G. cambogia extract affected cell proliferation during MCE and to identify target molecules for any anti-adipogenic activity. G. cambogia extract suppressed isobutylmethylxanthine and dexamethasone-and-insulin (MDI)-induced adipogenesis at an early stage by attenuating MCE. In G. cambogia extract-treated preadipocytes, MDI-induced cell proliferation and cell cycle progression were inhibited by G0 /G1 arrest due to an increase in p21 and p27 expression, and inhibition of cyclin-dependent kinase 2, cyclin E1 expression, and retinoblastoma (Rb) phosphorylation. In addition, the MDI-induced phosphorylation and subsequent translocation into the nucleus of p90 ribosomal S6 kinase (p90RSK) and signal transducer and activator of transcription (Stat) 3 were suppressed. Specific inhibitors of p90RSK (FMK) and Stat3 (stattic) regulated cell proliferation and adipogenesis. In conclusion, this study demonstrated that G. cambogia extract inhibited MCE by regulating p90RSK, Stat3, and cell cycle proteins, leading to G0 /G1 arrest. These findings provide new insight into the mechanism by which G. cambogia suppresses adipocyte differentiation and show that p90RSK is critical for adipogenesis as a new molecular target.

Sodium propionate exerts anticancer effect in mice bearing breast cancer cell xenograft by regulating JAK2/STAT3/ROS/p38 MAPK signaling.

Propionate is a short-chain fatty acid (SCFA) mainly produced from carbohydrates by gut microbiota. Sodium propionate (SP) has shown to suppress the invasion in G protein-coupled receptor 41 (GPR41) and GPR43-overexpressing breast cancer cells. In this study we investigated the effects of SP on the proliferation, apoptosis, autophagy, and antioxidant production of breast cancer cells. We showed that SP (5-20 mM) dose-dependently inhibited proliferation and induced apoptosis in breast cancer cell lines JIMT-1 (ER-negative and HER2-expressing) and MCF7 (ER-positive type), and this effect was not affected by PTX, thus not mediated by the GPR41 or GPR43 SCFA receptors. Meanwhile, we demonstrated that SP treatment increased autophagic and antioxidant activity in JIMT-1 and MCF7 breast cancer cells, which might be a compensatory mechanism to overcome SP-induced apoptosis, but were not sufficient to overcome SP-mediated suppression of proliferation and induction of apoptosis. We revealed that the anticancer effect of SP was mediated by inhibiting JAK2/STAT3 signaling which led to cell-cycle arrest at G0/G1 phase, and increasing levels of ROS and phosphorylation of p38 MAPK which induced apoptosis. In nude mice bearing JIMT-1 and MCF7 cells xenograft, administration of SP (20 mg/mL in drinking water) significantly suppressed tumor growth by regulating STAT3 and p38 in tumor tissues. These results suggest that SP suppresses proliferation and induces apoptosis in breast cancer cells by inhibiting STAT3, increasing the ROS level and activating p38. Therefore, SP is a candidate therapeutic agent for breast cancer.

Statistical Design of Sustained-Release Tablet Garcinia cambogia Extract and Bioconverted Mulberry Leaf Extract for Anti-Obesity.

Obesity is a major health concern worldwide, and it is leading to worsening disease morbidity and mortality. Herbal supplements and diet-based therapies have attracted interest in the treatment of obesity. It is known that Garcinia cambogia (GA) and mulberry leaf, which contain polyphenols, have anti-obesity activity. Herein, we developed a combined tablet consisting of GA extract and bioconverted mulberry leaf extract (BMUL) using a statistical design approach. The ratio and amount of sustained polymers were set as factors. In the cell study, the combination of GA and BMUL showed synergistic anti-obesity activity. In a statistical model, the optimized amounts of hydroxypropyl methylcellulose 2208 (HPMC 2208) and polyethylene oxide 303 (POLYOX 303) were 41.02% and 58.98%, respectively. Additionally, the selected ratio of microcrystalline cellulose (MCC) was 0.33. When the release, hardness, and friability of the GABMUL tablet were evaluated, the error percentages of the response were lower than 10%. This indicates that the GABMUL tablet was successfully prepared.

Hepatoprotective effects of an Acer tegmentosum Maxim extract through antioxidant activity and the regulation of autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Acer tegmentosum Maxim (AT), the East Asian stripe maple, is an herb used to treat liver disease and is approved as a functional food in Korea. AT protects against hepatic disorders, atopic dermatitis, and diabetes mellitus. AIM OF THE STUDY: We explored the mechanism of the hepatoprotective effects of AT extract in in vitro and in vivo levels. MATERIALS AND METHODS: AT extract from Acer tegmentosum Maxim was extracted by hot water. Hepatoprotective effects of AT extract were confirmed using carbon tetrachloride (CCl4)- or alcohol-induced mouse model, and H2O2- or alcohol-induced HepG2 (liver hepatocellular carcinoma cell line) cells by measuring GOT, GPT, TG, and MDA levels. Hematoxylin and eosin (H&E) staining was used to observe the pathological analysis. Cytotoxicity or protective effect of AT extract was confirmed using MTT assay in HepG2 cells. Antioxidant effect of AT extract was measured using DPPH or H2DCFDA assay. Mechanism study of antioxidant and autophagy was carried out using western blotting and immunofluorescence analysis. RESULTS: AT extract increased the viability of HepG2 cells treated with H2O2 and ethanol, and protected the liver against damage induced by CCl4 and alcohol. The AT extract increased the levels of nuclear respiratory factor 2 (Nrf2) and heme oxygenase-1 (HO-1). The level of microtubule-associated protein light chain 3 (LC3)-Ⅱ, beclin-1, autophagy-related genes (Atg) such as Atg3 and Atg12-5 as markers of autophagy activation was also increased. Moreover, the AT extract increased activation of mitogen-activated protein kinase (MAPK), which regulated autophagy and HO-1. CONCLUSION: Therefore, these results indicate that the AT extract has a hepatoprotective effect by increasing antioxidant activity and inducing autophagy.

Effects of unaltered and bioconverted mulberry leaf extracts on cellular glucose uptake and antidiabetic action in animals.

BACKGROUND: Mulberry is a Korean medicinal herb that shows effective prevention and treatment of obesity and diabetes. Bioconversion is the process of producing active ingredients from natural products using microorganisms or enzymes. METHODS: In this study, we prepared bioconverted mulberry leaf extract (BMLE) with Viscozyme L, which we tested in insulin-sensitive cells (i.e., skeletal muscle cells and adipocytes) and insulin-secreting pancreatic ß-cells, as well as obese diabetic mice induced by co-administration of streptozotocin (100 mg/kg, IP) and nicotinamide (240 mg/kg, IP) and feeding high-fat diet, as compared to unaltered mulberry leaf extract (MLE). RESULTS: BMLE increased the glucose uptake in C2C12 myotubes and 3 T3-L1 adipocytes and increased glucose-stimulated insulin secretion in HIT-T15 pancreatic ß-cells. The fasting blood glucose levels in diabetic mice treated with BMLE or MLE (300 and 600 mg/kg, PO, 7 weeks) were significantly lower than those of the vehicle-treated group. At the same concentration, BMLE-treated mice showed better glucose tolerance than MLE-treated mice. Moreover, the blood concentration of glycated hemoglobin (HbA1C) in mice treated with BMLE was lower than that in the MLE group at the same concentration. Plasma insulin levels in mice treated with BMLE or MLE tended to increase compared to the vehicle-treated group. Treatment with BMLE yielded significant improvements in insulin resistance and insulin sensitivity. CONCLUSION: These results indicate that in the management of diabetic condition, BMLE is superior to unaltered MLE due to at least, in part, high concentrations of maker compounds (trans-caffeic acid and syringaldehyde) in BMLE.

Anti-apoptotic effects of autophagy via ROS regulation in microtubule-targeted and PDGF-stimulated vascular smooth muscle cells.

Autophagy has been studied as a therapeutic strategy for cardiovascular diseases. However, insufficient studies have been reported concerning the influence of vascular smooth muscle cells (VSMCs) through autophagy regulation. The aim of the present study was to determine the effects of VSMCs on the regulation of autophagy under in vitro conditions similar to vascular status of the equipped microtubule target agent-eluting stent and increased release of platelet-derived growth factor-BB (PDGF-BB). Cell viability and proliferation were measured using MTT and cell counting assays. Immunofluorescence using an anti-α-tubulin antibody was performed to determine microtubule dynamic formation. Cell apoptosis was measured by cleavage of caspase-3 using western blot analysis, and by nuclear fragmentation using a fluorescence assay. Autophagy activity was assessed by microtubule-associated protein light chain 3-II (LC-II) using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured using H2DCFDA. The proliferation and viability of VSMCs were inhibited by microtubule regulation. Additionally, microtubule-regulated and PDGF-BB-stimulated VSMCs increased the cleavage of caspase-3 more than only the microtubule-regulated condition, similar to that of LC3-II, implying autophagy. Inhibitory autophagy of microtubule-regulated and PDGF-BB-stimulated VSMCs resulted in low viability. However, enhancement of autophagy maintained survival through the reduction of ROS. These results suggest that the apoptosis of conditioned VSMCs is decreased by the blocking generation of ROS via the promotion of autophagy, and proliferation is also inhibited. Thus, promoting autophagy as a therapeutic target for vascular restenosis and atherosclerosis may be a good strategy.

Cucurbitane Triterpenoids from the Fruits of Momordica Charantia Improve Insulin Sensitivity and Glucose Homeostasis in Streptozotocin-Induced Diabetic Mice.

SCOPE: Momordica charantia (M. charantia) has antidiabetic effects, and cucurbitane-type triterpenoid is one of the compounds of M. charantia. This study aims to investigate whether the new cucurbitane-type triterpenoids affect insulin sensitivity both in vitro and in vivo, and the underlying mechanisms. METHODS AND RESULTS: Four compounds (C1-C4) isolated from the ethanol extract of M. charantia enhance glucose uptake in C2C12 myotubes via insulin receptor substrate-1 (IRS-1) rather than via adenosine monophosphate-activated protein kinase. The most potent, compound 2 (C2), significantly increases the activation of IRS-1 and downstream signaling pathways, resulting in glucose transporter 4 translocation. Furthermore, these C2-induced in vitro effects are blocked by specific signal inhibitors. We further evaluate the antidiabetic effect of C2 using a streptozotocin (STZ)-induced diabetic mouse model. Consistent with in vitro data, treatment with C2 (1.68 mg kg-1 ) significantly decreases blood glucose level and enhances glycogen storage in STZ-injected mice. These effects appear to be mediated by the IRS-1 signaling pathway in skeletal muscle, not in adipose and liver tissues, suggesting that C2 improves hyperglycemia by increasing glucose uptake into skeletal muscle. CONCLUSION: Our findings demonstrate that the new cucurbitane-type triterpenoids have potential for prevention and management of diabetes by improving insulin sensitivity and glucose homeostasis.

Alleviation of ascorbic acid-induced gastric high acidity by calcium ascorbate in vitro and in vivo.

Ascorbic acid is one of the most well-known nutritional supplement and antioxidant found in fruits and vegetables. Calcium ascorbate has been developed to mitigate the gastric irritation caused by the acidity of ascorbic acid. The aim of this study was to compare calcium ascorbate and ascorbic acid, focusing on their antioxidant activity and effects on gastric juice pH, total acid output, and pepsin secretion in an in vivo rat model, as well as pharmacokinetic parameters. Calcium ascorbate and ascorbic acid had similar antioxidant activity. However, the gastric fluid pH was increased by calcium ascorbate, whereas total acid output was increased by ascorbic acid. In the rat pylorus ligation-induced ulcer model, calcium ascorbate increased the gastric fluid pH without changing the total acid output. Administration of calcium ascorbate to rats given a single oral dose of 100 mg/kg as ascorbic acid resulted in higher plasma concentrations than that from ascorbic acid alone. The area under the curve (AUC) values of calcium ascorbate were 1.5-fold higher than those of ascorbic acid, and the Cmax value of calcium ascorbate (91.0 ng/ml) was higher than that of ascorbic acid (74.8 ng/ml). However, their Tmax values were similar. Thus, although calcium ascorbate showed equivalent antioxidant activity to ascorbic acid, it could attenuate the gastric high acidity caused by ascorbic acid, making it suitable for consideration of use to improve the side effects of ascorbic acid. Furthermore, calcium ascorbate could be an appropriate antioxidant substrate, with increased oral bioavailability, for patients with gastrointestinal disorders.