Single oral administration of quercetin glycosides prevented acute hyperglycemia by promoting GLUT4 translocation in skeletal muscles through the activation of AMPK in mice.

Quercetin is a natural flavonol and has various health beneficial functions. Our pervious study demonstrated that long-term feeding (13 weeks) of quercetin and its glycosides, isoquercitrin, rutin, and enzymatically modified isoquercitrin, which is a mixture of quercetin monoglycoside and its oligoglycosides, prevented hyperglycemia and adiposity in mice fed a high-fat diet but not standard diet. It is, however, unclear whether a single administration of these compounds prevent postprandial hyperglycemia or not. In the present study, we estimated their prevention effect on acute hyperglycemia by an oral glucose tolerance test in ICR mice and investigated its mechanism. It was found that quercetin glycosides, but not the aglycone, suppressed acute hyperglycemia and isoquercitrin showed the strongest effect among the glycosides. As the underlying mechanism, quercetin glycosides promoted translocation of glucose transporter 4 to the plasma membrane of skeletal muscle of mice through phosphorylation of adenosine monophosphate-activated protein kinase and its upstream Ca2+/calmodulin-dependent protein kinase kinase ß without activating the insulin- and JAK/STAT-signal pathways. In conclusion, single oral administration of quercetin glycosides prevented a blood sugar spike by promoting glucose transporter 4 translocation through activating the CAMKKß/AMPK signaling pathway.

Androgen receptor suppresses ß-adrenoceptor-mediated CREB activation and thermogenesis in brown adipose tissue of male mice.

Thermoregulation is a process by which core body temperature is maintained in mammals. Males typically have a lower body temperature than females. However, the effects of androgens, which show higher levels in males, on adrenergic receptor-mediated thermogenesis remain unclear. Here, we demonstrate that androgen-androgen receptor (AR) signaling suppresses the ß-adrenergic agonist-induced rise of core body temperature using castrated and AR knockout (ARKO) male mice. Furthermore, in vitro mechanistic studies show that activated AR inhibits cAMP response element (CRE)-mediated transcription by suppressing cAMP response element-binding protein (CREB) phosphorylation. The elevation of body temperature induced by the ß-adrenergic agonist CL316243 was higher in ARKO and castrated mice than in the control mice. Similarly, CL316243 induced a greater increase in Uncoupling protein 1 (Ucp1) expression and CREB phosphorylation in the brown adipose tissue of ARKO mice than in that of controls. We determined that activation of AR by dihydrotestosterone suppressed ß3-agonist- or forskolin-induced CRE-mediated transcription, which was prevented by AR antagonist. AR activation also suppressed CREB phosphorylation induced by forskolin. Moreover, we found AR nuclear localization, but not transcriptional activity, was necessary for the suppression of CRE-mediated transcription. Finally, modified mammalian two-hybrid and immunoprecipitation analyses suggest nuclear AR and CREB form a protein complex both in the presence and absence of dihydrotestosterone and forskolin. These results suggest androgen-AR signaling suppresses ß-adrenoceptor-induced UCP1-mediated brown adipose tissue thermogenesis by suppressing CREB phosphorylation, presumably owing to a protein complex with AR and CREB. This mechanism explains sexual differences in body temperature, at least partially.

All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion.

All-trans retinoic acid (ATRA) promotes myoblast differentiation into myotubes. Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) is a candidate ATRA-responsive gene; however, its role in skeletal muscles remains unclear. Here, we demonstrated that during the differentiation of murine C2C12 myoblasts into myotubes, Lgr6 mRNA expression transiently increased before the increase in the expression of the mRNAs encoding myogenic regulatory factors, such as myogenin, myomaker, and myomerger. The loss of LGR6 decreased the differentiation and fusion indices. The exogenous expression of LGR6 up to 3 and 24 h after the induction of differentiation increased and decreased the mRNA levels of myogenin, myomaker, and myomerger, respectively. Lgr6 mRNA was transiently expressed after myogenic differentiation in the presence of a retinoic acid receptor α (RARα) agonist and an RARγ agonist in addition to ATRA, but not in the absence of ATRA. Furthermore, a proteasome inhibitor or Znfr3 knockdown increased exogenous LGR6 expression. The loss of LGR6 attenuated the Wnt/ß-catenin signaling activity induced by Wnt3a alone or in combination with Wnt3a and R-spondin 2. These results indicate that LGR6 promotes myogenic differentiation and that ATRA is required for the transient expression of LGR6 during differentiation. Furthermore, LGR6 expression appeared to be downregulated by the ubiquitin-proteasome system involving ZNRF3.

Curcumin activates G protein-coupled receptor 97 (GPR97) in a manner different from glucocorticoid.

Curcumin is a yellow pigment in turmeric (Curcuma longa) with various physiological effects in the body. To elucidate the molecular mechanisms by which bioactive compounds exert their function, identification of their molecular targets is crucial. In this study, we show that curcumin activates G protein-coupled receptor 97 (GPR97). Curcumin dose-dependently activated serum-response element-, but not serum-response factor-response element-, nuclear factor of activated T-cell-response element-, or cAMP-response element-, mediated transcription in cells overexpressed with GPR97. The structure-activity relationship indicated that (i) the double-bonds of the central 7-carbon chain were essential for activation; (ii) a methoxy group on the aromatic ring was required for maximal activity; (iii) the addition of glucuronic acid moiety or a methoxy group to the aromatic ring, but not the methylation of the aromatic p-hydroxy group, eliminated the activity; (iv) the stability of curcumin would be related to receptor activation. Both mutant GPR97(T250A) lacking the cleavage at GPCR proteolysis site and mutant GPR97(ΔN) lacking the N-terminal extracellular region were activated by curcumin and its related compounds similar to wild-type GPR97. In contrast, the synthetic glucocorticoid beclomethasone dipropionate and l-Phe activated wild-type GPR97 and GPR97(T250A), but not GPR97(ΔN). Moreover, curcumin exerted an additive effect on the activation of wild-type GPR97 with beclomethasone dipropionate, but not with l-Phe. Taken together, these results indicate that curcumin activates GPR97 coupled to Gi/Go subunit, and suggest that curcumin and glucocorticoid activate GPR97 in a different manner.

Dietary oleamide attenuates obesity induced by housing mice in small cages.

Physical inactivity due to prolonged sedentary behavior induces obesity. Therefore, we investigated whether housing mice in small cages to mimic sedentary behavior induced obesity and whether dietary oleamide (cis-9,10-octadeceneamide) suppressed the induced obesity. A single oral administration of oleamide (50 mg/kg) to mice resulted in the accumulation of the exogenous oleamide in abdominal visceral fat. Next, mice were housed in small cages and oleamide (50 mg/kg/d) was orally administered for 12 weeks. Housing mice in small cages impaired glucose tolerance and increased food efficiency. It also increased body weight and abdominal fat mass. Dietary oleamide improved the impairment and inhibited their increase in mice housed in small cages. Furthermore, dietary oleamide suppressed the mRNA expression of inflammation-related factors in the abdominal fat of mice housed in small cages. Hence, these results indicate that although housing mice in small cages induces obesity and increases abdominal fat mass, dietary oleamide suppresses the obesity.

Carotenoid transporter CD36 expression depends on hypoxia-inducible factor-1α in mouse soleus muscles.

Dietary ß-carotene induces muscle hypertrophy and prevents muscle atrophy in red slow-twitch soleus muscles, but not in white fast-twitch extensor digitorum longus (EDL) muscles and gastrocnemius muscles. However, it remains unclear why these beneficial effects of ß-carotene are elicited in soleus muscles. To address this issue, we focused on carotenoid transporters in skeletal muscles. In mice, Cd36 mRNA levels were higher in red muscle than in white muscle. The siRNA-mediated knockdown of CD36 decreased ß-carotene uptake in C2C12 myotubes. In soleus muscles, CD36 knockdown inhibited ß-carotene-induced increase in muscle mass. Intravenous injection of the hypoxia marker pimonidazole produced more pimonidazole-bound proteins in soleus muscles than in EDL muscles, and the hypoxia-inducible factor-1 (HIF-1) α protein level was higher in soleus muscles than in EDL muscles. In C2C12 myotubes, hypoxia increased the expression of CD36 and HIF-1α at the protein and mRNA levels, and HIF-1α knockdown reduced hypoxia-induced increase in Cd36 mRNA level. In soleus muscles, HIF-1α knockdown reduced Cd36 mRNA level. These results indicate that CD36 is predominantly involved in ß-carotene-induced increase in soleus muscle mass of mice. Furthermore, we demonstrate that CD36 expression depends on HIF-1α in the soleus muscles of mice, even under normal physiological conditions.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.

Role of gut microbiota in sex- and diet-dependent metabolic disorders that lead to early mortality of androgen receptor-deficient male mice.

The gut microbiota is involved in metabolic disorders induced by androgen deficiency after sexual maturation in males (late-onset hypogonadism). However, its role in the energy metabolism of congenital androgen deficiency (e.g., androgen-insensitive syndrome) remains elusive. Here, we examined the link between the gut microbiota and metabolic disease symptoms in androgen receptor knockout (ARKO) mouse by administering high-fat diet (HFD) and/or antibiotics. HFD-fed male, but not standard diet-fed male or HFD-fed female, ARKO mice exhibited increased feed efficiency, obesity with increased visceral adipocyte mass and hypertrophy, hepatic steatosis, glucose intolerance, insulin resistance, and loss of thigh muscle. In contrast, subcutaneous fat mass accumulated in ARKO mice irrespective of the diet and sex. Notably, all HFD-dependent metabolic disorders observed in ARKO males were abolished after antibiotics administration. The ratios of fecal weight-to-food weight and cecum weight-to-body weight were specifically reduced by ARKO in HFD-fed males. 16S rRNA sequencing of fecal microbiota from HFD-fed male mice revealed differences in microbiota composition between control and ARKO mice. Several genera or species (e.g., Turicibacter and Lactobacillus reuteri, respectively) were enriched in ARKO mice, and antibiotics treatment spoiled the changes. Furthermore, the life span of HFD-fed ARKO males was shorter than that of control mice, indicating that androgen deficiency causes metabolic dysfunctions leading to early death. These findings also suggest that AR signaling plays a role in the prevention of metabolic dysfunctions, presumably by influencing the gut microbiome, and improve our understanding of health consequences in subjects with hypogonadism and androgen insensitivity.

6-(Methylsulfinyl)hexyl isothiocyanate protects acetaldehyde-caused cytotoxicity through the induction of aldehyde dehydrogenase in hepatocytes.

In the body, alcohol dehydrogenase rapidly converts ethanol to its toxic metabolite, acetaldehyde, which is further metabolized to non-toxic acetic acid by aldehyde dehydrogenase (ALDH). 6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC), a major bioactive compound in Wasabi (Wasabia japonica) has various physiological effects such as anti-oxidative, anti-inflammatory and anti-cancer effects. However, the effect of 6-MSITC on alcohol metabolism has not been studied. In this study, we investigated the effects of 6-MSITC on hepatic ALDH activity and protein expression both in vitro and in vivo. 6-MSITC inhibited ethanol- and acetaldehyde-induced cytotoxicity. Treatment with 6-MSITC to HepG2 cells enhanced ALDH activity through the induction of mitochondrial ALDH2 expression, but not cytosolic ALDH1A1. Knockdown of Nrf2 canceled the 6-MSITC-induced ALDH2 expression, indicating that Nrf2 regulated ALDH2 expression. Moreover, 6-MSITC increased the nuclear translocation of Nrf2 and the expression levels of HO-1 and SOD2, Nrf2-regulated phase II drug-metabolizing enzymes. Oral administration of 6-MSITC increased the mitochondrial ALDH2 activity and its expression in the liver of C57BL/6J mice. These results suggested that 6-MSITC is possible to protect acetaldehyde toxicity in hepatocytes by induction of mitochondrial ALDH2 expression through Nrf2/ARE pathway.

Enzymatically synthesized glycogen protects inflammation induced by urban particulate matter in normal human epidermal keratinocytes.

Urban particulate matters (PM) exposure is significantly correlated with extrinsic skin aging signs and skin cancer incidence. PM contains polycyclic aromatic hydrocarbons, and they act as the agonists of aryl hydrocarbon receptor (AhR). Activation of AhR promotes generation of intracellular reactive oxygen species (ROS) and inflammation. Enzymatically synthesized glycogen (ESG), which is synthesized from starch, possesses various functions, such as anti-tumor, anti-obesity and antioxidant. However, the effects of ESG on PM-induced skin inflammation remain unclear. In this study, we investigated whether ESG has a protective effect on PM-induced oxidative stress and inflammation in human epidermal keratinocytes. ESG inhibited PM-induced expression of inflammatory cytokines IL6, TNFA and PTGS2. ESG also inhibited PM-induced phosphorylation of MAPKs and ROS accumulation. However, ESG had no effect on PM-induced expression of CYP1A1, one of the target proteins of AhR. On the other hand, ESG increased nuclear translocation of Nrf2 and expression of antioxidant proteins, HO-1 and NQO1. These results suggest that ESG suppressed PM-induced inflammation by decreasing ROS accumulation through the Nrf2 pathway.

Enzymatically synthesized glycogen prevents ultraviolet B-induced cell damage in normal human epidermal keratinocytes.

Enzymatically synthesized glycogen is a product from starch. Enzymatically synthesized glycogen has been reported to possess various health beneficial effects such as anti-oxidative and anti-inflammatory effects. In this study, we investigated the effect of enzymatically synthesized glycogen on ultraviolet B-induced oxidative stress and apoptosis in normal human epidermal keratinocytes. Treatment with enzymatically synthesized glycogen suppressed ultraviolet B-induced reactive oxygen species, caspase-3 activity, and DNA fragmentation in normal human epidermal keratinocytes. Furthermore, enzymatically synthesized glycogen increased in the expression level of heme oxygenase-1, NAD(P)H: quinone oxidoreductase 1, and NF-E2-related factor 2, a transcriptional factor for heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1. Although enzymatically synthesized glycogen did not increase in its mRNA expression level of NF-E2-related factor 2, enzymatically synthesized glycogen retained its protein degradation. Knockdown of heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1 canceled enzymatically synthesized glycogen-suppressed reactive oxygen species accumulation in normal human epidermal keratinocytes. It is, therefore, concluded that enzymatically synthesized glycogen inhibited ultraviolet B-induced oxidative stress through increasing the expression level of heme oxygenase-1 and NAD(P)H: quinone oxidoreductase 1 through the NF-E2-related factor 2 pathway in normal human epidermal keratinocytes.

Bisacurone suppresses hepatic lipid accumulation through inhibiting lipogenesis and promoting lipolysis.

Turmeric and its components have various health beneficial functions. However, little is known about function of bisacurone, which is one of the sesquiterpenes in turmeric, at the compound level. In this study, we investigated the preventive effect of bisacurone on hepatic lipid accumulation and its mechanism in HepG2 cells and ICR mice. In HepG2 cells, bisacurone significantly inhibited fatty acid-induced intracellular lipid accumulation in a dose-dependent manner. Bisacurone at 10 µM increased protein expression of peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase-1A accompanied by phosphorylation of AMP-activated protein kinase. In the liver of ICR mice, bisacurone decreased total lipids, triglyceride, and cholesterol contents. Bisacurone at 10 mg/kg body weight increased phosphorylation of AMP-activated protein kinase, and its downstream acetyl-CoA carboxylase as a rate-limiting enzyme for lipogenesis, while it decreased the nuclear translocation level of sterol regulatory element-binding protein 1 and carbohydrate-responsive element-binding protein as the major transcription factors for lipogenesis. On the other hand, bisacurone promoted lipolysis by up-expression of peroxisome proliferator-activated receptor α and carnitine palmitoyltransferase-1A. Thus, bisacurone might be a valuable food factor for preventing hepatic lipid accumulation by inhibiting lipogenesis and promoting lipolysis through phosphorylation of AMP-activated protein kinase.

Enzymatically synthesized glycogen inhibited degranulation and inflammatory responses through stimulation of intestine.

The patients of type I allergic diseases were increased in the developed countries. Recently, many studies have focused on food factors with anti-allergic activities. Enzymatically synthesized glycogen, a polysaccharide with a multi-branched α-1,4 and α-1,6 linkages, is a commercially available product from natural plant starch, and has immunostimulation activity. However, effect of enzymatically synthesized glycogen on the anti-allergic activity was unclear yet. In this study, we investigated that enzymatically synthesized glycogen inhibited allergic and inflammatory responses using a co-culture system consisting of Caco-2 and RBL-2H3 cells. Enzymatically synthesized glycogen inhibited antigen-induced ß-hexosaminidase release and production of TNF-α and IL-6 in RBL-2H3 cells in the co-culture system. Furthermore, enzymatically synthesized glycogen inhibited antigen-induced phosphorylation of tyrosine kinases, phospholipase C γ1/2, mitogen-activated protein kinases and Akt. Anti-allergic and anti-inflammatory activities of enzymatically synthesized glycogen were indirect action through stimulating Caco-2 cells, but not by the direct interaction with RBL-2H3 cells, because enzymatically synthesized glycogen did not permeate Caco-2 cells. These findings suggest that enzymatically synthesized glycogen is an effective food ingredient for prevention of type I allergy through stimulating the intestinal cells.

Prevention effect of quercetin and its glycosides on obesity and hyperglycemia through activating AMPKα in high-fat diet-fed ICR mice.

Quercetin and its glycosides possess various health beneficial functions, but comparative study of them on energy metabolism in different tissues are not well studied. In this study, we investigated AMP-activated protein kinase regulated glucose metabolism in the skeletal muscle and lipid metabolism in the white adipose tissue and liver to compare the effectiveness of quercetin and its glycosides, namely isoquercitrin, rutin, and enzymatically modified isoquercitrin, in male ICR mice. The mice were fed a standard or high-fat diet supplemented with 0.1% quercetin and its glycosides for 13 weeks. Quercetin glycosides, but not quercetin, decreased body weight gain and fat accumulation in the mesenteric adipose tissue in high-fat groups. All compounds decreased high-fat diet-increased plasma glucose and insulin levels. Moreover, all compounds significantly increased AMP-activated protein kinase phosphorylation in either standard or high-fat diet-fed mice in all tissues tested. As its downstream events, all compounds induced glucose transporter 4 translocation in the muscle. In the white adipose tissue and liver, all compounds increased lipogenesis while decreased lipolysis. Moreover, all compounds increased browning markers and decreased differentiation markers in adipose tissue. Therefore, quercetin and its glycosides are promising food components for prevention of adiposity and hyperglycemia through modulating AMP-activated protein kinase-driven pathways.

A physiological concentration of luteolin induces phase II drug-metabolizing enzymes through the ERK1/2 signaling pathway in HepG2 cells.

The flavon luteolin has various health-promoting activities including cardiovascular protection, anti-inflammatory activity and anticancer activity. A serum concentration of about 100 nM luteolin is reached by dietary habit. However, little is known about the function of luteolin over its physiological concentration range. In this study, we investigated whether a physiological concentration of luteolin could activate nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated expression of phase II drug-metabolizing enzymes in human hepatoma HepG2 cells. Interestingly, less than 1 nM of luteolin could induce phase II drug-metabolizing enzymes, such as GSTs, HO-1, and NQO1. Both 1 and 100 nM luteolin increased expression and activity of ALDH2, which metabolized toxic acetaldehyde into nontoxic acetic acid. Luteolin increased nuclear accumulation of Nrf2 and enhanced the ARE-binding complex through increasing the stability of the Nrf2 protein. Luteolin increased phosphorylation of Nrf2 at Ser40, and MEK inhibitors (U0126 and PD98059) canceled luteolin-induced phosphorylation of Nrf2. Furthermore, luteolin increased modified Keap1. In conclusion, a physiological concentration of luteolin induces the expression of phase II drug-metabolizing enzymes by enhancement of Nrf2 nuclear accumulation through MEK1/2-ERK1/2-mediated phosphorylation of Nrf2, increasing Nrf2 stability and inducing a conformational change of Keap1.

Lactoferrin induces tropoelastin expression by activating the lipoprotein receptor-related protein 1-mediated phosphatidylinositol 3-kinase/Akt pathway in human dermal fibroblasts.

Dermal fibroblasts generate the extracellular matrix component elastin, which is synthesized as tropoelastin (TE) and play a critical role in maintaining skin elasticity. Lactoferrin (Lf), an 80-kDa iron-binding glycoprotein, has biological functions such as anti-bacterial, -inflammatory, and -cancer activities. We previously reported that bovine Lf increases TE mRNA expression in human dermal fibroblasts. However, it remains unclear how Lf up-regulates TE expression. Here, we investigated molecular mechanisms underlying this effect. Lf promoted the phosphorylation of Akt1 and extracellular signal-regulated protein kinase (ERK)1/2. As expected, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the MAPK inhibitor U0126 inhibited Lf-induced phosphorylation of Akt1 and ERK1/2, respectively. In contrast, LY294002, but not U0126, inhibited Lf-induced TE expression. Human dermal fibroblasts expressed lipoprotein receptor-related protein 1 (LRP-1) mRNA, and the LRP1 inhibitor receptor-associated protein attenuated Lf-induced increases in TE expression. Furthermore, siRNA-mediated knockdown of LRP-1 significantly suppressed Lf-increased TE expression and Lf-induced Akt1 phosphorylation. Iron-saturated Lf (holo-Lf) increased TE expression and promoted Akt1 phosphorylation, when compared to those parameters in cells treated with iron-free Lf (apo-Lf). Transforming growth factor (TGF)-ß1 also increased TE expression. LY294002 inhibited TGF-ß1-mediated TE upregulation, whereas TGF-ß1 activated Akt2, but not Akt1, phosphorylation. These results indicate that holo-Lf, but not apo-Lf, increases TE expression through LRP-1 in human dermal fibroblasts and suggest that holo-Lf and TGF-ß1 enhance TE expression by activating the PI3K/Akt1 and PI3K/Akt2 pathways, respectively.

The collagen derived dipeptide hydroxyprolyl-glycine promotes C2C12 myoblast differentiation and myotube hypertrophy.

The majority of studies on possible roles for collagen hydrolysates in human health have focused on their effects on bone and skin. Hydroxyprolyl-glycine (Hyp-Gly) was recently identified as a novel collagen hydrolysate-derived dipeptide in human blood. However, any possible health benefits of Hyp-Gly remain unclear. Here, we report the effects of Hyp-Gly on differentiation and hypertrophy of murine skeletal muscle C2C12 cells. Hyp-Gly increased the fusion index, the myotube size, and the expression of the myotube-specific myosin heavy chain (MyHC) and tropomyosin structural proteins. Hyp-Gly increased the phosphorylation of Akt, mTOR, and p70S6K in myoblasts, whereas the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited their phosphorylation by Hyp-Gly. LY294002 and the mammalian target of rapamycin (mTOR) inhibitor rapamycin repressed the enhancing effects of Hyp-Gly on MyHC and tropomyosin expression. The peptide/histidine transporter 1 (PHT1) was highly expressed in both myoblasts and myotubes, and co-administration of histidine inhibited Hyp-Gly-induced phosphorylation of p70S6K in myoblasts and myotubes. These results indicate that Hyp-Gly can induce myogenic differentiation and myotube hypertrophy and suggest that Hyp-Gly promotes myogenic differentiation by activating the PI3K/Akt/mTOR signaling pathway, perhaps depending on PHT1 for entry into cells.

A Natural Chalcone Cardamonin Inhibited Transformation of Aryl Hydrocarbon Receptor Through Binding to the Receptor Competitively.

SCOPE: Chalcones are widely present in most plants and have various health beneficial functions. This study investigates the suppressive effect of 13 natural and synthetic chalcones on transformation of aryl hydrocarbon receptor (AhR) induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC) in a cell-free system, Hepa-1c1c7 cells, and liver of ICR mice. METHODS AND RESULTS: In the cell-free system, cardamonin dose-dependently inhibits AhR transformation. Chalcones with substitution on 2' and/or 6' position is important for the suppressive effect, while the substitution on 4' position is negatively for the effect. Moreover, cardamonin and 2'-hydroxychalcone competitively inhibit the binding of [3H]-3-MC to the AhR. In Hepa-1c1c7 cells, cardamonin inhibits AhR transformation and expression of cytochrome P4501A1 (CYP1A1) in a dose-dependent manner through suppressing TCDD-induced phosphorylation of both AhR and AhR nuclear translocator, heterodimerization of them, and nuclear translocation of AhR. In the liver of mice, oral administered cardamonin also inhibits 3-MC-induced AhR translocation and expression of CYP1A1. CONCLUSION: Among used chalcones, a natural chalcone cardamonin competitively binds to AhR and suppresses its transformation. Thus, cardamonin is an effective food factor for suppression of the dioxin-caused biochemical alterations and toxicities.

Insulin reduces endoplasmic reticulum stress-induced apoptosis by decreasing mitochondrial hyperpolarization and caspase-12 in INS-1 pancreatic ß-cells.

Pancreatic ß-cell mass is a critical determinant of insulin secretion. Severe endoplasmic reticulum (ER) stress causes ß-cell apoptosis; however, the mechanisms of progression and suppression are not yet fully understood. Here, we report that the autocrine/paracrine function of insulin reduces ER stress-induced ß-cell apoptosis. Insulin reduced the ER-stress inducer tunicamycin- and thapsigargin-induced cell viability loss due to apoptosis in INS-1 ß-cells. Moreover, the effect of insulin was greater than that of insulin-like growth factor-1 at physiologically relevant concentrations. Insulin did not attenuate the ER stress-induced increase in unfolded protein response genes. ER stress did not induce cytochrome c release from mitochondria. Mitochondrial hyperpolarization was induced by ER stress and prevented by insulin. The protonophore/mitochondrial oxidative phosphorylation uncoupler, but not the antioxidants N-acetylcysteine and α-tocopherol, exhibited potential cytoprotection during ER stress. Both procaspase-12 and cleaved caspase-12 levels increased under ER stress. The caspase-12 inhibitor Z-ATAD-FMK decreased ER stress-induced apoptosis. Caspase-12 overexpression reduced cell viability, which was diminished in the presence of insulin. Insulin decreased caspase-12 levels at the post-translational stages. These results demonstrate that insulin protects against ER stress-induced ß-cell apoptosis in this cell line. Furthermore, mitochondrial hyperpolarization and increased caspase-12 levels are involved in ER stress-induced and insulin-suppressed ß-cell apoptosis.

Mogrol stimulates G-protein-coupled bile acid receptor 1 (GPBAR1/TGR5) and insulin secretion from pancreatic ß-cells and alleviates hyperglycemia in mice.

Target identification is a crucial step in elucidating the mechanisms by which functional food components exert their functions. Here, we identified the G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) as a target of the triterpenoid mogrol, a class of aglycone mogroside derivative from Siraitia grosvenorii. Mogrol, but not mogrosides, activated cAMP-response element-mediated transcription in a TGR5-dependent manner. Additionally, mogrol selectively activated TGR5 but not the other bile acid-responsive receptors (i.e., farnesoid X receptor, vitamin D receptor, or muscarinic acetylcholine receptor M3). Several amino acids in TGR5 (L71A2.60, W75AECL1, Q77AECL1, R80AECL1, Y89A3.29, F161AECL2, L166A5.39, Y240A6.51, S247A6.58, Y251A6.62, L262A7.35, and L266A7.39) were found to be important for mogrol-induced activation. Mogrol activated insulin secretion under low-glucose conditions in INS-1 pancreatic ß-cells, which can be inhibited by a TGR5 inhibitor. Similar effects of mogrol on insulin secretion were observed in the isolated mouse islets. Mogrol administration partially but significantly alleviated hyperglycemia in KKAy diabetic mice by increasing the insulin levels without affecting the ß-cell mass or pancreatic insulin content. These results suggest that mogrol stimulates insulin secretion and alleviates hyperglycemia by acting as a TGR5 agonist.