Further delineation of the phenotypic and metabolomic profile of ALDH1L2-related neurodevelopmental disorder.

ALDH1L2, a mitochondrial enzyme in folate metabolism, converts 10-formyl-THF (10-formyltetrahydrofolate) to THF (tetrahydrofolate) and CO2. At the cellular level, deficiency of this NADP+-dependent reaction results in marked reduction in NADPH/NADP+ ratio and reduced mitochondrial ATP. Thus far, a single patient with biallelic ALDH1L2 variants and the phenotype of a neurodevelopmental disorder has been reported. Here, we describe another patient with a neurodevelopmental disorder associated with a novel homozygous missense variant in ALDH1L2, Pro133His. The variant caused marked reduction in the ALDH1L2 enzyme activity in skin fibroblasts derived from the patient as probed by 10-FDDF, a stable synthetic analog of 10-formyl-THF. Additional associated abnormalities in these fibroblasts include reduced NADPH/NADP+ ratio and pool of mitochondrial ATP, upregulated autophagy and dramatically altered metabolomic profile. Overall, our study further supports a link between ALDH1L2 deficiency and abnormal neurodevelopment in humans.

Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

Pear pomace alleviated atopic dermatitis in NC/Nga mice and inhibited LPS-induced inflammation in RAW 264.7 macrophages.

BACKGROUND/OBJECTIVES: Poorly regulated inflammation is believed to be the most predominant factor that can result in a wide scope of diseases including atopic dermatitis (AD). Despite many studies on the effect of pear pomace in obesity-related disorders including dysregulated gut microbiota, the protective effect of pear pomace in AD is still unknown. This study aimed to evaluate the effect of pear pomace ethanol extract (PPE) on AD by inhibiting inflammation. MATERIALS/METHODS: In the in vivo experiment, 2, 4-dinitrochlorobenzene (DNCB) was applied to NC/Nga mice to induce AD-like skin lesions. After the induction, PPE was administered daily by oral gavage for 4 weeks. The clinical severity score, serum IgE levels, spleen weight, histological changes in dorsal skin, and inflammation-related proteins were measured. In the cell study, RAW 264.7 cells were pretreated with PPE before stimulation with lipopolysaccharide (LPS). Nitrite oxide (NO) production and nuclear factor kappa B (NF-kB) protein expression were detected. RESULTS: Compared to the AD control (AD-C) group, IgE levels were dramatically decreased via PPE treatment. PPE significantly reduced scratching behavior, improved skin symptoms, and decreased ear thickness compared to the AD-C group. In addition, PPE inhibited the DNCB-induced expression of inducible nitrite oxide synthase (iNOS), the receptor for advanced glycation end products, extracellular signal-regulated kinase (ERK) 1/2, and NF-κB. PPE inhibited the LPS-induced overproduction of NO and the enhanced expression of iNOS and cyclooxygenase-2. Moreover, the phosphorylation of ERK1/2 and NF-κB in RAW 264.7 cells was suppressed by PPE. CONCLUSIONS: These results suggest that PPE could be explored as a therapeutic agent to prevent AD.

Red Pepper Seeds Inhibit Hepatic Lipid Accumulation by Inducing Autophagy via AMPK Activation.

Although the red pepper and its seeds have been studied for metabolic diseases, the effects and potential mechanisms of red pepper seed extract (RPS) on hepatic lipid accumulation are not yet completely understood. This study aimed to evaluate the inhibitory effect of RPS on hepatic lipid accumulation via autophagy. C57BL/6 mice were fed a high-fat diet (HFD) or a HFD supplemented with RPS. RPS treatment inhibited hepatic lipid accumulation by suppressing lipogenesis, inducing hepatic autophagic flux, and activating AMPK in HFD-fed mice. To investigate the effect of RPS on an oleic acid (OA)-induced hepatic steatosis cell model, HepG2 cells were incubated in a high-glucose medium and OA, followed by RPS treatment. RPS treatment decreased OA-induced lipid accumulation and reduced the expression of lipogenesis-associated proteins. Autophagic flux dramatically increased in the RPS-treated group. RPS phosphorylated AMPK in a dose-dependent manner, thereby dephosphorylated mTOR. Autophagy inhibition with 3-methyladenine (3-MA) antagonized RPS-induced suppression of lipogenesis-related protein expressions. Moreover, the knockdown of endogenous AMPK also antagonized the RPS-induced regulation of lipid accumulation and autophagy. Our findings provide new insights into the beneficial effects of RPS on hepatic lipid accumulation through the AMPK-dependent autophagy-mediated downregulation of lipogenesis.

Visceral adipose tissue remodeling in pancreatic ductal adenocarcinoma cachexia: the role of activin A signaling.

Pancreatic ductal adenocarcinoma (PDAC) patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue. Activin A levels were measured in serum and analyzed in tumor specimens of both a cohort of Stage IV PDAC patients and the genetically engineered KPC mouse model. Our data revealed that serum activin A levels were significantly elevated in Stage IV PDAC patients in comparison to age-matched non-cancer patients. Little is known about the role of activin A in adipose tissue wasting in the setting of PDAC cancer cachexia. We established a correlation between elevated activin A and remodeling of visceral adipose tissue. Atrophy and fibrosis of visceral adipose tissue was examined in omental adipose tissue of Stage IV PDAC patients and gonadal adipose tissue of an orthotopic mouse model of PDAC. Remarkably, white visceral adipose tissue from both PDAC patients and mice exhibited decreased adipocyte diameter and increased fibrotic deposition. Strikingly, expression of thermogenic marker UCP1 in visceral adipose tissues of PDAC patients and mice remained unchanged. Thus, we propose that activin A signaling could be relevant to the acceleration of visceral adipose tissue wasting in PDAC-associated cachexia.

Development of ovarian tumour causes significant loss of muscle and adipose tissue: a novel mouse model for cancer cachexia study.

BACKGROUND: Cancer-associated cachexia (CAC) is a complex syndrome of progressive muscle wasting and adipose loss with metabolic dysfunction, severely increasing the morbidity and mortality risk in cancer patients. However, there are limited studies focused on the underlying mechanisms of the progression of CAC due to the complexity of this syndrome and the lack of preclinical models that mimics its stagewise progression. METHODS: We characterized the initiation and progression of CAC in transgenic female mice with ovarian tumours. We measured proposed CAC biomarkers (activin A, GDF15, IL-6, IL-1ß, and TNF-α) in sera (n = 6) of this mouse model. The changes of activin A and GDF15 (n = 6) were correlated with the decline of bodyweight over time. Morphometry and signalling markers of muscle atrophy (n ≥ 6) and adipose tissue wasting (n ≥ 7) were assessed during CAC progression. RESULTS: Cancer-associated cachexia symptoms of the transgenic mice model used in this study mimic the progression of CAC seen in humans, including drastic body weight loss, skeletal muscle atrophy, and adipose tissue wasting. Serum levels of two cachexia biomarkers, activin A and GDF15, increased significantly during cachexia progression (76-folds and 10-folds, respectively). Overactivation of proteolytic activity was detected in skeletal muscle through up-regulating muscle-specific E3 ligases Atrogin-1 and Murf-1 (16-folds and 14-folds, respectively) with decreasing cross-sectional area of muscle fibres (P < 0.001). Muscle wasting mechanisms related with p-p38 MAPK, FOXO3, and p-AMPKα were highly activated in concurrence with an elevation in serum activin A. Dramatic fat loss was also observed in this mouse model with decreased fat mass (n ≥ 6) and white adipocytes sizes (n = 6) (P < 0.0001). The adipose tissue wasting was based on thermogenesis, supported by the up-regulation of uncoupling protein 1 (UCP1). Fibrosis in adipose tissue was also observed in concurrence with adipose tissue loss (n ≥ 13) (p < 0.0001). CONCLUSIONS: Our novel preclinical CAC mouse model mimics human CAC phenotypes and serum biomarkers. The mouse model in this study showed proteolysis in muscle atrophy, browning in adipose tissue wasting, elevation of serum activin A and GDF15, and atrophy of pancreas and liver. This mouse line would be the best preclinical model to aid in clarifying molecular mediators of CAC and dissecting metabolic dysfunction and tissue atrophy during the progression of CAC.

Essential role of systemic iron mobilization and redistribution for adaptive thermogenesis through HIF2-α/hepcidin axis.

Iron is an essential biometal, but is toxic if it exists in excess. Therefore, iron content is tightly regulated at cellular and systemic levels to meet metabolic demands but to avoid toxicity. We have recently reported that adaptive thermogenesis, a critical metabolic pathway to maintain whole-body energy homeostasis, is an iron-demanding process for rapid biogenesis of mitochondria. However, little information is available on iron mobilization from storage sites to thermogenic fat. This study aimed to determine the iron-regulatory network that underlies beige adipogenesis. We hypothesized that thermogenic stimulus initiates the signaling interplay between adipocyte iron demands and systemic iron liberation, resulting in iron redistribution into beige fat. To test this hypothesis, we induced reversible activation of beige adipogenesis in C57BL/6 mice by administering a ß3-adrenoreceptor agonist CL 316,243 (CL). Our results revealed that CL stimulation induced the iron-regulatory protein-mediated iron import into adipocytes, suppressed hepcidin transcription, and mobilized iron from the spleen. Mechanistically, CL stimulation induced an acute activation of hypoxia-inducible factor 2-α (HIF2-α), erythropoietin production, and splenic erythroid maturation, leading to hepcidin suppression. Disruption of systemic iron homeostasis by pharmacological HIF2-α inhibitor PT2385 or exogenous administration of hepcidin-25 significantly impaired beige fat development. Our findings suggest that securing iron availability via coordinated interplay between renal hypoxia and hepcidin down-regulation is a fundamental mechanism to activate adaptive thermogenesis. It also provides an insight into the effects of adaptive thermogenesis on systemic iron mobilization and redistribution.

Dietary Iron Deficiency Modulates Adipocyte Iron Homeostasis, Adaptive Thermogenesis, and Obesity in C57BL/6 Mice.

BACKGROUND: Adaptive thermogenesis is an iron-demanding pathway, significantly contributing to whole-body energy expenditure. However, the effects of iron-deficient diets on adaptive thermogenesis and obesity remain unknown. OBJECTIVES: We aimed to determine the impact of dietary iron deficiency on iron homeostasis in adipocytes, adaptive thermogenic capacity, and metabolic consequences in obesity. METHODS: C57BL/6 male mice were assigned to either the iron-adequate (IA, 35 ppm) or the iron-deficient group (ID, 3 ppm) at weaning. Upon 8 wk of age, both IA and ID groups received an isocaloric high-fat diet (45% kcal from fat) for 10 wk, maintaining the same iron content. Mice (n = 8) were used to determine the iron status at the systemic and tissue levels and lipid metabolism and inflammatory signaling in adipose tissue. The same mice were used to evaluate cold tolerance (4°C) for 3 h. For assessing adaptive thermogenesis, mice (n = 5) received an intraperitoneal injection of ß3-adrenoceptor agonist CL316243 (CL) for 5 d. RESULTS: Compared with the IA group, the ID group had nonanemic iron deficiency, lower serum ferritin (42.8%, P < 0.01), and greater weight gain (8.67%, P < 0.05) and insulin resistance (159%, P < 0.01), partly due to reduced AMP-activated protein kinase activation (61.0%, P < 0.05). Upon cold exposure, the ID group maintained a core body temperature 2°C lower than the IA group. The ID group had lower iron content (47.0%, P < 0.01) in the inguinal adipose tissue (iWAT) than the IA group, which was associated with impaired adaptive thermogenesis. In response to CL, ID mice showed decreased heat production (P < 0.01) and defective upregulation of beige adipocyte-specific markers, including uncoupling protein 1 (41.1%, P < 0.001), transferrin receptor 1 (47.5%, P < 0.001), and mitochondrial respiratory chain complexes (P < 0.05) compared with IA mice. CONCLUSIONS: Dietary iron deficiency deregulates iron balance in the iWAT and impairs adaptive thermogenesis, thereby escalating the diet-induced weight gain in C57BL/6 mice.

The thermogenic characteristics of adipocytes are dependent on the regulation of iron homeostasis.

The development of thermogenic adipocytes concurs with mitochondrial biogenesis, an iron-dependent pathway. Iron regulatory proteins (IRP) 1 and 2 are RNA-binding proteins that regulate intracellular iron homeostasis. IRPs bind to the iron-response element (IRE) of their target mRNAs, balancing iron uptake and deposition at the posttranscriptional levels. However, IRP/IRE-dependent iron regulation in adipocytes is largely unknown. We hypothesized that iron demands are higher in brown/beige adipocytes than white adipocytes to maintain the thermogenic mitochondrial capacity. To test this hypothesis, we investigated the IRP/IRE regulatory system in different depots of adipose tissue. Our results revealed that 1) IRP/IRE interaction was increased in proportional to the thermogenic function of the adipose depot, 2) adipose iron content was increased in adipose tissue browning upon ß3-adrenoceptor stimulation, while decreased in thermoneutral conditions, and 3) modulation of iron content was linked with mitochondrial biogenesis. Moreover, the iron requirement was higher in HIB1B brown adipocytes than 3T3-L1 white adipocytes during differentiation. The reduction of the labile iron pool (LIP) suppressed the differentiation of brown/beige adipocytes and mitochondrial biogenesis. Using the 59Fe-Tf, we also demonstrated that thermogenic stimuli triggered cell-autonomous iron uptake and mitochondrial compartmentalization as well as enhanced mitochondrial respiration. Collectively, our work demonstrated that IRP/IRE signaling and subsequent adaptation in iron metabolism are a critical determinant for the thermogenic function of adipocytes.

St. John's Wort Suppresses Growth in Triple-Negative Breast Cancer Cell Line MDA-MB-231 by Inducing Prodeath Autophagy and Apoptosis.

The rational regulation of programmed cell death by means of autophagy and apoptosis has been considered a potential treatment strategy for cancer. We demonstrated the inhibitory effect of St. John's Wort (SJW) on growth in the triple-negative breast cancer (TNBC) cell line and xenografted mice and its target mechanism concerning autophagic and apoptotic cell death. SJW ethanol extract (SJWE) inhibited proliferation in a dose-dependent manner. SJWE treatment dramatically increased autophagy flux and apoptosis compared with the control. The autophagy inhibitor, 3-methyladenine (3-MA), reversed the SJWE-induced inhibition of cell proliferation and regulation of autophagy and apoptosis, indicating that SJWE induced apoptosis through prodeath autophagy. Furthermore, SJWE inhibited tumor growth and induced autophagy and apoptosis in the tumor of MDA-MB-231 xenografted athymic nude mice. Our results indicate that SJWE might have great potential as a new anticancer therapy for triple-negative breast cancer by inducing prodeath autophagy and apoptosis.

Red pepper seed water extract suppresses high-fat diet-induced obesity in C57BL/6 mice.

In this study, the protective effect of red pepper seed water extract (RPS) against the obesity in high fat diet (HFD)-fed mice was investigated (HFD control group, and HFD group treated with 100 or 200 mg/kg body weight of RPS for 13 weeks). The application of RPS partially reversed the HFD-induced increases in body weight and adipose tissue weight. The patterns of the adipose tissue weights were parallel to the patterns of fat area, as measured in DXA procedure. In the adipose tissue, RPS suppressed the expression of adipogenic transcription factors and adipose marker genes. AMP-activated protein kinase activation was observed in the adipose tissue by RPS treatment. In addition, RPS improved high homeostasis model assessment of insulin resistance and hyperlipidemia in HFD fed mice. These findings suggest that RPS can be used as a potential therapeutic substance for reducing body fat and obesity related diseases.

Alpha-Linolenic Acid-Enriched Butter Promotes Fatty Acid Remodeling and Thermogenic Activation in the Brown Adipose Tissue.

Supplementation with n-3 long-chain (LC) polyunsaturated fatty acids (PUFA) is known to promote thermogenesis via the activation of brown adipose tissue (BAT). Agricultural products that are biofortified with α-linolenic acid (ALA), the precursor of n-3 LC PUFA, have been launched to the market, but their impact on BAT function is unknown. This study aimed to evaluate the effects of ALA-biofortified butter on lipid metabolism and thermogenic functions in the BAT. C57BL/6 mice were fed a high-fat diet containing ALA-biofortified butter (n3Bu, 45% calorie from fat) for ten weeks in comparison with the isocaloric high-fat diets prepared from conventional butter or margarine. The intake of n3Bu significantly reduced the whitening of BAT and increased the thermogenesis in response to acute-cold treatment. Also, n3Bu supplementation is linked with the remodeling of BAT by promoting bioconversion into n-3 LC PUFA, FA elongation and desaturation, and mitochondrial biogenesis. Taken together, our results support that ALA-biofortified butter is a novel source of n-3 PUFA, which potentiates the BAT thermogenic function.

Red Raspberry Polyphenols Attenuate High-Fat Diet-Driven Activation of NLRP3 Inflammasome and its Paracrine Suppression of Adipogenesis via Histone Modifications.

SCOPE: The authors aim to investigate the mechanisms by which red raspberry (RR) polyphenolic fractions regulate obesity and inflammation with an emphasis on the crosstalk between adipose tissue macrophages (ATM) and adipocyte progenitors. METHODS AND RESULTS: C57BL/6 male mice are fed either a high-fat (HF) diet or an HF diet supplemented with a RR polyphenolic fraction from whole fruit, pulp, or seed. Supplementation with pulp significantly increases energy expenditure and reduces HF-diet-induced obesity and insulin resistance. The pulp, and to a lesser extent, whole polyphenols, decreases the recruitment of ATM, activation of the nod-like receptor protein 3 (NLRP3) inflammasome, and adipocyte hypertrophy, which is associated with epigenetic modulation of adipogenesis (e.g., H3K27Ac, H3K9Ac). Results from an IL-1ß reporter assay in J774 macrophages recapitulate the inhibitory role of RR polyphenols on NLRP3 inflammasome activation. Using conditioned media from macrophages, it is demonstrated that RR polyphenols reverse the IL-1ß-mediated epigenetic suppression of H3K27Ac in adipocyte progenitor cells. CONCLUSIONS: RR polyphenols from pulp and whole fruit serve as an inhibitor for NLRP3 inflammasome activation and an epigenetic modifier to regulate adipogenesis, which confers resistance against diet-induced obesity and metabolic dysfunction.

α-Linolenic acid-enriched butter attenuated high fat diet-induced insulin resistance and inflammation by promoting bioconversion of n-3 PUFA and subsequent oxylipin formation.

α-Linolenic acid (ALA) is an essential fatty acid and the precursor for long-chain n-3 PUFA. However, biosynthesis of n-3 PUFA is limited in a Western diet likely due to an overabundance of n-6 PUFA. We hypothesized that dietary reduction of n-6/n-3 PUFA ratio is sufficient to promote the biosynthesis of long-chain n-3 PUFA, leading to an attenuation of high fat (HF) diet-induced obesity and inflammation. C57BL/6 J mice were fed a HF diet from ALA-enriched butter (n3Bu, n-6/n-3=1) in comparison with isocaloric HF diets from either conventional butter lacking both ALA and LA (Bu, n-6/n-3=6), or margarine containing a similar amount of ALA and abundant LA (Ma, n-6/n-3=6). Targeted lipidomic analyses revealed that n3Bu feeding promoted the bioconversion of long-chain n-3 PUFA and their oxygenated metabolites (oxylipins) derived from ALA and EPA. The n3Bu supplementation attenuated hepatic TG accumulation and adipose tissue inflammation, resulting in improved insulin sensitivity. Decreased inflammation by n3Bu feeding was attributed to the suppression of NF-κB activation and M1 macrophage polarization. Collectively, our work suggests that dietary reduction of the n-6/n-3 PUFA ratio, as well as total n-3 PUFA consumed, is a crucial determinant that facilitates n-3 PUFA biosynthesis and subsequent lipidomic modifications, thereby conferring metabolic benefits against obesity-induced inflammation and insulin resistance.

Pear pomace water extract reduces adiposity in vivo and in vitro by activating the AMPK-dependent pathway

Objective: To explore the inhibitory effect of water extract from pear pomace on abdominal fat accumulation and its underlying mechanism in high fat diet-fed animals. Methods: Three groups of male C57BL/6J mice were fed with a 60％ kcal fat diet for 8 weeks. Pear pomace water extract (200 or 400 mg/kg body weight) was administered once daily via oral gavage. To confirm the possibility of the water extract of pear pomace acting as an activator of adenosine 5'-monophosphate-activated protein kinase (AMPK), differentiation of 3T3-L1 preadipocytes was induced in the presence of the water extract of pear pomace with or without compound C. Body weight, food efficacy ratio, insulin resistance, and adipogenic protein expression were measured. Moreover, in the 3T3-L1 cells, lipid content and lipogenesis-related proteins were measured using Oil Red O staining and Western blotting analysis. Results: Body weight gain and total abdominal fat weight were reduced in mice treated with pear pomace water extract. Pear pomace water extract reduced fasting blood glucose and insulin, thereby reducing the homeostatic model assessment of insulin resistance. It also resulted in dose-dependent decreases in triglyceride, total cholesterol, and low-density lipoprotein-cholesterol. The protein expression of p-AMPK increased, while the expression of AMPK-downstream proteins including PPAR-γ, C/EBPα, SREBP-1c, ACC, and FAS decreased in the adipose tissue of mice treated with pear pomace water extract. Furthermore, the inhibition of AMPK by compound C blocked pear pomace water extract-induced reduction of lipid content and the expression of lipogenesis-related genes. Conclusions: Pear pomace water extract prevents fat accumulation both in vivo and in vitro by activating AMPK.

Red pepper seed water extract inhibits preadipocyte differentiation and induces mature adipocyte apoptosis in 3T3-L1 cells.

BACKGROUND/OBJECTIVES: Reducing the number of adipocytes by inducing apoptosis of mature adipocytes as well as suppressing differentiation of preadipocytes plays an important role in preventing obesity. This study examines the anti-adipogenic and pro-apoptotic effect of red pepper seed water extract (RPS) prepared at 4℃ (RPS4) in 3T3-L1 cells. MATERIALS/METHODS: Effect of RPS4 or its fractions on lipid accumulation was determined in 3T3-L1 cells using oil red O (ORO) staining. The expressions of AMP-activated protein kinase (AMPK) and adipogenic associated proteins [peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBP α), sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC)] were measured in 3T3-L1 cells treated with RPS4. Apoptosis and the expression of Akt and Bcl-2 family proteins [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated death promoter (Bad), Bcl-2 like protein 4 (Bax), Bal-2 homologous antagonist/killer (Bak)] were measured in mature 3T3-L1 cells treated with RPS4. RESULTS: Treatment of RPS4 (0-75 µg/mL) or its fractions (0-50 µg/mL) for 24 h did not have an apparent cytotoxicity on pre and mature 3T3-L1 cells. RPS4 significantly suppressed differentiation and cellular lipid accumulation by increasing the phosphorylation of AMPK and reducing the expression of PPAR-γ, C/EBP α, SREBP-1c, FAS, and ACC. In addition, all fractions except ethyl acetate fraction significantly suppressed cellular lipid accumulation. RPS4 induced the apoptosis of mature adipocytes by hypophosphorylating Akt, increasing the expression of the pro-apoptotic proteins, Bak, Bax, and Bad, and reducing the expression of the anti-apoptotic proteins, Bcl-2 and p-Bad. CONCLUSIONS: These finding suggest that RPS4 can reduce the numbers as well as the size of adipocytes and might useful for preventing and treating obesity.

St. John's Wort Regulates Proliferation and Apoptosis in MCF-7 Human Breast Cancer Cells by Inhibiting AMPK/mTOR and Activating the Mitochondrial Pathway.

St. John's Wort (SJW) has been used as an estrogen agonist in the systems affected by menopause. Also, hypericin, a bioactive compound of SJW, has been used as a photosensitizer in photodynamic therapy. In the present study, we investigate the anti-proliferative and pro-apoptotic effects of SJW to demonstrate the chemo-preventive effect in human breast cancer cells. MCF-7 cells were cultured with DMSO or various concentrations of SJW ethanol extract (SJWE). Cell viability, proliferation, apoptosis, the expression of proteins involved in cell growth and apoptosis, and caspase-3/7 activity were examined. SJWE dose-dependently suppressed cell growth and induced apoptosis of MCF-7 cells. Mechanistically, SJWE enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and decreased the expression of p-mammalian target of rapamycin (p-mTOR) and p-eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). Also, SJWE inhibited the phosphorylation of protein kinase B (Akt) and showed increases in the expression of pro-apoptotic proteins Bax and Bad with decreases in the expression of anti-apoptotic proteins including B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), and p-Bcl-2-associated death promoter (p-Bad). SJWE at 50 µg/mL showed markedly enhanced caspase-7 activation. Taken together, our results provide evidence that SJWE shows anti-proliferative and pro-apoptotic effects via inhibition of AMPK/mTOR and activation of a mitochondrial pathway. Therefore, SJWE can be used as a chemo-preventive agent without photo-activation.

Red Pepper Seed Inhibits Differentiation of 3T3-L1 Cells during the Early Phase of Adipogenesis via the Activation of AMPK.

Obesity is the main risk factor for metabolic syndromes and there has been an upsurge in demand for effective therapeutic strategies. This study investigated the effect of red pepper seed water extract (RPS) on the process of differentiation in 3T3-L1 adipocytes. RPS treatment significantly suppressed cellular lipid accumulation and reduced the expression of adipocytes-associated proteins, peroxisome proliferator-activated receptor-[Formula: see text] (PPAR-[Formula: see text]), CCAAT/enhancer-binding proteins [Formula: see text] (C/EBP [Formula: see text]), sterol regulatory element binding protein-1c (SREBP-1c), as well as fatty acid synthase (FAS), and fatty acid binding protein 4 (FABP4). The inhibitory effect of RPS on differentiation was mainly through the modulation of the C/EBP [Formula: see text] and C/EBP [Formula: see text] expression at the early phase of differentiation. Moreover, at the early phase of differentiation, RPS markedly increased the phosphorylation of AMP-activated protein kinase (AMPK). Such enhancing effect of RPS was abolished in the presence of compound C. Our results suggest that activation of AMPK at early stage of adipogenesis is involved in the anti-adipogenesis effect of RPS.

Pear pomace ethanol extract improves insulin resistance through enhancement of insulin signaling pathway without lipid accumulation.

BACKGROUND/OBJECTIVES: The anti-diabetic activity of pear through inhibition of α-glucosidase has been demonstrated. However, little has been reported about the effect of pear on insulin signaling pathway in obesity. The aims of this study are to establish pear pomace 50% ethanol extract (PPE)-induced improvement of insulin sensitivity and characterize its action mechanism in 3T3-L1 cells and high-fat diet (HFD)-fed C57BL/6 mice. MATERIALS/METHODS: Lipid accumulation, monocyte chemoattractant protein-1 (MCP-1) secretion and glucose uptake were measure in 3T3-L1 cells. Mice were fed HFD (60% kcal from fat) and orally ingested PPE once daily for 8 weeks and body weight, homeostasis model assessment of insulin resistance (HOMA-IR), and serum lipids were measured. The expression of proteins involved in insulin signaling pathway was evaluated by western blot assay in 3T3-L1 cells and adipose tissue of mice. RESULTS: In 3T3-L1 cells, without affecting cell viability and lipid accumulation, PPE inhibited MCP-1 secretion, improved glucose uptake, and increased protein expression of phosphorylated insulin receptor substrate 1 [p-IRS-1, (Tyr632)], p-Akt, and glucose transporter type 4 (GLUT4). Additionally, in HFD-fed mice, PPE reduced body weight, HOMA-IR, and serum lipids including triglyceride and LDL-cholesterol. Furthermore, in adipose tissue, PPE up-regulated GLUT4 expression and expression ratio of p-IRS-1 (Tyr632)/IRS, whereas, down-regulated p-IRS-1 (Ser307)/IRS. CONCLUSIONS: Our results collectively show that PPE improves glucose uptake in 3T3-L1 cells and insulin sensitivity in mice fed a HFD through stimulation of the insulin signaling pathway. Furthermore, PPE-induced improvement of insulin sensitivity was not accompanied with lipid accumulation.

Pear pomace water extract suppresses hepatic lipid peroxidation and protects against liver damage in rats fed a high fat/cholesterol diet.

The protective effect of pear pomace water extract (PPWE) against hepatic lipid peroxidation was investigated in rats fed a 41% kcal fat diet containing 0.21% cholesterol (HFCD). For 5 weeks, 200 or 400 mg/kg of PPWE was administrated once daily via oral gavage. Body weights were lower in the PPWE-treated group than in the control group. Serum total antioxidant capacity increased, whereas hepatic thiobarbituric acid reactive substances significantly decreased after the administration of PPWE. PPWE recovered the HFCD-induced reduction of hepatic glutathione S-transferase and glutathione peroxidase activity. The serum alanine aminotransferase and aspartate aminotransferase activities significantly decreased on PPWE treatment. The present investigation suggests that PPWE represents a valuable natural antioxidant source for use in the health food industry.