Sulforaphane attenuates obesity by inhibiting adipogenesis and activating the AMPK pathway in obese mice.

Obesity is associated with metabolic disorders. Sulforaphane, an isothiocyanate, inhibits adipogenesis and the occurrence of cardiovascular disease. In this study, we investigated whether sulforaphane could prevent high-fat diet (HFD)-induced obesity in C57BL/6N mice. Mice were fed a normal diet (ND), HFD or HFD plus 0.1% sulforaphane (SFN) for 6 weeks. Food efficiency ratios and body weight were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN attenuated HFD-induced visceral adiposity, adipocyte hypertrophy and fat accumulation in the liver. Serum total cholesterol and leptin, and liver triglyceride levels were lower in HFD-SFN-fed mice than in HFD-fed mice. SFN decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and leptin in the adipose tissue of HFD-SFN mice and increased adiponectin expression. Phosphorylation of AMP-activated protein kinase α (AMPKα) and acetyl-CoA carboxylase in the adipose tissue of HFD-SFN-fed mice was elevated, and HMG-CoA reductase expression was decreased compared with HFD-fed mice. Thus, these results suggest that SFN may induce antiobesity activity by inhibiting adipogenesis through down-regulation of PPARγ and C/EBPα and by suppressing lipogenesis through activation of the AMPK pathway.

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells.

Echinacea purpurea has been shown to have anti-diabetic activities; for example, it activates peroxisome proliferator-activated receptor γ (PPARγ) and increases insulin-stimulated glucose uptake. Adipogenesis has been used to study the insulin signaling pathway and to screen anti-diabetic compounds. The present study was conducted to investigate the effects of an ethanol extract of E. purpurea (EEEP) and its constituents on the insulin-induced adipocyte differentiation of 3T3-L1 preadipocytes. When adipocyte differentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation of lipid droplets and the cellular triglyceride content were significantly increased by EEEP. The expressions of PPARγ and C/EBPα in adipocytes treated with EEEP were gradually increased as compared with control cells. Fat accumulation and triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly increased as compared with control cells. The expressions of PPARγ and C/EBPα in adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly higher than in control cells. These results suggest EEEP promotes the adipogenesis that is partially induced by insulin and that dodeca-2(E),4(E)-dienoic acid isobutylamide appears to be responsible for EEEP-enhanced adipocyte differentiation.

Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment.

Hinokitiol (1), a tropolone-related natural compound, induces apoptosis and has anti-inflammatory, antioxidant, and antitumor activities. In this study, the inhibitory effects of 1 were investigated on human colon cancer cell growth and tumor formation of xenograft mice. HCT-116 and SW-620 cells derived from human colon cancers were found to be similarly susceptible to 1, with IC50 values of 4.5 and 4.4 µM, respectively. Compound 1 induced S-phase arrest in the cell cycle progression and decreased the expression levels of cyclin A, cyclin E, and Cdk2. Conversely, 1 increased the expression of p21, a Cdk inhibitor. Compound 1 decreased Bcl-2 expression and increased the expression of Bax, and cleaved caspase-9 and -3. The effect of 1 on tumor formation when administered orally was evaluated in male BALB/c-nude mice implanted intradermally separately with HCT-116 and SW-620 cells. Tumor volumes and tumor weights in the mice treated with 1 (100 mg/kg) were decreased in both cases. These results suggest that the suppression of tumor formation by compound 1 in human colon cancer may occur through cell cycle arrest and apoptosis.

Green tomato extract attenuates high-fat-diet-induced obesity through activation of the AMPK pathway in C57BL/6 mice.

Obesity is a risk factor for numerous metabolic disorders. Recently, natural compounds that may be beneficial for improving obesity have received increasing attention. In this study, we investigated whether red and green tomato extracts attenuate high-fat-diet-induced obesity in C57BL/6 mice. The mice were maintained on a normal diet (ND) or high-fat diet (HFD) for 4 weeks and then fed ND, HFD, HFD plus 2% red tomato extract (RTE) or HFD plus 2% green tomato extract (GTE) for 13 weeks. The weekly food intakes among the groups were not significantly different. Body weight of mice fed HFD plus GTE was significantly decreased to the level of mice fed ND, but the body weight was only slightly reduced in mice fed HFD plus RTE. Epididymal adipose tissue and liver weights were significantly decreased in mice fed HFD plus GTE compared to those in HFD. Serum total cholesterol and low-density lipoprotein cholesterol levels in mice fed GTE were modestly reduced, and liver total cholesterol level was strongly decreased in HFD plus GTE-fed mice compared to that in HFD-fed mice. Adenosine-monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase phosphorylation in liver from HFD plus GTE-fed mice was significantly elevated, and HMG-CoA reductase expression was also significantly decreased. GTE strongly decreased the expression of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha and perilipin in the adipose tissue of mice fed HFD plus GTE. Our results indicate that the antiobesity effects of GTE may be associated with activation of the AMPK pathway.

Rhamnetin-induced suppression of clonal expansion during early stage of adipogenesis.

Adipocyte differentiation plays a pivotal role in the progression of obesity which is a major risk factor for several diseases such as diabetes, hypertension and coronary heart disease. In this study, the inhibitory effect of rhamnetin, a flavonoid compound, on adipogenesis in 3T3-L1 cells was investigated. Rhamnetin decreased the accumulation of lipid droplets, and inhibited the elevation of triglyceride content in the adipocytes (IC(50) = 17.3 µM). The expressions of PPARγ, C/EBPα, and perilipin, adipocyte differentiation markers, were significantly reduced by rhamnetin. Triglyceride biosynthesis and clonal expansion of adipocytes were completely inhibited during the early stage by rhamnetin. Additionally, rhamnetin significantly decreased the expression of C/EBPß, an early stage marker. Our results indicate that suppression of clonal expansion during the early stage of adipogenesis by rhamnetin may be associated with inhibition of the C/EBPß, C/EBPα, and PPARγ pathways.

Quantitative analysis of sphingomyelin by high-performance liquid chromatography after enzymatic hydrolysis.

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is mostly present in the plasma membrane. A new analytical method using high-performance liquid chromatography (HPLC) was developed to quantify sphingomyelin in mouse plasma and tissues, 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells. Sphingomyelin and dihydrosphingomyelin, an internal standard, were separated by high-performance thin-layer chromatography and simultaneously hydrolyzed with sphingolipid ceramide N-deacylase and sphingomyelinase to release sphingosine and dihydrosphingosine, respectively. Sphingomyelin content was measured by HPLC following o-phthalaldehyde derivatization. Sphingomyelin concentrations in 3T3-L1 cells, rat aortic smooth muscle cells, and HT-29 cells were 60.10 ± 0.24, 62.69 ± 0.08, and 58.38 ± 0.37 pmol/µg protein, respectively, whereas those in brain, kidney, and liver of ICR mice were 55.60 ± 0.43, 43.75 ± 0.21, and 22.26 ± 0.14 pmol/µg protein. The sphingomyelin concentration in mouse plasma was 407.40 ± 0.31 µM. The limits of detection and quantification for sphingomyelin were 5 and 20 pmol, respectively, in the HPLC analysis with fluorescence detection. This sensitivity was sufficient for analyzing sphingomyelin in biological samples. In conclusion, this analytical method is a sensitive and specific technique for quantifying sphingomyelin and was successfully applied to diverse biological samples with excellent reproducibility.

Myriocin, a serine palmitoyltransferase inhibitor, suppresses tumor growth in a murine melanoma model by inhibiting de novo sphingolipid synthesis.

Advanced melanoma is the most virulent form of cancer and has a poor prognosis. In a previous study, myriocin, an inhibitor of serine palmitoyltransferase, was found to suppress melanoma cell proliferation by cell cycle arrest at the G 2/M phase through decreased sphingolipid levels and increased p53 and p21 (waf1/cip1) expression. ( 1) In the present study, myriocin (1 mg/kg, every other day for 3 weeks) was administered intradermally or intraperitoneally to melanoma mice. Tumor formation was significantly inhibited by intradermal and intraperitoneal administration of myriocin. The expression of Cdc25C, Cdc2 and cyclin B1 was decreased in tumor tissues from myriocin-treated mice, while the expression of p53 and p21 (waf1/cip1) was increased compared with that of the controls. The levels of sphingolipids in serum, liver and tumor tissue from myriocin-treated mice were decreased compared with those of controls. The decreased levels of sphingolipids in serum and liver of melanoma mice treated with myriocin suggests that myriocin may be accessible to tumor tissues of advanced melanoma. Taken together, the suppression of sphingolipid synthesis by myriocin inhibits the expression of Cdc25C or activates the expression of p53 and p21 (waf1/cip1) . This is followed by Cdc2 and cyclin B1 inhibition which results in the suppression of tumor growth.

Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest.

Obesity is a risk factor for numerous metabolic disorders such as type 2 diabetes, hypertension, and coronary heart disease. Adipocyte differentiation is triggered by adipocyte hyperplasia, which leads to obesity. In this study, the inhibitory effect of sulforaphane, an isothiocyanate, on adipogenesis in 3T3-L1 cells was investigated. Sulforaphane decreased the accumulation of lipid droplets stained with Oil Red O and inhibited the elevation of triglycerides in the adipocytes (half-maximal inhibitory concentration = 7.3 µmol/l). The expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), major transcription factors for adipocyte differentiation, was significantly reduced by sulforaphane. The major effects of sulforaphane on the inhibition of adipocyte differentiation occurred during the early stage of adipogenesis. Thus, the expression of C/EBPß, an early-stage biomarker of adipogenesis, decreased in a concentration-dependent manner when the adipocytes were exposed to sulforaphane (0, 5, 10, and 20 µmol/l). The proliferation of adipocytes treated with 20 µmol/l sulforaphane for 24 and 48 h was also suppressed. These results indicate that sulforaphane may specifically affect mitotic clonal expansion to inhibit adipocyte differentiation. Sulforaphane arrested the cell cycle at the G(0)/G(1) phase, increased p27 expression, and decreased retinoblastoma (Rb) phosphorylation. Additionally, sulforaphane modestly decreased the phosphorylation of ERK1/2 and Akt. Our results indicate that the inhibition of early-stage adipocyte differentiation by sulforaphane may be associated with cell cycle arrest at the G(0)/G(1) phase through upregulation of p27 expression.

Inverse relationship between adipocyte differentiation and ceramide level in 3T3-L1 cells.

Adipocyte differentiation has been a target in anti-obesity strategies and is known to be closely related to lipid metabolism. Ceramide, a major sphingolipid metabolite, has been implicated in differentiation. In this study, we investigated whether ceramide biosynthesis is related to adipogenesis in 3T3-L1 cells. Preadipocytes can be differentiated synchronously by a mixture of adipogenic inducers including 3-isobutyl-1-methylxanthine, dexamethasone and insulin. The number of lipid droplets and the triglyceride content, which are differentiation biomarkers, gradually increased during adipogenesis. Interestingly, ceramide and sphingosine contents in the differentiated cells were decreased compared to those in preadipocytes. When the preadipocytes were treated with an 3-isobutyl-1-methylxanthine- or dexamethasone- or insulin-deficient mixture of inducers, the cellular ceramide levels were significantly increased compared with those in cells treated with the complete set of inducers. When preadipocytes were treated with 0, 0.1 or 1 µg/ml insulin along with 3-isobutyl-1-methylxanthine and dexamethasone, the ceramide levels were decreased and the triglyceride content was increased in a concentration-dependent manner. When the cells were treated with epigallocatechin gallate, an adipocyte differentiation inhibitor, during adipogenesis, the ceramide levels of adipocytes were increased and the fat content was decreased. In conclusion, our findings demonstrate that cellular ceramide levels are inversely correlated with adipocyte differentiation.

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells.

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [(3)H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.

Simultaneous HPLC analysis of ceramide and dihydroceramide in human hairs.

Ceramide, a major class of hair lipid, can help determine the physicochemical properties of human hairs such as the chemical diffusion barrier and water retention. In this study, we developed a quantitation method for ceramide and dihydroceramide, a saturated form of ceramide, in human hairs. Lipids were extracted with ethanol from human hairs spiked with N-oleoyl-D-erythro-C(17) sphingosine, an internal standard. Ceramide and dihydroceramide were resolved by TLC and deacylated by sphingolipid-ceramide deacylase to release sphingosine and dihydrosphingosine, respectively. The hair content of ceramide was measured by HPLC following derivatization with o-phthalaldehyde. The limits of detection and quantification for ceramide extracted from hair fibers were 0.1 and 1 pmol, respectively. The linear range of hair weight for determining ceramide and dihydroceramide contents was 1 to 50 mg, with R(2) values of 0.9695 and 0.9898, respectively. The recoveries of ceramide and dihydroceramide from intra-day and interday assays were 99.55% to 98.53%, respectively. Concentrations of dihydroceramide from the hair roots to distal tip ends ranged from 10.42 +/- 2.19 to 1.20 +/- 0.11 nmol/g hair, while those of ceramide ranged from 2.27 +/- 0.22 to 1.47 +/- 0.15 nmol/g hair. The present analytical method provides a simultaneous and reproducible quantification of ceramide and dihydroceramide, and may be used as a potential biomarker for lipid abnormality-related diseases.

Elevation of sphingoid base 1-phosphate as a potential contributor to hepatotoxicity in fumonisin B1-exposed mice.

Fumonisins are causative agents of diseases in mice and rats, including liver and renal toxicities, as well as cancer, and are specific inhibitors of ceramide synthase in the metabolism of sphingolipid. The purpose of this study was to determine whether an elevated level of sphingoid base 1-phosphate was related to the expressions of metabolism enzymes in the liver of fumonisin B1 (FB1)-treated mice and acted as a contributing factor to hepatotoxicity. In our previous study, FB1 was confirmed to be toxic to both liver and kidneys, coupled with simultaneous elevation of sphinganine 1-phosphate. ICR mice were treated intraperitoneally with 10 mg/kg/day FB1 for 5 days, with the concentrations of sphingolipid metabolites in the serum and liver measured using HPLC following Bligh-Dyer extraction. The levels of sphingoid bases and their 1-phosphates in the serum and liver were markedly elevated in response to treatment with FB1. In the liver, FB1 increased the expression of sphingosine kinase and inhibited the expression of sphingosine 1-phosphate lyase. The cleaved form of caspase-3 was detected in the liver of FB1-treated mice, indicating the occurrence of apoptosis in the liver following exposure to FB1. The expressions of proapoptotic signaling molecules, such as phosphorylated forms of c-Jun N-terminus kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), were increased in the liver of FB1-treated mice. In conclusion, these results suggest the elevation of sphingoid base 1-phosphate, as a result of the activation of sphingosine kinase and the inhibition of sphingosine 1-phosphate lyase, may be a major target for FB1-induced hepatotoxicity via the activation of an apoptotic signaling pathway.

Endogenous sphingolipid metabolites related to the growth in Sphingomonas chungbukensis.

Sphingolipids are present in animals, plants, fungi, yeasts and some bacteria. In mammalian cells sphingolipids act as lipid mediators for cell growth, differentiation, apoptosis and angiogenesis. In contrast, in bacteria the biological significance of sphingolipids has not been fully elucidated and sphingolipid metabolism has not been investigated. The aim of this study was to compare the pattern of sphingolipid metabolites in HIT-T15 beta cells originating from hamster pancreas to that in the bacterial strain Sphingomonas chungbukensis DJ77, under various culture conditions. It was found that the concentration of cellular sphinganine (Sa) in S. chungbukensis was higher than that of sphingosine (So), while the level of cellular So in HIT-T15 cells was higher than that of Sa. Aeration and shaking during culture increased bacterial growth in S. chungbukensis, and the contents of So and Sa were also elevated. These results indicate that a de novo sphingolipid pathway appeared to be active in bacteria and that bacterial growth may be closely related to Sa levels.

Protection of LLC-PK1 cells against hydrogen peroxide-induced cell death by modulation of ceramide level.

Oxidative stress has been reported to elevate ceramide level during cell death. The purpose of the present study was to modulate cell death in relation to cellular glutathione (GSH) level and GST (glutathione S-transferase) expression by regulating the sphingolipid metabolism. LLC-PK1 cells were treated with H2O2 in the absence of serum to induce cell death. Subsequent to exposure to H2O2, LLC-PK1 cells were treated with desipramine, sphingomyelinase inhibitor, and N-acetylcysteine (NAC), GSH substrate. Based on comparative visual observation with H2O2-treated control cells, it was observed that 0.5 microM of desipramine and 25 mM of NAC exhibited about 90 and 95% of cytoprotection, respectively, against H2O2-induced cell death. Desipramine and NAC lowered the release of LDH activity by 36 and 3%, respectively, when compared to 71% in H2O2-exposed cells. Cellular glutathione level in 500 microM H2O2-treated cells was reduced to 890 pmol as compared to control level of 1198 pmol per mg protein. GST P1-1 expression was decreased in H2O2-treated cells compared to healthy normal cells. In conclusion, it has been inferred that H2O2-induced cell death is closely related to cellular GSH level and GST P1-1 expression in LLC-PK1 cells and occurs via ceramide elevation by sphingomyelinase activation.

Altered de novo sphingolipid biosynthesis is involved in the serum deprivation-induced cell death in LLC-PK1 cells.

Fumonisin B1, a specific inhibitor of ceramide synthase, and ISPI (Myriocin), a serine palmitoyltransferase inhibitor, modulate the de novo sphingolipid biosynthesis pathway. This study was conducted to determine whether serum deprivation-induced cell death is regulated by de novo sphingolipid biosynthesis in pig kidney LLC-PK1 cells. Serum withdrawal from the culture medium produced cell death in LLC-PK1 cells. Fumonisin B1 at concentrations ranging from 5 M to 30 M delayed until 48 h this cell death resulting from the absence of fetal bovine serum (FBS) in cell culture. Pretreatment of cultured cells with fumonisin B1 in the presence of serum for 24 h increased by approximately 70% this cytoprotective activity of fumonisin B1 against serum deprivation-induced cell death. Serum deprivation increased sphingolipid biosynthesis threefold compared to 5% serum-enriched culture. Fumonisin B1 at 5-30 M lowered the content of total complex sphingolipids to levels of 50% and 77% of the content in serum-enriched culture, although the concentration of intracellular free sphinganine was elevated. ISP1 alone at greater than 1 nM concentration reduced total complex sphingolipid content to values in LLC-PK1 cells grown in the presence of 5% FBS. The results suggest that the de novo complex sphingolipid biosynthesis modulated by either fumonisin B1 or ISP1 may regulate serum deprivation-induced cell death in LLC-PK1 cells.

Mechanics and fatigability of the rat soleus muscle during early reloading.

In order to elucidate muscle functional changes by acute reloading, contractile and fatigue properties of the rat soleus muscle were investigated at three weeks of hindlimb suspension and the following 1 hr, 5 hr, 1 d, and 2 weeks of reloading. Compared to age-matched controls, three weeks of unloading caused significant changes in myofibrillar alignments, muscle mass relative to body mass (-43%), normalized tension (-35%), shortening velocity (+143%), and response times. Further significant changes were not observed during early reloading, because the transitional reverse process was gradual rather than abrupt. Although most of the muscle properties returned to the control level after two weeks of reloading, full recovery of the tissue would require more than the two-week period. Delayed recovery due to factors such as myofibrillar arrangement and fatigue resistance was apparent, which should be considered for rehabilitation after a long-term spaceflight or bed-rest.