Amelioration of Hyperglycemia-Induced Nephropathy by 3,3'-Diindolylmethane in Diabetic Mice.

Type 1 diabetes mellitus (insulin-dependent diabetes) is characterized by hyperglycemia caused by an insulin deficiency. Diabetic nephropathy is a major complication of hyperglycemia. 3,3'-diindolylmethane (DIM)-a natural compound produced from indole-3-carbinol, found in cruciferous vegetables-enhances glucose uptake by increasing the activation of the insulin signaling pathway in 3T3-L1 adipocytes. In this study, we investigated whether DIM could improve insulin-dependent diabetes and nephropathy in streptozotocin (STZ)-induced diabetic mice. In mice, STZ induced hyperglycemia, hunger, thirst, and abnormally increased kidney weight and serum creatinine, which is a renal functional parameter. DIM decreased STZ-increased high blood glucose levels and food and water intake in diabetic mice. DIM also improved diabetic nephropathy by inhibiting the expression of PKC-α, the marker of albuminuria, and TGF-ß1, an indicator of renal hypertrophy, in diabetic mice. Our findings suggest that DIM may ameliorate hyperglycemia and diabetic nephropathy through the inhibition of PKC-α and TGF-ß1 signaling.

3,3'-Diindolylmethane Enhances Glucose Uptake Through Activation of Insulin Signaling in 3T3-L1 Adipocytes.

OBJECTIVE: Indole-3-carbinol (I3C), a naturally occurring compound found in cruciferous vegetables, and its metabolite 3,3'-diindolylmethane (DIM) reduce body mass and serum glucose levels in high-fat-diet-induced obese mice. This study aimed to determine whether I3C or DIM could increase glucose uptake via enhanced insulin sensitivity in 3T3-L1 adipocytes, as well as the mechanism involved. METHODS: 3T3-L1 preadipocytes were differentiated by using a mixture of adipogenic inducers, including a suboptimal concentration of insulin. RESULTS: DIM, but not I3C, increased adipocyte differentiation through upregulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α. DIM also enhanced glucose uptake by increasing expression of glucose transporter 4 in adipocytes. This was associated with DIM-enhanced phosphorylation of the signaling intermediates Akt, insulin receptor substrate-1, and insulin receptor early in differentiation. CONCLUSIONS: Our findings suggest that DIM may improve insulin sensitivity through the activation of the insulin signaling pathway, leading to enhanced glucose uptake.

Enhancement of Glucose Uptake by Meso-Dihydroguaiaretic Acid through GLUT4 Up-Regulation in 3T3-L1 Adipocytes.

Type 2 diabetes is characterized by insulin resistance, which leads to increased blood glucose levels. Adipocytes are involved in the development of insulin resistance, resulting from the dysfunction of the insulin signaling pathway. In this study, we investigated whether meso-dihydroguaiaretic acid (MDGA) may modulate glucose uptake in adipocytes, and examined its mechanism of action. MDGA enhanced adipogenesis through up-regulation of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α in 3T3-L1 adipocytes partially differentiated with sub-optimal concentrations of insulin. MDGA also increased glucose uptake by stimulating expression and translocation of glucose transporter 4 (GLUT4) in adipocytes. These results suggest that MDGA may increase GLUT4 expression and its translocation by promoting insulin sensitivity, leading to enhanced glucose uptake.

Dodeca-2(E),4(E)-dienoic acid isobutylamide enhances glucose uptake in 3T3-L1 cells via activation of Akt signaling.

Dodeca-2(E),4(E)-dienoic acid isobutylamide (DDI), an alkamide derived from the plant Echinacea purpurea, promotes adipocyte differentiation and activates peroxisome proliferator-activated receptor γ, which is associated with enhanced insulin sensitivity. In the present study, we investigated whether DDI may increase glucose uptake through activation of the insulin signaling pathway in 3T3-L1 adipocytes. DDI increased insulin-stimulated glucose uptake, and expression and translocation of glucose transporter 4 in adipocytes treated with sub-optimal levels of insulin. Additionally, DDI enhanced Akt phosphorylation, whereas phosphoinositide 3-kinase/Akt inhibitors suppressed DDI-induced glucose uptake. These results suggest that DDI may improve insulin sensitivity through the activation of Akt signaling, which leads to enhanced glucose uptake.

Anti-Obesity Effect of 6,8-Diprenylgenistein, an Isoflavonoid of Cudrania tricuspidata Fruits in High-Fat Diet-Induced Obese Mice.

Obesity, which is characterized by excessive fat accumulation, is associated with several pathological disorders, including metabolic diseases. In this study, the anti-obesity effect of 6,8-diprenylgenistein (DPG), a major isoflavonoid of Cudrania tricuspidata fruits was investigated using high fat-diet (HFD)-induced obese mice at the doses of 10 and 30 mg/kg for six week. The body weight of the DPG-treated groups was significantly lower compared to the HFD-treated group. In addition, fat accumulation in epididymal adipose tissue and liver was dramatically decreased in the HFD + DPG groups. The food efficiency ratios of the HFD + DPG groups were also lower compared to the HFD group with the same food intake. Metabolic parameters that had increased in the HFD group were decreased in the HFD + DPG groups. Further studies demonstrate that DPG efficiently reduces lipogenic genes by regulation of transcription factors, such as peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), and hormones, such as leptin and adiponection. DPG also regulates acetyl-CoA carboxylase (ACC) and hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) by AMP-activated protein kinase (AMPK) activation. Taken together, DPG is beneficial for the regulation of obesity, especially resulting from high fat intake.

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.

Secoiridoids from the stem barks of Fraxinus rhynchophylla with pancreatic lipase inhibitory activity.

Pancreatic lipase digests dietary fats by hydrolysis, which is a key enzyme for lipid absorption. Therefore, reduction of fat absorption by the inhibition of pancreatic lipase is suggested to be a therapeutic strategy for obesity. From the EtOAc-soluble fraction of the stem barks of Fraxinus rhynchophylla (Oleaceae), four secoiridoids such as ligstroside (1), oleuropein (2), 2"-hydroxyoleuropein (3) and hydroxyframoside B (4) were isolated. The inhibitory activity of these compounds on pancreatic lipase was assessed using porcine pancreatic lipase as an in vitro assay system. Compound 4 showed the strongest inhibition on pancreatic lipase, which followed by compounds 1-3. In addition, compound 4 exerted inhibitory effect on pancreatic lipase in a mixed mechanism of competitive and noncompetitive manner. Taken together, F. rhynchophylla and its constituents might be beneficial to obesity.

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.

Hydroxyframoside B, a secoiridoid of Fraxinus rhynchophylla, inhibits adipocyte differentiation in 3T3-L1 cells.

Fraxinus rhynchophylla showed significant inhibitory activity on adipocyte differentiation in the 3T3-L1 preadipocyte cell line as assessed by measuring fat accumulation using Oil Red O staining. Further fractionation led to the isolation of two secoiridoids, oleuropein and hydroxyframoside B. Hydroxyframoside B significantly reduced fat accumulation and triglyceride content in differentiated 3T3-L1 cells without affecting cell viability, whereas oleuropein showed little effect. Further studies with interval treatment demonstrated that hydroxyframoside B exerted inhibitory activity on adipocyte differentiation when treated within 2 days (days 0-2) after differentiation induction. In addition, hydroxyframoside B significantly blocked the induction of adipogenic transcription factors such as C/EBP α, C/EBP ß, and PPAR γ. Taken together, these results suggest that hydroxyframoside B inhibited early/middle stage of adipogenic differentiation, in part, via inhibition of C/EBP α, C/EBP ß, and PPAR γ-dependent pathways.

Inhibitory effects of coumarins from the stem barks of Fraxinus rhynchophylla on adipocyte differentiation in 3T3-L1 cells.

In the course of screening anti-adipogenic activity of natural products employing the preadipocyte cell line, 3T3-L1 as an in vitro assay system, the EtOAc fraction of the stem barks of Fraxinus rhynchophylla DENCE (Oleaceae) showed significant inhibitory activity on adipocyte differentiation as assessed by measuring fat accumulation using Oil Red O staining. Activity-guided fractionation led to the isolation of six coumarins such as esculetin (1), scopoletin (2), fraxetin (3), fraxidin (4) esculin (5) and fraxin (6). Among the six coumarins isolated, esculetin (1) showed the most potent inhibitory activity on adipocyte differentiation, followed by fraxetin (3). Further studies with interval treatment demonstrated that esculetin (1) exerted inhibitory activity on adipocyte differentiation when treated within 2 d (days 0-2) after differentiation induction. We further investigated the effect of esculetin (1) on peroxisome proliferator activated receptor gamma (PPARgamma), one of the early adipogenic transcription factors. Esculetin (1) significantly blocked the induction of PPARgamma protein expression and inhibited adipocyte differentiation induced by troglitazone, a PPARgamma agonist. Taken together, these results suggest that esculetin (1), an active compound from F. rhynchophylla, inhibited early stage of adipogenic differentiation, in part, via inhibition of PPARgamma-dependent pathway.

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.

Oxygen vacancy clustering and electron localization in oxygen-deficient SrTiO(3): LDA + U study.

We find, using a local density approximation +Hubbard U method, that oxygen vacancies tend to cluster in a linear way in SrTiO(3), a prototypical perovskite oxide, accompanied by strong electron localization at the 3d state of the nearby Ti transition metal ion. The vacancy clustering and the associated electron localization lead to a profound impact on materials properties, e.g., the reduction in free-carrier densities, the appearance of characteristic optical spectra, and the decrease in vacancy mobility. The high stability against the vacancy migration also suggests the physical reality of the vacancy cluster.