Anti-adipogenic Properties Research Articles

Lactobacillus rhamnosus Cultured Milk Isolate may Reduce Adipogenesis

Obesity has reached epidemic levels around the world. There is a need for a better understanding of the different factors in the digestive tract environment that may modulate adipogenesis. Our goal is to understand the influence of potential probiotics on the differentiation and subsequent lipid storage of adipose cells. This study was performed to test the in vitro effects of a Lactobacillus rhamnosus isolate from “amabere amaruranu”, a traditional Kenyan cultured milk, on 3T3‐L1 cell line. Cultures of 3T3‐L1 cells were treated with bacterial isolate cell extract, filtered supernatant from overnight bacterial culture or with a PBS control. Cells were allowed to differentiate in the presence of different dose levels of appropriate treatments for 10 days. Cells were collected, lysed and the resultant lysate analyzed for adipogenic‐related transcription factors using western blot. Protein expression levels indicated that the treatment with bacterial isolate cell extract, but not the bacterial culture supernatant, downregulated the expression of various adipogenic‐related transcription factors in a dose‐dependent manner. The highest dose of the bacterial cell extract downregulated peroxisome proliferator‐activated receptor‐γ, sterol regulatory element‐binding protein 1, and adipose triglyceride lipase by 1.6, 1.5, and 1.8 fold, respectively. The cell extract did not affect the level of expression of CCAAT/enhancer binding protein‐α. In conclusion, bacterial cell extract from, Lactobacillus rhamnosus, from “amabere amaruranu” cultured milk showed anti‐adipogenic properties, and could be used for anti‐obesity management. This study shows that the L. rhamnosus isolate warrants further studies in vivo.

Inhibitory effects of oxyresveratrol and cyanomaclurin on adipogenesis of 3T3-L1 cells

Many phenolics are known to possess anti-obesity properties through mechanisms that inhibit adipogenesis, stimulate lipolysis or induce apoptosis. This study aimed to examine the anti-adipogenic effects of two phenolics, oxyresveratrol and cyanomaclurin in 3T3-L1 cells. At doses that they did not induce cytotoxicity (based on the lactate dehydrogenase assay), the exposure of oxyresveratrol (0–100 µM) or cyanomaclurin (0–600 µM) in the first three days of differentiation led to a dose-dependent decrease of triacylglycerol accumulation on day 9 post-confluent. Results of cell proliferation and cell cycle study demonstrated that both compounds inhibited differentiation through inducing cell cycle arrest by retaining the preadipocytes in the G1 phase during the first two days post-confluent. Their anti-adipogenic properties were also attributed to the down-regulation of protein expression of major transcriptional factors in adipogenesis, including peroxisome proliferator-activated receptor γ (PPAR γ) and CCAAT/Enhancer-binding proteins α (C/EBP α), and the induction of cell cycle arrest through regulating cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase inhibitor 1B (P27/kip1) expression during the mitotic clonal expansion period.

Carnosic Acid Inhibits Lipid Accumulation in 3T3-L1 Adipocytes Through Attenuation of Fatty Acid Desaturation.

Background:Excess body fat accumulation contributes to the development of metabolic disorders that can cause adverse health effects. Carnosic acid (CA), a major bioactive component of rosemary (Rosemarinus officinalis), has been suggested to possess anti-adipogenic properties. The present study was conducted to elucidate the mechanism underlying the anti-adipogenic effects of CA.Methods:3T3-L1 pre-adipocytes were treated with CA (0.1, 1, and 10 μM) from day 0 to day 8 of differentiation. On day 8, biochemical markers of lipid accumulation and the degree of fatty acid desaturation were measured.Results:Oil Red O staining results, triglyceride (TG) accumulation, and glycerol 3-phosphate dehydrogenase activity suggested that CA significantly inhibited lipid accumulation in 3T3-L1 adipocytes. CA significantly decreased mRNA expression of peroxisome proliferator-activated receptor-γ, sterol regulatory element-binding protein 1, and CCAAT/enhancer binding protein-α in a dose-dependent manner. Moreover, it decreased the ratio of both C16:1/C16:0 and C18:1/C18:0, with reduced expression of stearoyl CoA desaturase 1 mRNA and protein.Conclusions:These results suggest that CA efficiently suppressed adipogenesis in 3T3-L1 adipocytes and its action, at least in part, is associated with the downregulation of adipogenesis-related genes and the fatty acid composition of TG accumulated in adipocytes.

Protective efficacy of carnosic acid against hydrogen peroxide induced oxidative injury in HepG2 cells through the SIRT1 pathway.

Carnosic acid (CA), found in rosemary, has been reported to have antioxidant and antiadipogenic properties. Here, we investigate the molecular mechanism by which CA inhibits hydrogen peroxide (H2O2)-induced injury in HepG2 cells. Cells were pretreated with 2.5-10 μmol/L CA for 2 h and then exposed to 3 mmol/L H2O2 for an additional 4 h. CA dose-dependently increased cell viability and decreased lactate dehydrogenase activities. Pretreatment with CA completely attenuated the inhibited expression of manganese superoxide dismutase (MnSOD) and the B-cell lymphoma-extra large (Bcl-xL), and reduced glutathione activity caused by H2O2, whereas it reversed reactive oxygen species accumulation and the increase in cleaved caspase-3. Importantly, sirtuin 1 (SIRT1), a NAD(+)-dependent deacetylase, was significantly increased by CA. Considering the above results, we hypothesized that SIRT1 may play important roles in the protective effects of CA in injury induced by H2O2. As expected, SIRT1 suppression by Ex527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) and siRNA-mediated SIRT1 silencing (si-SIRT1) significantly aggravated the H2O2-induced increased level of cleaved caspase-3 but greatly reduced the decreased expression of MnSOD and Bcl-xL. Furthermore, the positive regulatory effect of CA was inhibited by si-SIRT1. Collectively, the present study indicated that CA can alleviate H2O2-induced hepatocyte damage through the SIRT1 pathway.

Effects of black adzuki bean (Vigna angularis) extract on proliferation and differentiation of 3T3-L1 preadipocytes into mature adipocytes.

The aim of this work was to investigate the effects of black adzuki bean (BAB) extract on adipocytes, and to elucidate the cellular mechanisms. In order to examine the proliferation of preadipocytes and differentiating adipocytes, cell viability and DNA content were measured over a period of time. Lipid accumulation during cell differentiation and the molecular mechanisms underlying the effects of BAB on the transcriptional factors involved, with their anti-adipogenic effects, were also identified. We observed that BAB exhibits anti-adipogenic effects through the inhibition of proliferation, thereby lowering mRNA expression of C/EBPβ and suppressing adipogenesis during the early stage of differentiation. This, in turn, resulted in a reduction of TG accumulation in a dose- and time-dependent manner. Treating the cells with BAB not only suppressed the adipogenesis-associated key transcription factors PPARγ and C/EBPα but also significantly decreased the mRNA expression of GLUT4, FABP4, LPL and adiponectin. The expression of lipolytic genes like ATGL and HSL were higher in the treatment group than in the control. Overall, the black adzuki bean extract demonstrated an anti-adipogenic property, which makes it a potential dietary supplement for attenuation of obesity.

Mechanism of the antiadipogenic-antiobesity effects of a rice hull smoke extract in 3T3-L1 preadipocyte cells and in mice on a high-fat diet.

The present study investigated the inhibitory effects of a rice hull smoke extract (RHSE) against adipogenesis in 3T3-L1 preadipocyte cells and in mice fed high fat (HFD) and normal (ND) diets. At concentrations of 0.1% and 0.5%, RHSE was shown to reduce the cellular lipid content in MDI-induced 3T3-L1 cells by about 72% and 88%, respectively, compared to that in control cells without RHSE, indicating a strong antiadipogenic effect. This result was supported by the finding that the expression of the adipocyte differentiation marker adiponectin was suppressed. MTT and lactate dehydrogenase (LDH) release assays showed that RHSE doses of up to 0.5% (v/v) were not cytotoxic to the 3T3-L1 cells. RHSE activates AMP-activated protein kinase (AMPK) through raising the phosphorylated ratio during the early phase of cell differentiation, and western blot analysis showed that it dose-dependently inhibited the expression of the peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT-enhancer binding protein (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) at the late stage of differentiation. The antiadipogenic properties of RHSE were confirmed in vivo using experimental obese mice on a high-fat diet. Dietary supplementations of 0.5% and 1% RHSE resulted in a reduction at the end of the 7-week feeding study of body weight gain of 66.9% and 72.5%, respectively, a reduction of epididymal white adipose tissue weight by up to 87.9%, restoration of elevated total cholesterol and triglyceride levels in plasma and liver to those observed in the ND-fed mice, normalization of distorted serum leptin and adiponectin levels, and restoration of liver weight and glutamate oxaloacetate transaminase/glutamate pyruvate transaminase (GOT/GPT) enzymes, blood urea, and serum creatinine to the normal control levels observed in the ND-fed mice. As was found in the 3T3-L1 cells, RHSE up-regulated AMPK phosphorylation and down-regulated PPARγ, C/EBPα and SREBP-1c protein expression in the epididymal white adipose tissues. These results indicate that RHSE inhibits the AMPK signaling pathway in mice and might serve as an antiobesity multifunctional food additive.

참산부추(Allium sacculiferum Max.) 메탄올 추출물의 지방세포 내 ROS 생성 및 지질 축적 억제 효능

Allium sacculiferum Max. (ASM) is a perennial plant of the Liliaceae family and grows over the entire regions of Korea. Obesity is a serious health problem worldwide and has currently become a prevalent chronic disease. Adipocytes produced by preadipocyte differentiation during adipogenesis and adipocytes combined with abnormal accu- mulation cause obesity. Recently, intracellular reactive oxygen species (ROS) were shown to accelerate lipid accumu- lation in 3T3-L1 cells. In this study, we investigated the effects of ASM methanol extracts on ROS production and lipid accumulation in 3T3-L1 adipocytes. Our results indicate that the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of ASM methanol extracts increased in a dose-dependent manner. ASM methanol extracts suppressed ROS production and lipid accumulation during adipogenesis. In addition, ASM methanol extracts inhibited the mRNA ex- pression of both pro-oxidant enzymes such as glucose-6-phosphate dehydrogenase as well as the transcription factors, including sterol regulatory element-binding proteins 1c, peroxisome proliferator-activated receptor γ, and CCAAT/en- hancer-binding protein α. Our results suggest that ASM methanol extracts inhibit ROS production and lipid accumulation by controlling ROS regulatory genes and adipogenic transcription factors. Thus, ASM has potent natural antioxidant, anti-adipogenic properties and have potential in the development of a potent anti-obesity agent.

3T3-L1 지방전구세포에서 홍국균 균사체-고체발효 원두커피 추출물의 지방축적 억제효과

Obesity is the leading metabolic disease in industrialized countries and is closely associated with coronary heart disease, hypertension, diabetes, and cancer. The objective of this study was to evaluate the anti-adipogenic effects of two roasted coffee beans, Vietnam robusta (VR) and Ethiopia Mocha Sidamo G2 (ES), as well as fermented coffee beans with Monascus ruber (MR) mycelium on differentiation of 3T3-L1 preadipocytes. Treatments with 1,000 μg/mL of hot water extract from coffee beans significantly reduced intracellular lipid accumulation. In addition, VR more effectively inhibited transcription factors such as PPARγ, C/EBPα, FAS, and aP2 compared to ES. Further, ES fermented with MR showed more effective anti-adipogenic activity than non-fermented ES. These results suggest that VR and ES inhibit adipocyte differentiation which may contribute to their anti-adipogenic properties.

Antioxidant and antiadipogenic activities of galkeun-tang, a traditional korean herbal formula.

Galkeun-tang (GKT; Galgen-tang in Chinese and Kakkon-to in Japanese), a traditional herbal formula, has been used for treatment of the common cold. Here, we report in vitro antioxidant and antiadipogenic effects of GKT. GKT increased the activities of scavenging 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. GKT also significantly reduced the malondialdehyde (MDA) generation during low-density lipoprotein (LDL) oxidation and the electrophoretic mobility of oxidized LDL, indicating inhibitory effects of GKT on Cu2+-mediated oxidation of LDL. Regarding antiadipogenic activity, GKT treatment significantly suppressed lipid accumulation, triglyceride production, and glycerol-3-phosphate dehydrogenase (GPDH) activity in differentiated 3T3-L1 adipocytes. Consistent with this, GKT significantly reduced the secretion of leptin, a major adipokine, in differentiated 3T3-L1 adipocytes. Overall, our findings suggest that GKT has the potential for antioxidative and antiadipogenic properties.

COH-SR4 Reduces Body Weight, Improves Glycemic Control and Prevents Hepatic Steatosis in High Fat Diet-Induced Obese Mice

Obesity is a chronic metabolic disorder caused by imbalance between energy intake and expenditure, and is one of the principal causative factors in the development of metabolic syndrome, diabetes and cancer. COH-SR4 (“SR4”) is a novel investigational compound that has anti-cancer and anti-adipogenic properties. In this study, the effects of SR4 on metabolic alterations in high fat diet (HFD)-induced obese C57BL/J6 mice were investigated. Oral feeding of SR4 (5 mg/kg body weight.) in HFD mice for 6 weeks significantly reduced body weight, prevented hyperlipidemia and improved glycemic control without affecting food intake. These changes were associated with marked decreases in epididymal fat mass, adipocyte hypertrophy, increased plasma adiponectin and reduced leptin levels. SR4 treatment also decreased liver triglycerides, prevented hepatic steatosis, and normalized liver enzymes. Western blots demonstrated increased AMPK activation in liver and adipose tissues of SR4-treated HFD obese mice, while gene analyses by real time PCR showed COH-SR4 significantly suppressed the mRNA expression of lipogenic genes such as sterol regulatory element binding protein-1c (Srebf1), acetyl-Coenzyme A carboxylase (Acaca), peroxisome proliferator-activated receptor gamma (Pparg), fatty acid synthase (Fasn), stearoyl-Coenzyme A desaturase 1 (Scd1), carnitine palmitoyltransferase 1a (Cpt1a) and 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr), as well as gluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose-6-phosphatase (G6pc) in the liver of obese mice. In vitro, SR4 activates AMPK independent of upstream kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ). Together, these data suggest that SR4, a novel AMPK activator, may be a promising therapeutic compound for treatment of obesity, fatty liver disease, and related metabolic disorders.

The mechanisms of carnosic acid attenuates tumor necrosis factor‐α‐mediated inflammation and insulin resistance in 3T3‐L1 adipocytes

Insulin resistance has been linked to a low-grade chronic inflammatory response. Carnosic acid (CA), which is found in rosemary, has been reported to have antioxidant, anti-inflammation, and anti-adipogenic properties. Here, we examined the effects of CA on inflammation and insulin resistance in 3T3-L1 adipocytes treated with tumor necrosis factor-α (TNF-α). CA attenuated the TNF-α-induced mRNA expression of inflammatory genes, including IL-6 and monocyte chemoattractant protein-1. CA also attenuated the TNF-α-mediated activation of extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and c-Jun; the phosphorylation of inhibitor-κB (IκB) kinase (IKK)α/β, the phosphorylation and degradation of IκBα, the nuclear translocation of p65, and the DNA-binding activity of NF-κB and AP-1. CA or PP242 (an mTOR inhibitor) suppressed the TNF-α-induced protein expression of mTOR, p70S6K, eIF4E, and IL-6. Moreover, CA attenuated the TNF-α-mediated suppression of peroxisome proliferator-activated receptor γ, adiponectin, and adipocyte protein 2. CA reversed the TNF-α-mediated suppression of insulin-stimulated glucose uptake and the phosphorylation of Tyr(632) insulin receptor substrate-1 (IRS-1), Akt, and FoxO1, but decreased the TNF-α-induced phosphorylation of Ser(307) IRS-1 and total FoxO1. CA attenuates TNF-α-mediated inflammation via inhibition of NF-κB and AP-1 pathways and insulin resistance via Akt-dependent FoxO1 signaling in 3T3-L1 adipocytes.

High conjugated linoleic acid enriched ghee (clarified butter) increases the antioxidant and antiatherogenic potency in female Wistar rats

BackgroundHypercholesterolemia and oxidative stress are the main stimulating factors responsible for coronary artery disease and progression of atherosclerosis. Dairy food products are rich in conjugated linoleic acid (CLA) which is considered as an important component due to its potential health benefits such as anticarcinogenic, antiatherogenic, antidiabetic and antiadipogenic properties. In the present study, the effect of CLA enriched ghee on the antioxidant enzyme system and antiatherogenic properties in Wistar rats has been studied.MethodsFemale Wistar rats of 21 days were taken for the study and fed with soybean diet (Control diet), low CLA diet and high CLA ghee diet (treatments) for thirty five days for studying antioxidative enzymes and sixteen weeks in case of antiatherogenic studies.ResultsFeeding of high CLA enhanced ghee during pubescent period in rats lead to an increase in catalase (CAT) and superoxide dismutase (SOD) enzyme activities in blood and increased CAT, SOD and glutathione transferase (GST) enzymes activities in liver by 27, 130 and 168 percent, respectively. Plasma nitrate concentration and Haemoglobin levels remained the same in all the treatments. Feeding of high CLA ghee resulted in lower (P < 0.01) plasma cholesterol & triglyceride level (52.17 and 30.27%), and higher high density lipoproteins (33.26%) than feeding of soybean oil (control group) and thus manifested in decreased (P < 0.05) atherogenic index (from 0.472 to 0.244). Lesser cholesterol and triglyceride levels were observed in the liver and aorta of high CLA fed rats than in those of the other groups. Histopathological studies of liver showed normal hepatic cords with portal triad in the high CLA ghee fed rats whereas fatty degeneration of hepatocytes containing fat vacuoles was observed in the liver of the other groups.ConclusionThis paper is the first report of the antioxidant and antiatherogenic properties of the high CLA enriched ghee suggesting that high CLA ghee can be used as a potential food for decreasing the risk of cardiovascular diseases, particularly in India, where, ghee is widely used for culinary and medicinal purposes.

Regulation of TCF/LEF promoter activity by soy phytoestrogens in 3T3‐L1 fibroblasts

The canonical Wnt pathway inhibits adipogenesis through activation of the transcription factor, TCF/LEF1. Soy phytoestogens, such as genistein (GEN) and daidzein (DAID), are paradoxically shown to both induce and repress adipogenesis. To determine if the concentration of soy phytoestrogen effects adipogenic regulation, 3T3‐L1 fibroblasts were transfected with either a estrogen receptor (ER) or TCF/LEF lentiviral reporter construct. Transfected cells were then treated with GEN or DAID at 100 pM, nM, or uM concentration. Our data showed that both ER and TCF/LEF promoter activity was 2–3 fold greater in cells treated with 100 uM of GEN or DAID (vs. vehicle). In contrast, fibroblasts treated with the pM or nM concentrations exhibited limited promoter activity. Collectively, these data indicate that the anti‐adipogenic properties of soy phytoestrogen may be attributed to high pharmacological doses.

A comparative analysis of the adipogenic potential in human mesenchymal stromal cells from cord blood and other sources

Mesenchymal stromal cells (MSCs) from umbilical cord blood (CB) attract attention by significantly impaired or absent adipogenic differentiation compared with MSCs derived from bone marrow (BM) and adipose tissue (AT). The diverging adipogenic propensity between the developmentally younger CB-MSCs and MSCs of the adult AT and BM resembles the age-dependent process in the BM, where adipose tissue increases with advancing age, accompanied by loss of bone stability. Thus, MSCs appeal as an attractive model to study the adipogenic process with respect to tissue sources and developmental ages. We followed the expression of main adipogenic transcription factors, genes and protein markers in CB-, BM- and AT-MSCs under adipogenic induction, after silencing of preadipocyte factor 1 (Pref-1, PREF1) and after incubation with CB-plasma supplemented adipogenic media. An inverse relation in the expression of adipogenesis-associated markers and PREF1 in CB-MSCs suggested an inhibitory role of Pref-1 toward adipogenesis. However, Pref-1 protein was rarely detected in CB-MSCs, and siRNA silencing of Pref-1 failed to induce adipogenic differentiation in CB-MSCs. Thus, the impaired adipogenic differentiation of CB-MSCs in vitro was unrelated to endogenous Pref-1 protein expression. Nevertheless CB-plasma containing Pref-1 protein revealed an anti-adipogenic effect on AT-MSCs. Because Pref-1 is vastly abundant in CB-plasma and confers anti-adipogenic properties, Pref-1 in association with the ontogenic milieu probably induces long-lasting unresponsiveness toward adipogenic stimuli in CB-MSCs.

Depressed Levels of Prostaglandin F2α in Mice Lacking Akr1b7 Increase Basal Adiposity and Predispose to Diet-Induced Obesity

Negative regulators of white adipose tissue (WAT) expansion are poorly documented in vivo. Prostaglandin F2α (PGF2α) is a potent antiadipogenic factor in cultured preadipocytes, but evidence for its involvement in physiological context is lacking. We previously reported that Akr1b7, an aldo-keto reductase enriched in adipose stromal vascular fraction but absent from mature adipocytes, has antiadipogenic properties possibly supported by PGF2α synthase activity. To test whether lack of Akr1b7 could influence WAT homeostasis in vivo, we generated Akr1b7−/− mice in 129/Sv background. Akr1b7−/− mice displayed excessive basal adiposity resulting from adipocyte hyperplasia/hypertrophy and exhibited greater sensitivity to diet-induced obesity. Following adipose enlargement and irrespective of the diet, they developed liver steatosis and progressive insulin resistance. Akr1b7 loss was associated with decreased PGF2α WAT contents. Cloprostenol (PGF2α agonist) administration to Akr1b7−/− mice normalized WAT expansion by affecting both de novo adipocyte differentiation and size. Treatment of 3T3-L1 adipocytes and Akr1b7−/− mice with cloprostenol suggested that decreased adipocyte size resulted from inhibition of lipogenic gene expression. Hence, Akr1b7 is a major regulator of WAT development through at least two PGF2α-dependent mechanisms: inhibition of adipogenesis and lipogenesis. These findings provide molecular rationale to explore the status of aldo-keto reductases in dysregulations of adipose tissue homeostasis.

Effect of nano-encapsulated (-) epigallocatechin gallate on triglyceride accumulation in 3T3-L1 adipocytes

Background Obesity is considered the major risk factor for type 2 diabetes mellitus (T2DM), cardio-vascular diseases like hypertension and atherosclerosis [1]. Catechins are the major source of flavonols and they are comprised of various types of catechins such as epigallo catechin 3 gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) [2]. Numbers of in vivo studies have reported weight loss after drinking green tea for several months. To support these in vivo findings, many in vitro studies demonstrated effect of pure EGCG which is an important biologically active green tea catechin in reducing triglyceride accumulation in adipocytes [3]. However, these antiadipogenic properties of EGCG are greatly dependent on bio-availability which is very low in animals as well as in humans. One study showed that, peak plasma concentrations in humans were 1.3 μM for EGCG after a single dose of 1.5 mmol through oral administration [4]. Nanoencapsulated EGCG (Nano-EGCG) may help to increase bioavailability and to exert maximum anti-adipogenic effects.

In Vitro Hepatotoxicity and Cytochrome P450 Induction and Inhibition Characteristics of Carnosic Acid, a Dietary Supplement with Antiadipogenic Properties

Carnosic acid is a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), which may have anticancer, antiadipogenic, and anti-inflammatory properties. Recently, carnosic acid was shown to prevent weight gain and hepatic steatosis in a mouse model of obesity and type II diabetes. Based on these results, carnosic acid has been suggested as a potential treatment for obesity and nonalcoholic fatty liver disease; however, little is known about the safety of carnosic acid at doses needed to elicit a pharmacological effect. For this reason, hepatotoxicity and cytochrome P450 inhibition and induction studies were performed in primary human hepatocytes and microsomes. Measuring cellular ATP, carnosic acid showed a dose-dependent increase in hepatotoxicity with an EC(50) value of 94.8 ± 36.7 μM in three human hepatocyte donors without a concurrent increase in the apoptosis markers caspase-3/7. In human liver microsomes, carnosic acid did not exhibit significant time-dependent inhibition for any of the cytochrome P450 enzymes investigated, although it did inhibit CYP2C9- and CYP3A4-catalyzed reactions with K(i) values of 9.2 and 4.3 μM, respectively. Carnosic acid also induced CYP2B6 and CYP3A4 mRNA and enzyme activity in a dose-dependent manner. At 10 μM, carnosic acid increased CYP2B6 enzyme activity 61.6 and 49.3% in two donors compared with phenobarbital, and it increased CYP3A enzyme activity 82.6 and 142% compared with rifampicin. These results indicate the potential for drug interactions with carnosic acid and illustrate the need for an appropriate safety assessment before being used as a weight loss supplement.

Piceatannol, Natural Polyphenolic Stilbene, Inhibits Adipogenesis via Modulation of Mitotic Clonal Expansion and Insulin Receptor-dependent Insulin Signaling in Early Phase of Differentiation

Piceatannol, a natural stilbene, is an analog and a metabolite of resveratrol. Despite a well documented health benefit of resveratrol in intervention of the development of obesity, the role of piceatannol in the development of adipose tissue and related diseases is unknown. Here, we sought to determine the function of piceatannol in adipogenesis and elucidate the underlying mechanism. We show that piceatannol inhibits adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner at noncytotoxic concentrations. This anti-adipogenic property of piceatannol was largely limited to the early event of adipogenesis. In the early phase of adipogenesis, piceatannol-treated preadipocytes displayed a delayed cell cycle entry into G(2)/M phase at 24 h after initiation of adipogenesis. Furthermore, the piceatannol-suppressed mitotic clonal expansion was accompanied by reduced activation of the insulin-signaling pathway. Piceatannol dose-dependently inhibited differentiation mixture-induced phosphorylation of insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/Akt pathway in the early phase of adipogenesis. Moreover, we showed that piceatannol is an inhibitor of IR kinase activity and phosphatidylinositol 3-kinase (PI3K). Our kinetics study of IR further identified a K(m) value for ATP of 57.8 μm and a K(i) value for piceatannol of 28.9 μm. We also showed that piceatannol directly binds to IR and inhibits IR kinase activity in a mixed noncompetitive manner to ATP, through which piceatannol appears to inhibit adipogenesis. Taken together, our study reveals an anti-adipogenic function of piceatannol and highlights IR and its downstream insulin signaling as novel targets for piceatannol in the early phase of adipogenesis.

Additive Effects of Sonic Hedgehog and Nell-1 Signaling in Osteogenic Versus Adipogenic Differentiation of Human Adipose-Derived Stromal Cells

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

Carnosic acid (CA) prevents lipid accumulation in hepatocytes through the EGFR/MAPK pathway

Carnosic acid (CA), found in rosemary, has been reported to have antioxidant and anti-adipogenic properties. We recently demonstrated that CA protects against steatosis in ob/ob mice. In the present report, we investigated the molecular mechanism by which CA inhibits lipids accumulation both in vivo and in vitro. In the in vivo study, ob/ob mice were fed a standard chow diet with or without CA for 5 weeks, then their hepatocyte lipid accumulation was determined. The serum concentrations of cytokines, the levels of lipid regulatory mediators, and the hepatic metabolic and signaling molecules were also evaluated. In the in vitro study, HepG2 cells were used to further clarify the effects of CA on cellular lipid accumulation and to confirm the signaling pathways involved in these effects. CA significantly reduced hepatocyte lipid accumulation. This effect was associated with repressed levels of hepatic PPARγ, reduced expression of inflammatory cytokines such as IL-1β, IL-12, IL-17, IFN-γ, MCP-1, and MIP-1β, and increased ATP, acetyl CoA, NAD(P)(+), and NAD(P)H. Other signaling molecules, such as EGFR, MAPK, AMPK, and ACC, which regulate lipid metabolism, were activated in mice fed the CA diet. CA inhibited palmitate-induced cellular lipid accumulation and stimulated the phosphorylation of both EGFR and MAPK. Pretreatment with either the EGFR inhibitor AG1478 or the MEK-specific inhibitor U0126 abolished the effects of CA on cellular lipid accumulation and decreased both the protein expression and activity of PPARγ. EGFR/MAPK signaling plays an important role in the inhibitory effect of CA on hepatocyte lipid accumulation.