Cysteine deprivation prevents induction of peroxisome proliferator-activated receptor gamma-2 and adipose differentiation of 3T3-L1 cells

Plasma cysteine is strongly associated with body fat mass in human cohorts and diets low in cysteine prevents fat accumulation in mice. It is unclear if plasma cysteine affects fat development or if fat accumulation raises plasma cysteine. To determine if cysteine affects adipogenesis, we differentiated 3T3-L1 preadipocytes in medium with reduced cysteine. Cells incubated in media with 10–20 μM cysteine exhibited reduced capacity to differentiate into triacylglycerol-storing mature adipocytes compared with cells incubated with 50 μM cysteine. Low cysteine severely reduced expression of peroxisome proliferator-activated receptor gamma2 (Pparγ2) and its target genes perlipin1 (Plin1) and fatty acid binding protein-4 (Fabp4). Expression of stearoyl-CoA desaturase-1 (Scd1), known to be repressed with cysteine depletion, was also reduced with low cysteine. Medium depletion of the essential amino acids leucine, valine, and isoleucine had only a modest effect on adipocyte specific gene expression and differentiation. Stimulation with the PPARγ agonist BRL-49653 or addition of a hydrogen sulfide donor enhanced differentiation of 3T3-L1 cells cultured in low cysteine. This demonstrates that the ability to induce PPARγ expression is preserved when cells are cultured in low cysteine. It therefore appears that cysteine depletion inhibits adipogenesis by specifically affecting molecular pathways required for induction of PPARγ expression, rather than through a general reduction of global protein synthesis. In conclusion, we show that low extracellular cysteine reduces adipocyte differentiation by interfering with PPARγ2 and PPARγ target gene expression. Our results provide further evidence for the hypothesis that plasma cysteine is a casual determinant for body fat mass.     

Anti-adipogenic activity of compounds isolated from Idesia polycarpa on 3T3-L1 cells

Recently, obesity is a complex multifactorial chronic disease increasing the risk for type 2 diabetes, coronary heart disease and hypertension, and has become a major worldwide health problem. In the course of screening natural products employing 3T3-L1 cells as an in vitro system, the methanol extract of Idesia polycarpa Maxim. Fruits (Flacourtiaceae) significantly inhibited adipocyte differentiation by measuring lipid contents using oil red O staining. One new compound, 6-(oxymethyl)-2-hydroxyphenyl-O-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranoside (8), was isolated along with nine known compounds (1–7 and 9–10) from CHCl₃ and n-BuOH fractions of the methanol extract of I. polycarpa fruits. Among them, idescarpin (1) with 1-hydroxy-6-oxo-2-cyclohexenecarboxylate moiety showed the most potent inhibitory activity on adipocyte differentiation with IC₅₀ values of 23.2 μM. Idescarpin (1) dramatically suppressed the induction of C/EBPα expression, whereas it significantly increased the induction of PPARγ expression, supported by quantitative real time PCR and Western blot analysis. The down-regulation in mRNA levels of SREBP1c, SCD-1, and FAS by idescarpin (1) during adipocyte differentiation revealed that the inhibition of adipocyte differentiation was mediated by the regulation of lipogenesis. Taken together, we suggest that idescarpin (1) shows a great potential against obesity and diabetes though the anti-adipogenic activity and the up-regulation of PPARγ.     

Fish-oil-derived n-3 polyunsaturated fatty acids reduce NLRP3 inflammasome activity and obesity-related inflammatory cross-talk between adipocytes and CD11b+ macrophages

Adipocyte–macrophage cross-talk propagates immune responses in obese adipose tissue (AT). Long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) mitigate inflammation, partly through up-regulation of adiponectin; however, specific mechanisms are unclear. We determined if adipocyte–macrophage cross-talk could be mitigated by dietary LC n-3 PUFA and if this was dependent on adiponectin-mediated signaling. We utilized an in vitro co-culture model mimicking the ratio of adipocytes:macrophages in obese AT, whereby 3T3-L1 adipocytes were co-cultured with splenic CD11b⁺ macrophages from C57BL/6 mice fed high-fat control (HF-CON; 34% w/w fat) or fish oil diets (HF-FO; 34% w/w fat containing 7.6% w/w FO), as well as mice fed low-fat control (LF-CON; 10% w/w fat) or FO diets (LF-FO; 10% w/w fat containing 3% w/w FO). Co-culture conditions tested effects of soluble mediator-driven mechanisms (trans-well system), cell contact and low-dose lipopolysaccharide (LPS) mimicking acute or chronic inflammatory conditions. HF-FO macrophages from acute LPS-stimulated trans-well co-cultures had decreased mRNA expression of Casp1, Il1β and Il18, as well as cellular caspase-1 activity compared to HF-CON macrophages (P ≤ .05). Moreover, adipocytes from acute LPS-stimulated HF-FO co-cultures had decreased caspase-1 activity and decreased IL-1β/IL-18 levels following chronic LPS pretreatment compared to HF-CON co-cultures (P ≤ .05). Additionally, in contact co-cultures with adiponectin-neutralizing antibody, the FO-mediated modulation of NFκB activity and decrease in phosphorylated p65 NFκB, expression of NLRP3 inflammasome genes, M1 macrophage marker genes and inflammatory cytokine/chemokine secretion were controlled partly through adiponectin, while cellular caspase-1 activity and IL-1β/1L-18 levels were decreased independently of adiponectin (P ≤ .05). LC n-3 PUFA may decrease the intensity of adipocyte–macrophage cross-talk to mitigate obesity-associated pathologies.     

The effect of limited hydrolysis with Neutrase and ultrafiltration on the anti-adipogenic activity of soy protein

Limited hydrolysis of isolated soy protein (ISP) with Neutrase for 4 h to obtain the hydrolysate (NH4h) revealed the ability to suppress glycerol-3-phosphate dehydrogenase (GPDH) activity and relative lipid accumulation (RLA) in 3T3-L1 cells during differentiation. Lower GPDH activity or RLA indicates higher anti-adipogenic activity. Sequentially fractionating NH4h with 30–1 kDa (kilo-daltons) molecular weight cut-off (MWCO) membranes to obtain the 1 kDa concentrate resulted in further enhancing its anti-adipogenic activity in the cells. The GPDH activity significantly decreased from 280 to 100 U/mg protein (p < 0.05). When comparing the high-performance size-exclusion chromatography (HPSEC) profiles, the most active peptide for the anti-adipogenic activity was primarily composed of peptides with molecular weight between 1300 and 2200 Da. The in vitro effect of gastrointestinal protease on the anti-adipogenic activity of 1 kDa concentrate was also investigated. The results suggested that gastrointestinal proteases have very little effect on anti-adipogenic activity of the concentrate. According to the Western immunoblot analysis, 1 kDa concentrate inhibits adipogenesis by affecting the expression of peroxisome proliferators-activated receptor γ (PPARγ) and the CCAAT/enhancer binding protein α (C/EBPα) during 3T3-L1 cells differentiation.     

Cystathionine γ lyase–hydrogen sulfide increases peroxisome proliferator-activated receptor γ activity by sulfhydration at C139 site thereby promoting glucose uptake and lipid storage in adipocytes

Adipocytes express the cystathionine γ lyase (CSE)–hydrogen sulfide (H₂S) system. CSE–H₂S promotes adipogenesis but ameliorates adipocyte insulin resistance. We investigated the mechanism of how CSE–H₂S induces these paradoxical effects. First, we confirmed that an H₂S donor or CSE overexpression promoted adipocyte differentiation. Second, we found that H₂S donor inhibited but CSE inhibition increased phosphodiesterase (PDE) activity. H₂S replacing isobutylmethylxanthine in the differentiation program induced adipocyte differentiation in part. Inhibiting PDE activity by H₂S induced peroxisome proliferator activated receptor γ (PPARγ) protein and mRNA expression. Of note, H₂S directly sulfhydrated PPARγ protein. Sulfhydrated PPARγ increased its nuclear accumulation, DNA binding activity and adipogenesis gene expression, thereby increasing glucose uptake and lipid storage, which were blocked by the desulfhydration reagent DTT. H₂S induced PPARγ sulfhydration, which was blocked by mutation of the C139 site of PPARγ. In mice fed a high-fat diet (HFD) for 4 weeks, the CSE inhibitor decreased but H₂S donor increased adipocyte numbers. In obese mice fed an HFD for 13 weeks, H₂S treatment increased PPARγ sulfhydration in adipose tissues and attenuated insulin resistance but did not increase obesity. In conclusion, CSE–H₂S increased PPARγ activity by direct sulfhydration at the C139 site, thereby changing glucose into triglyceride storage in adipocytes. CSE–H₂S-mediated PPARγ activation might be a new therapeutic target for diabetes associated with obesity.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling,down-regulation of PPARγ and induction of iNOS

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC₅₀ of 10 μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.     

Anti adipogenic activity of Aegle marmelos Correa

In continuation of evaluating the anti-obesity effect of Aegle marmelos, we have screened the n-hexane, dichloro methane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts of the leaves at the concentration of 25, 50, 75 and 100 μg/ml for adipogenesis inhibition in the adipocytes. Nile red staining with the help of fluorometry was used as indicator of the antiobesity activity. The most active DCM extract showed the 33.98 ± 3.55% lipid content at 100 μg/ml and was selected for the further isolation. 14 compounds were isolated from DCM extract of A. marmelos leaves. The compounds were screened for the adipogenesis inhibition at 50 and 100 μM concentrations. Out of the 14 compounds, halfordinol, ethyl ether aegeline and esculetin were showing 10.04 ± 0.52, 16.29 ± 0.85 and 25.09 ± 1.31% lipid content respectively at 100 μM. We hereby report the adipogenesis inhibition by A. marmelos as one of the pathway for its antiobesity effect.     

Discovery of novel dual functional agent as PPARγ agonist and 11β-HSD1 inhibitor for the treatment of diabetes

PPARγ and 11β-HSD1 are attractive therapeutic targets for type 2 diabetes. However, PPARγ agonists induce adipogenesis, which causes the side effect of weight gain, whereas 11β-HSD1 inhibitors prevent adipogenesis and may be beneficial for the treatment of obesity in diabetic patients. For the first time, we designed, synthesized a series of α-aryloxy-α-methylhydrocinnamic acids as dual functional agents which activate PPARγ and inhibit 11β-HSD1 simultaneously. The compound 11e exhibited the most potent inhibitory activity compared to that of the lead compound 2, with PPARγ (EC₅₀ = 6.76 μM) and 11β-HSD1 (IC₅₀ = 0.76 μM) in vitro. Molecular modeling study for compound 11e was also presented. Compound 11e showed excellent efficacy for lowering glucose, triglycerides, body fat, in well established mice and rats models of diabetes and obesity and had a favorable ADME profile.     

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of pNPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.     

The cardioprotective effect of an aqueous extract of fermented rooibos (Aspalathus linearis) on cultured cardiomyocytes derived from diabetic rats

Diabetic cardiomyopathy (DCM) is a disorder of the heart muscle that contributes to cardiovascular deaths in the diabetic population. Excessive generation of free radicals has been directly implicated in the pathogenesis of DCM. The use of antioxidants, through dietary supplementation, to combat increased cellular oxidative stress has gained popularity worldwide. Aspalathus linearis (rooibos) is a popular herbal tea that contains a novel antioxidant, aspalathin. Literature has reported on the antidiabetic, anti-inflammatory and free radical scavenging effects of rooibos. However, its protective effect against DCM has not been established. Therefore, this study investigated whether chronic exposure to an aqueous extract of fermented rooibos (FRE) has an ex vivo cardioprotective effect on hearts obtained from streptozotocin (STZ) induced diabetic rats. Adult Wistar rats were injected with 40 mg/kg of STZ. Two weeks after STZ injection, cardiomyocytes were isolated and cultured. Cultured cardiomyocytes were treated with FRE (1 and 10 μg/ml), vitamin E (50 μg/ml), and n-acetyl cysteine (1 mM) for 6 h, before exposure to either hydrogen peroxide (H₂O₂) or an ischemic solution. Cardiomyocytes exposed to H₂O₂ or an ischemic solution showed a decrease in metabolic activity and glutathione content with a concomitant increase in apoptosis and intracellular reactive oxygen species. Pretreatment with FRE was able to combat these effects and the observed amelioration was better than the known antioxidant vitamin E. This study provides evidence that an aqueous extract of fermented rooibos protects cardiomyocytes, derived from diabetic rats, against experimentally induced oxidative stress and ischemia.     

Insulin-sensitizing activities of tanshinones,diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1–10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents.     

Side-by-side comparison of horizontal subsurface flow and free water surface flow constructed wetlands and artificial neural network (ANN) modelling approach

A horizontal subsurface flow (HSSF) and a free water surface flow (FWSF) constructed wetlands (4 m² of each) were set up on the campus of Harran University, Sanliurfa, Turkey. The main objective of the research was to compare the performance of two systems to decide the better one for future planning of wastewater treatment system on the campus. Both of the wetland systems were planted with Phragmites australis and Canna indica. During the observation period (10 months), environmental conditions such as pH, temperature and total chemical oxygen demand (COD), soluble COD, total biochemical oxygen demand (BOD), soluble BOD, total suspended solids (TSS), total phosphate (TP), total nitrogen (TN) removal efficiencies of the systems were determined. According to the results, average yearly removal efficiencies for the HSSF and the FWSF, respectively, were as follows: total COD (75.7% and 69.9%), soluble COD (85.4% and 84.3%), total BOD (79.6% and 87.6%), soluble BOD (87.7% and 95.3%), TN (33.2% and 39.4%), and TP (31.5% and 6.5%). Soluble COD and BOD removal efficiencies of both systems increased gradually since the start-up. After nine months of operation, above 90% removal of organic matters were observed. The treatment performances of the HSSF were better than that of the FWSF with regard to the removal of suspended solids and total COD at especially high temperatures. In FWSF systems, COD concentrations extremely exceeded the discharge limit values due to high concentrations of algae in spring months.The performance of the two systems was modelled using an artificial neural network-back-propagation algorithm. The ANN model was competent at providing reasonable match between the measured and the predicted concentrations of total COD (R = 0.90 for HSSF and R = 0.96 for FWSF), soluble COD (R = 0.90 for HSSF and R = 0.74 for FWSF) and total BOD (R = 0.94 for HSSF and R = 0.84 for FWSF) in the effluents of constructed wetlands.     

Lactobacillus plantarum effects on silage fermentation and in vitro microbial yield

In four parallel experiments, herbage [three harvests of alfalfa (308 to 379 g dry matter (DM)/kg), one of whole-plant corn (331 g DM/kg)] was ensiled with three different treatments: no inoculant (control), Lactobacillus plantarum (LP) or formic acid (FA), in 1-L mini-silos and fermented for 60 d at room temperature (22 °C). Mini-silos were opened and analyzed for fermentation characteristics and soluble N fractions. A subsample of wet silage from each mini-silo was ground to 4 mm and stored at ?20 °C. Silages were thawed and subjected to 9 h ruminal in vitro incubations to measure gas production and volatile fatty acid (VFA) production as well as microbial biomass yield (MBY) and microbial non-ammonia N (MNAN) formation using ¹⁵N as a marker. In all four experiments, silage fermentation products and pH indicated good preservation across all treatments. Analysis of data showed that FA- and LP-treated silages had lower concentrations of ammonia-N and free amino acids N than control. The FA treatment was lower in soluble N, but higher in peptide-N, than control. Silage pH was lowest in FA (4.25), followed by LP (4.28), and control (4.38). Ruminal in vitro gas production and VFA concentrations were not different among treatments (P>0.05). Compared to control, FA- and LP-treated silage yielded greater MNAN and MBY. These findings suggested that L. plantarum preserved more true protein during silage fermentation than control, which in turn increased in vitro ruminal microbial growth.     

Chemical constituents from Ainsliaea acerifolia as potential anti-obesity agents

Bioassay-guided isolation of an ethanolic extract of the aerial parts from Ainsliaea acerifolia using pancreatic lipase inhibitory assay led to the isolation of a new guaianolide-type sesquiterpene lactone (1), a new dicaffeoylquinic acid derivative (4), as well as eight known secondary metabolites. Structures of the two new compounds were elucidated on the basis of spectroscopic methods. All isolated compounds were evaluated for their inhibitory effects against pancreatic lipase, and compound 2 exhibited significant inhibitory activity with an IC₅₀ value of 15.3 ± 0.7 μM. Furthermore, compound 2 also exhibited potent inhibitory effects against 3T3-L1 adipocyte cells.