Differentiated 3T3-L1 Adipocytes Research Articles

Radish Seed Exerts Anti-Diabetic and Obesity-Reducing Effects in Mice by Promoting the Activation of Uncoupling Protein 1 and Peroxisome Proliferator-Activated Receptor-γ Coactivator 1-α.

Obesity is primarily due to excessive energy intake and lipid accumulation, leading to type 2 diabetes. Studies showed radish seed extract (RSE) can impede weight gain in mice, but the mechanism was unclear. We hypothesized that RSE inhibits obesity by stimulating adipocyte browning. Radish seeds were water-extracted, yielding a sulforaphene (SE) concentration of 1.381 ± 0.005 mg/g RSE. In 3T3-L1 adipocyte differentiation experiments, RSE and SE increased the expression of beige adipocyte markers uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α). In C57BL/6 mice, RSE and SE mitigated weight increase, averted fatty liver, and diminished fat accumulation. In the adipose tissue, we also noted the enhanced browning of white adipocytes through elevated expression of UCP1 and PGC1α. Increased mitochondrial numbers in treated adipocytes supported this effect. Additionally, RSE and SE improved glucose homeostasis and insulin sensitivity in high-fat diet-fed mice, indicating RSE's potential to prevent obesity and diabetes by enhancing adipocyte thermogenesis.

Effect of H2O2 induced oxidative stress on volatile organic compounds in differentiated 3T3-L1 cells

Oxidative stress (OS) refers to the disruption in the balance between free radical generation and antioxidant defenses, leading to potential tissue damage. Reactive oxygen species (ROS) can interact with biological components, triggering processes like protein oxidation, lipid peroxidation, or DNA damage, resulting in the generation of several volatile organic compounds (VOCs). Recently, VOCs provided new insight into cellular metabolism and can serve as potential biomarkers. The objective is to investigate the impact of OS on cell metabolism by analyzing the release or alterations of VOCs in the headspace of differentiated 3T3-L1 adipocytes. An OS model in differentiated 3T3-L1 cell lines was constructed using hydrogen peroxide (H2O2) treatment. The effect of OS on cell metabolism was analyzed by detecting VOCs in the headspace of the cells using solid phase micro extraction (SPME) and gas chromatography-mass spectrometry (GCMS). Our findings indicate that H2O2 concentrations exceeding 300 µM induce significant OS, leading to adipocyte apoptosis, as evidenced by various assays. Of the twenty VOCs identified, ten were upregulated in the cells. VOCs such as diphenyl ether, 1,3,5-trioxane, 5-methyl tridecane, 2-ethyl-1-hexanol, and 2,4-di-tert-butyl phenol emerged as potential biomarkers for OS. This study demonstrates that elevated OS alters VOC profiles in differentiated 3T3-L1 adipocytes, providing insights into the effects of OS on adipose tissue and identifying potential OS biomarkers.

GRK5 is required for adipocyte differentiation through ERK activation.

Previous studies have identified G protein-coupled receptor (GPCR) kinase 5 (GRK5) as a genetic factor contributing to obesity pathogenesis, but the underlying mechanism remains unclear. We demonstrate here that Grk5 mRNA is more abundant in stromal vascular fractions of mouse white adipose tissue, the fraction that contains adipose progenitor cells, or committed preadipocytes, than in adipocyte fractions. Thus, we generated a GRK5 knockout (KO) 3T3-L1 preadipocyte to further investigate the mechanistic role of GRK5 in regulating adipocyte differentiation. During adipogenic stimulation, GRK5 KO preadipocytes had decreased lipid accumulation and delayed mature adipocyte development compared to wildtype cells coupled with suppressed adipogenic and lipogenic gene expression. Beside GPCR signaling, RNA sequencing and pathway analysis identified insulin-like growth factor 1 (IGF-1) signaling to be one of the top 5 significantly dysregulated pathways in GRK5 KO cells. GRK5 KO cells also displayed decreased insulin-stimulated ERK phosphorylation, a downstream target of insulin-stimulated IGF-1 receptor activation, suggesting that GRK5 acts through this critical pathway to impact 3T3-L1 adipocyte differentiation. To find a more translational approach, we identified a new small molecule GRK5 inhibitor that was able to reduce 3T3-L1 adipogenesis. These data suggest that GRK5 is required for adipocyte differentiation through IGF-1 receptor/ERK activation and may be a promising translational target for obesity.

Inhibitory effects of cashew Anacardium occidentale L. kernel, apple, and shell extracts on lipid accumulation and adipogenesis in 3T3-L1 adipocytes

Obesity, a major risk factor for various metabolic diseases, often results in dysfunctional white adipose tissue and altered adipogenesis leading to ectopic fat accumulation, inflammation, and insulin resistance. On the other hand, cashew (Anacardium occidentale L.) nut worldwide consumption and production is increasing steadily, which augments the mass of byproducts to be discarded. Indeed, cashew apples and cashew shells have shown potent effects to lower adiposity weight in human and animal models. However, the direct effect on adipocyte differentiation still remains unexplored. Therefore, this study aimed to investigate the biological effect of cashew nut or kernel (CK), dried cashew apple (DA), and cashew shell (SH) ethanolic extracts on 3T3-L1 adipocyte differentiation and lipid accumulation. SH showed strong inhibition on adipocyte differentiation by downregulating transcription factors, PPARγ, C/EBPα, and SREBP-1. DA also inhibited the transcription factors accompanied by reduced lipid accumulation, while proteins for de novo lipogenesis were unchanged. Finally, CK did not alter any markers in adipocyte differentiation, however, interestingly adiponectin level was significantly increased. Concisely, our findings showed that CK ameliorates adiponectin production without interfering adipogenesis, while DA lowers lipid accumulation and SH suppresses adipogenesis.

Oyster Mushroom (Pleurotus ostreatus) Ethanolic Extract Inhibits Pparg Expression While Maintaining the Methylation of the Pparg Promoter During 3T3-L1 Adipocyte Differentiation.

This study aims to provide new insights into the potential of oyster mushroom (Pleurotus ostreatus) ethanolic extract in preventing obesity through the inhibition of Pparg expression and modulation of methylation level on Pparg promoter during 3T3-L1 adipocyte differentiation. This in vitro quantitative experimental study was conducted by treating the 3T3-L1 cell line differentiated using 0.5mM methyl-isobutyl-xanthine, 1μM dexamethasone, and 10μg/mL insulin-containing medium with oyster mushroom ethanolic extract. The extract was obtained from 80 g of dried oyster mushroom powder extracted three times with 800 mL of ethanol, filtered, evaporated, and reconstituted in dimethyl sulfoxide (DMSO) to final concentrations of 0, 25, 50, and 100µg/mL, with DMSO limited to 0.5% in all solutions. Pparg mRNA expression was quantified by qRT-PCR analysis and Pparg promoter methylation levels were measured quantitatively by pyrosequencing of bisulfite-treated DNA samples. The addition of 25µg/mL oyster mushroom ethanolic extract significantly suppressed Pparg mRNA expression with no significant change in the Pparg promoter methylation levels. Oyster mushroom ethanolic extract inhibited Pparg mRNA expression without altering Pparg promoter methylation, suggesting reduced adipocyte differentiation. This study emphasizes the potential of oyster mushroom in the prevention or treatment of obesity by inhibiting adipocyte differentiation.

Fulvic acid inhibits the differentiation of 3T3-L1 adipocytes by activating the Ca2+/CaMKⅡ/AMPK pathway.

Type 2 diabetes increases the risk of developing obesity. Although fulvic acid alleviates back fat thickness in pigs, the mechanism underlying its anti-obesity effect remains unclear. Therefore, we investigated the anti-obesity mechanism of fulvic acid using 3T3-L1 adipocytes. We examined the effects of fulvic acid on adipocyte differentiation, cell viability, and lipid accumulation using molecular techniques. Fulvic acid treatment significantly decreased intracellular lipid accumulation in 3T3-L1 cells during the differentiation compared with that in the control group. Western blotting revealed fulvic acid-induced downregulated expression of the adipocyte differentiation-related markers peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha, and sterol regulatory element-binding protein 1. The fulvic acid treatment decreased the expression of the lipid uptake-related markers fatty acid-binding protein 4 and the cluster of differentiation 36 in 3T3-L1 cells. Moreover, fulvic acid significantly increased cytosolic Ca2+ concentration via Ca2+ sequestration from the endoplasmic reticulum, enhanced Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, and upregulated AMP-activated protein kinase (AMPK), thereby reducing adipocyte differentiation. Conclusively, fulvic acid attenuates adipocyte differentiation by activating the Ca2+/CaMKⅡ/AMPK pathway, suggesting its anti-obesity potential.

Bacillus lipopeptides inhibit lipase activity and promote 3T3-L1 preadipocyte differentiation

Bacillus lipopeptides have been reported to display anti-obesity effects. In the present study, Lipopeptides from Bacillus velezensis FJAT-45028 that consisted of iturin, fengycin and surfactin were reported. The lipopeptides exhibited a strong lipase inhibition activity in a concentration-dependent manner with a half maximal inhibitory concentration of 0.012 mg/mL, and the inhibition mechanism and type were reversible and competitive, respectively. Results of CCK8 assay showed that 3T3-L1 preadipocyte cells were completely viable under treatment of 0.050–0.2 mg/mL lipopeptides for 24 or 48 h. It was found that the lipopeptides could increase lipid droplets in the differentiated 3T3-L1 adipocytes in tested concentration and suppress the expression of peroxisome proliferator-activated receptor gamma (PPARγ). These results indicated the potential anti-obesity mechanism of the tested lipopeptides might be to inhibit lipase activity but not to suppress lipid accumulation in the adipocytes. Moreover, the lipopeptides could elevate glucose utilisation by 14.43%–33.81% in the differentiated 3T3-L1 adipocytes.

Time-Course Strategy Reveals a Dual Potential of Perfluorooctanoic Acid and Its Alternative in Adipocyte Differentiation.

Exposure to perfluorooctanoic acid (PFOA) and hexafluoropropylene oxide dimer acid (HFPO-DA) was associated with adipogenesis. However, potential mechanisms remain to be elucidated. Herein, a 3T3-L1 adipocyte model was used to explore the dynamic changes in adipocyte differentiation (2, 4, and 8 days) under PFOA and HFPO-DA exposure. PFOA and HFPO-DA increased the adipocyte formation rate and intracellular levels of triglycerides (TG). Meanwhile, adipocyte browning was induced by PFOA and HFPO-DA, which was characterized by small lipid droplets, low levels of TG per adipocyte, increased ATP levels, and elevated mitochondrial respiration activities with time-dependent differentiation. The browning potency indexes of PFOA and HFPO-DA were approximately 1.5 times higher than those of the controls. Time-course transcriptomics analysis showed that PFOA and HFPO-DA activated the biological process of adipocyte browning but different gene expression patterns regulated adipocyte browning. Only overexpressed hydroxymethylglutaryl-CoA synthase (Hmgcs2) was shared between PFOA and HFPO-DA groups from 2 to 8 days. Hmgcs2 could regulate adipocyte browning induced by PFOA and HFPO-DA, and this observation was lost when Hmgcs2 was knocked down. Our study suggests that PFOA and HFPO-DA could play dual roles in the differentiation of 3T3-L1 adipocytes, and Hmgcs2 might be a target of PFOA- and HFPO-DA-induced adipocyte browning.

Mechanical Stretch Control of Adipocyte AKT Signaling and the Role of FAK and ROCK Mechanosensors.

Adipose tissue in vivo is physiologically exposed to compound mechanical loading due to bodyweight bearing, posture, and motion. The capability of adipocytes to sense and respond to mechanical loading milieus to influence metabolic functions may provide a new insight into obesity and metabolic diseases such as type 2 diabetes (T2D). Here, we evidenced physiological mechanical loading control of adipocyte insulin signaling cascades. We exposed differentiated 3T3-L1 adipocytes to mechanical stretching and assessed key markers of insulin signaling, AKT activation, and GLUT4 translocation, required for glucose uptake. We showed that cyclic stretch loading at 5% strain and 1 Hz frequency increases AKT phosphorylation and GLUT4 translocation to the plasma membrane by approximately two-fold increases compared to unstretched controls for both markers as assessed by immunoblotting (p < 0.05). These results indicate that cyclic stretching activates insulin signaling and GLUT4 trafficking in adipocytes. In the mechanosensing mechanism study, focal adhesion kinase (FAK) inhibitor (FAK14) and RhoA kinase (ROCK) inhibitor (Y-27632) impaired actin cytoskeleton structural formation and significantly suppressed the stretch induction of AKT phosphorylation in adipocytes (p < 0.001). This suggests the regulatory role of focal adhesion and cytoskeletal mechanosensing in adipocyte insulin signaling under stretch loading. Our finding on the impact of mechanical stretch loading on key insulin signaling effectors in differentiated adipocytes and the mediatory role of focal adhesion and cytoskeleton mechanosensors is the first of its kind to our knowledge. This may suggest a therapeutic potential of mechanical loading cue in improving conditions of obesity and T2D. For instance, cyclic mechanical stretch loading of adipose tissue could be explored as a tool to improve insulin sensitivity in patients with obesity and T2D, and the mediatory mechanosensors such as FAK and ROCK may be targeted to further invigorate stretch-induced insulin signaling activation.

Investigating the crosstalk between ABCC4 and ABCC5 in 3T3-L1 adipocyte differentiation

IntroductionThe plasma membrane-bound protein, multi-drug resistance-associated protein 4 (MRP4/ABCC4), has gained attention for its pivotal role in facilitating the efflux of a wide range of endogenous and xenobiotic molecules. Its significance in adipogenesis and fatty acid metabolism has been brought to light by recent studies. Notably, research on ABCC4 knockout (ABCC4−/−) mice has established a link between the absence of ABCC4 and the development of obesity and diabetes. Nevertheless, the specific contribution of ABCC4 within adipose tissue remains largely unexplored.MethodsTo address this gap, we conducted a study to elucidate the role of the ABCC4 transporter in mature adipocytes, using siRNA constructs to silence its gene function.ResultsThe successful knockdown of ABCC4 significantly altered lipid status and adipogenic gene expression in mature 3T3-L1 adipocytes. Intriguingly, this knockdown also altered the gene expression patterns of other ABCC transporter family members in 3T3-L1 cells. The downregulation of ABCC5 expression was particularly noteworthy, suggesting potential crosstalk between ABCC transporters in mature adipocytes. Additionally, knocking down ABCC5 resulted in significantly higher adipogenic and lipogenic gene expression levels. Oil Red O staining confirmed increased lipid accumulation following the knockdown of ABCC4 and ABCC5. Surprisingly, the simultaneous knockdown of both transporters did not show a cumulative effect on adipogenesis, rather it led to higher levels of intracellular cAMP and extracellular prostaglandin metabolite, both of which are essential signaling molecules in adipogenesis.ConclusionThese results highlight the complex interplay between ABCC4 and ABCC5 transporters in adipocyte function and suggest their individual contributions toward obesity and related disorders.

A Compressive Review on Formulation and Evaluation of Herbal Niosomes containing Phaseolus vulgaris and zingiber officinale for the Treatment of Diabetes Mellitus

This study aimed to formulate and evaluate niosomes containing Phaseolus vulgaris (PV) and Zingiber officinale (ZO) for their antidiabetic activity. Niosomes, non-ionic surfactant vesicles, were prepared using PV and ZO extracts via the thin-film hydration method. The formulations were characterized for particle size, morphology, entrapment efficiency, and in vitro release profile. Antidiabetic activity was assessed through in vitro α-amylase inhibition assay and glucose uptake studies using differentiated 3T3-L1 adipocytes. The formulated niosomes exhibited a mean particle size within the nanometer range, indicating their suitability for drug delivery. Morphological examination revealed spherical vesicles with uniform size distribution. Entrapment efficiency of the bioactive constituents from PV and ZO extracts was found to be satisfactory. In vitro release studies demonstrated sustained release profiles over time, indicative of controlled drug delivery potential. Antidiabetic activity assessment revealed significant α-amylase inhibition by the niosomal formulations compared to free extracts. Furthermore, glucose uptake studies demonstrated enhanced glucose uptake by 3T3-L1 adipocytes treated with niosomes containing PV and ZO extracts compared to untreated cells.

ATF3 suppresses 3T3-L1 adipocyte adipogenesis by transcriptionally repressing USP53.

Obesity is a widespread nutritional disorder, leading to a strong predisposition toward adverse health consequences. Activating transcription factor 3 (ATF3), a stress-induced transcription factor, has been documented as a therapeutic target for obesity. The intent of this project was to characterize the detailed role of ATF3 in adipogenesis in the context of obesity and its obscure downstream mechanism. After adipogenic differentiation, RT-qPCR and western blot examined ATF3 and ubiquitin-specific peptidase 53 (USP53) mRNA levels and protein levels. Adipogenesis was identified by Oil Red O staining, triglyceride (TG) levels, and western blot analysis. JASPAR database, ChIP and luciferase reporter assays predicted and validated the transcriptional regulation of USP53 by ATF3. Western blot also examined the protein levels of Ras homolog family member A (RhoA)/Rho-associated coiled-coil kinase (ROCK) pathway-involved proteins. ATF3 mRNA and protein levels were depleted in the differentiated 3T3-L1 adipocytes, and ATF3 elevation hindered the adipogenesis of 3T3-L1 adipocytes. ATF3 suppressed the transcription of USP53 as a transcription factor and lowered USP53 expression. Eventually, USP53 upregulation partially blunted the inhibitory role of ATF3 overexpression in adipogenesis and the RhoA/ROCK pathway. Consequently, ATF3 might transcriptionally inactivate USP53 to repress adipocyte adipogenesis and downregulate the RhoA/ROCK pathway.

Contribution of glucose and glutamine to hypoxia-induced lipid synthesis decreases, while contribution of acetate increases, during 3T3-L1 differentiation

The molecular mechanisms linking obstructive sleep apnea syndrome (OSA) to obesity and the development of metabolic diseases are still poorly understood. The role of hypoxia (a characteristic feature of OSA) in excessive fat accumulation has been proposed. The present study investigated the possible effects of hypoxia (4% oxygen) on de novo lipogenesis by tracking the major carbon sources in differentiating 3T3-L1 adipocytes. Gas-permeable cultuware was employed to cultivate 3T3-L1 adipocytes in hypoxia (4%) for 7 or 14 days of differentiation. We investigated the contribution of glutamine, glucose or acetate using 13C or 14C labelled carbons to the newly synthesized lipid pool, changes in intracellular lipid content after inhibiting citrate- or acetate-dependent pathways and gene expression of involved key enzymes. The results demonstrate that, in differentiating adipocytes, hypoxia decreased the synthesis of lipids from glucose (44.1 ± 8.8 to 27.5 ± 3.0 pmol/mg of protein, p < 0.01) and partially decreased the contribution of glutamine metabolized through the reverse tricarboxylic acid cycle (4.6% ± 0.2–4.2% ± 0.1%, p < 0.01). Conversely, the contribution of acetate, a citrate- and mitochondria-independent source of carbons, increased upon hypoxia (356.5 ± 71.4 to 649.8 ± 117.5 pmol/mg of protein, p < 0.01). Further, inhibiting the citrate- or acetate-dependent pathways decreased the intracellular lipid content by 58% and 73%, respectively (p < 0.01) showing the importance of de novo lipogenesis in hypoxia-exposed adipocytes. Altogether, hypoxia modified the utilization of carbon sources, leading to alterations in de novo lipogenesis in differentiating adipocytes and increased intracellular lipid content.

Weighted Gene Co-Expression Network Based on Transcriptomics: Unravelling the Differentiation Dynamics of 3T3-L1 Preadipocytes and the Regulatory Mechanism of Protopanaxatriol.

The intricate regulatory mechanisms governing adipocyte differentiation are pivotal in elucidating the complex pathophysiology underlying obesity. This study aims to explore the dynamic changes in gene expression during the differentiation of 3T3-L1 adipocytes using transcriptomics methods. Protopanaxatriol (PPT) significantly inhibited adipocyte differentiation. To uncover the molecular mechanisms, we conducted an extensive transcriptomic analysis of adipocytes throughout various differentiation stages, comparing gene expression profiles before and after PPT treatment. The construction of 16 co-expression modules was achieved using weighted gene co-expression network analysis (WGCNA). The 838 differentially expressed genes in the blue module were highly correlated with PPT treatment. Further analysis revealed that PIKfyve, STAT3, JAK1, CTTN, TYK2, JAK3, STAT2, STAT5b, SOCS3, and IRF9 were core genes closely associated with adipocyte differentiation. This discovery underscores the potential pivotal function of these ten genes in regulating adipocyte differentiation. This study elucidated that PPT, an active ingredient in ginseng, could reduce lipid accumulation by inhibiting the differentiation of adipocyte precursors through the negative regulation of genes such as PIKfyve, STAT3, and JAK1. Finally, molecular docking identified potential binding sites for PPT on PIKfyve and JAK1. This study provides potential drug targets for preventing obesity and related metabolic diseases.

Antioxidant activity and anti-adipogenic effect of ethyl acetate fraction of cumin seeds on 3T3-L1 adipocytes differentiation

Antioxidant activity and anti-adipogenic effect of ethyl acetate fraction of cumin seeds on 3T3-L1 adipocytes differentiation

1α,25-Dihydroxyvitamin D Downregulates Adipocyte Impact on Breast Cancer Cell Migration and Adipokine Release.

Excess adiposity is associated with a higher risk of breast cancer metastasis and mortality. Evidence suggests that dietary vitamin D inhibits breast cancer metastasis. However, the mechanistic link between vitamin D's regulation of adipocyte metabolism and metastasis has not been previously investigated. Therefore, the purpose of these experiments was to examine the effect of the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D), on adipocyte release of bioactive compounds and whether the impact on adipocytes leads to inhibition of breast cancer cell migration, an important step of metastasis. Differentiated 3T3-L1 adipocytes were treated with 1,25(OH)2D for two days, followed by either harvesting the adipocytes or collecting adipocyte-conditioned media without 1,25(OH)2D. A transwell migration assay was conducted with vehicle- or 1,25(OH)2D-conditioned media. In order to explore the mechanism underlying effects on breast cancer metastatic capability, the mRNA expression of leptin, adiponectin, insulin-like growth factor (IGF-1), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) was measured in adipocytes following either vehicle or 1,25(OH)2D treatment. Conditioned media from 1,25(OH)2D-treated adipocytes inhibited the migration of metastatic MDA-MB-231 breast cancer cells compared to conditioned media from vehicle-treated adipocytes. Treatment of adipocytes with 1,25(OH)2D decreased mRNA expression of leptin, adiponectin, IGF-1, IL-6, and MCP-1. Consistent with mRNA expression, concentrations of leptin, adiponectin, IGF-1, and IL-6 in adipocyte-conditioned media were decreased with 1,25(OH)2D treatment, although MCP-1 remained unchanged. In summary, these results suggest that 1,25(OH)2D alters adipocyte secretions to prevent breast cancer metastasis.

Assessment of Adipocyte Transduction Using Different AAV Capsid Variants.

Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry. Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated. AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5-2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 104 to 1.6 × 105 VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue. Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes.

Discovery of tetrahydro γ-carboline based novel glucose uptake agent for the treatment of diabetes and Alzheimer’s diseases

The emergence of diabetes mellitus (DM), one of the prevalent metabolic disorders, has increased at alarming rates around the globe in the past few decades. The early discovery of insulin paved the way for the broadened non-invasive anti-hyperglycemic drug therapies to manage and treat diabetes. Recently, insulin resistance in brain tissues has been considered a new hallmark of disease progression, leading to neurodegeneration. Tetrahydro γ-carbolines are privileged scaffolds with various activities against obesity, cancer, and central nervous system (CNS) diseases. The failure of Latrepirdine in clinical phase trials as anti-AD, prompted us to explore further the γ-Carboline derivatives with anti-diabetic activity. To address this, we have leveraged our expertise in drug design to develop tetrahydro γ-carboline derivatives and studied their effect on glucose uptake. All the compounds exhibited low cytotoxicity towards the mammalian cell line 3T3-L1. Next, we screened the 10 derivatives to identify the novel anti-diabetic activities using a fluorescent glucose analog 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)-2-deoxy-D-glucose) based on a glucose-uptake assay in differentiated 3T3-L1 adipocytes. Compound 11a has significantly increased glucose uptake at 100 µM compared to rH-Insulin. The dose optimization (1 nM-100 nM) of lead compounds for 2-NBDG-glucose uptake clearly indicated that 11a is superior compared to Insulin. The 11a is the close analog of Latrepirdine, an anti-histamine drug, reported for Alzheimer disease (AD). Further, to understand its anti-AD potential we carried out in silico docking studies with its probable targets, Acetylcholinesterase (AChE), 5-hydroxytryptamine (5HT6) receptor, Butyrylcholinesterase (BChE), and N-methyl-D-aspartate (NMDA) receptor. The docking studies revealed that 11a had stronger binding to these targets than Latrepirdine, correlating with the glucose uptake assay. Thus, 11a is a potential lead disease-modifying agent and could be considered for the management and treatment of type 2 DM and AD.

Vanillin, ferulic acid and their 1:1 combination inhibit lipid accumulation in 3T3 L1 adipocytes and 3D spheroids

BackgroundObesity is poised to be a major healthcare crisis worldwide. Genetic predisposition, inadequate activity, changing lifestyle and dietary patterns are cited as major causes for obesity. Even as a number of anti-obesity medications hit the market, there is still an ongoing quest to explore natural compounds, which are perceived as safer alternatives, for their anti-obesity activity. This study explores the anti-obesity potential of dietary polyphenols vanillin, ferulic acid and their combination using 3T3 L1 adipocytes and their 3D spheroids. MethodsStudies were conducted on differentiated 3T3 L1 adipocytes and their 3D spheroids. Assays conducted on 3T3 L1 adipocytes include Oil red O, fluorescent Nile Red staining and triglyceride quantification to assess effect on lipid droplet accumulation. 2 NBDG was used to assess glucose uptake following drug treatment. 3D spheroid cultures were generated and triglyceride content was quantified. Effect of drug treatment on gene expression was analysed using qRT-PCR. Results of monolayer culture were compared with 3D spheroid models. ResultsVanillin, ferulic acid and their combination lower intracellular triglyceride content and lipid droplet accumulation, inhibiting glucose uptake and conversion to triglycerides in 3T3 L1 adipocytes and their 3D spheroids. Compounds and their combination downregulated mRNA expression of C/EBP α and PPAR ɣ, FAS, ACC1, GLUT4, LPL, aP2. Vanillin treatment upregulated leptin mRNA expression. ConclusionVanillin, ferulic acid and their combination lower lipid accumulation and glucose uptake in 3T3 L1 adipocytes and 3D spheroids.

In Vitro and In Silico Analysis of PTP1B Inhibitors from Cleistocalyx operculatus Leaves and Their Effect on Glucose Uptake.

As part of our ongoing research on new anti-diabetic compounds from ethnopharmacologically consumed plants, two previously undescribed lupane-type triterpenoids (1 and 2) with dicarboxylic groups, an undescribed nor-taraxastane-type triterpenoid (3), and 14 known compounds (4-17) were isolated from the leaves of Cleistocalyx operculatus. Extensive spectroscopic analysis (IR, HRESIMS, 1D, and 2D NMR) was used for structure elucidation, while the known compounds were compared to reference data reported in the scientific literature. All the isolates (1-17) were evaluated for their inhibitory effects on the protein tyrosine phosphatase 1B (PTP1B) enzyme. Compounds 6, 9, and 17 showed strong PTP1B inhibitory activities. The mechanism of PTP1B inhibition was studied through enzyme kinetic experiments. A non-competitive mechanism of inhibition was determined using Lineweaver-Burk plots for compounds 6, 9, and 17. Additionally, Dixon plots were employed to determine the inhibition constant. Further insights were gained through a structure-activity relationship study and molecular docking analysis of isolated compounds with the PTP1B crystal structure. Moreover, all isolates (1-17) were tested for their stimulatory effects on the uptake of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) in differentiated 3T3-L1 adipocyte cells. Compounds 6, 13, and 17 exhibited strong glucose absorption stimulation activity in a dose-dependent manner.