3T3-L1 Cells Research Articles

Effectiveness and Safety of Boesenbergia rotunda Extract on 3T3-L1 Preadipocytes and Its Use in Capsaicin-Loaded Body-Firming Formulation: In Vitro Biological Study and In Vivo Human Study

Boesenbergia rotunda has been used as an antiobesity agent by suppressing adipogenesis. This study aimed to investigate the biological activity of B. rotunda on preadipocyte cells and to evaluate the effectiveness and safety of using B. rotunda extract in a capsaicin-loaded body-firming formulation. The antiadipogenesis of B. rotunda ethanolic extract was evaluated in 3T3-L1 preadipocyte cells. After the application of the B. rotunda extract-loaded body-firming formulation on the skin of volunteers for 28 d, thigh circumference, melanin index, and skin erythema were investigated. The results showed that the ethanolic extract of B. rotunda was not toxic toward 3T3-L1 cells at concentrations lower than 20 µg/mL, with antiadipogenesis of the B. rotunda extract occurring at a concentration of 1 µg/mL. The B. rotunda extract containing panduratin A was mixed with capsaicin body-firming products and successfully permeated into and through the skin. Applying this formulation to the thighs of the volunteers two times a day for 21 days led to a significant reduction in thigh circumference and melanin index. A slight elevation in skin erythema was observed, but there was no significant increase in redness or pain. In conclusion, the B. rotunda extract contained bioactive compounds that inhibited antiadipogenesis. The formulations containing B. rotunda extract and capsaicin showed potential as effective body-firming products.

Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake via Direct Inhibition of Mitochondrial Malate Dehydrogenase in Mouse 3T3-L1 Cells.

Differentiation-inducing factor 1 (DIF-1), found in Dictyostelium discoideum, has antiproliferative and glucose-uptake-promoting activities in mammalian cells. DIF-1 is a potential lead for the development of antitumor and/or antiobesity/antidiabetes drugs, but the mechanisms underlying its actions have not been fully elucidated. In this study, we searched for target molecules of DIF-1 that mediate the actions of DIF-1 in mammalian cells by identifying DIF-1-binding proteins in human cervical cancer HeLa cells and mouse 3T3-L1 fibroblast cells using affinity chromatography and liquid chromatography-tandem mass spectrometry and found mitochondrial malate dehydrogenase (MDH2) to be a DIF-1-binding protein in both cell lines. Since DIF-1 has been shown to directly inhibit MDH2 activity, we compared the effects of DIF-1 and the MDH2 inhibitor LW6 on the growth of HeLa and 3T3-L1 cells and on glucose uptake in confluent 3T3-L1 cells in vitro. In both HeLa and 3T3-L1 cells, DIF-1 at 10-40 μM dose-dependently suppressed growth, whereas LW6 at 20 μM, but not at 2-10 μM, significantly suppressed growth in these cells. In confluent 3T3-L1 cells, DIF-1 at 10-40 μM significantly promoted glucose uptake, with the strongest effect at 20 μM DIF-1, whereas LW6 at 2-20 μM significantly promoted glucose uptake, with the strongest effect at 10 μM LW6. Western blot analyses showed that LW6 (10 μM) and DIF-1 (20 μM) phosphorylated and, thus, activated AMP kinase in 3T3-L1 cells. Our results suggest that MDH2 inhibition can suppress cell growth and promote glucose uptake in the cells, but appears to promote glucose uptake more strongly than it suppresses cell growth. Thus, DIF-1 may promote glucose uptake, at least in part, via direct inhibition of MDH2 and a subsequent activation of AMP kinase in 3T3-L1 cells.

Examining the molecular mechanisms of topiramate in alleviating insulin resistance: A study on C2C12 myocytes and 3T3L-1 adipocytes.

Migraine, a severely debilitating condition, may be effectively managed with topiramate, known for its migraine prevention and weight loss properties due to changes in body muscle and fat composition and improved insulin sensitivity. However, the mechanism of topiramate in modulating insulin response in adipocytes and myocytes remains elusive. This study aims to elucidate these molecular mechanisms, offering insights into its role in weight management for migraine sufferers and underpinning its clinical application. Insulin resistance improvements were evaluated through glucose uptake measurements in C2C12 muscle cells and 3T3L-1 adipocytes, with Oil red O staining conducted on adipocytes. RNA-seq transcriptome analysis was used to identify the regulatory target genes of topiramate in these cells. The involvement of key genes and pathways was further validated through western blot analysis. Topiramate effectively reduced insulin resistance in C2C12 and 3T3L-1 cells. In C2C12 cells, it significantly lowered SORBS1 gene and protein levels. In 3T3L-1 cells, topiramate upregulated CTGF and downregulated MAPK8 and KPNA1 genes. Changes were notable in nuclear cytoplasmic transport and circadian signaling pathways. Furthermore, it caused downregulation of MKK7, pJNK1/ JNK1, BMAL1, and CLOCK proteins compared to the insulin-resistant model. This study provides preliminary insights into the mechanisms through which topiramate modulates insulin resistance in C2C12 myocytes and 3T3L-1 adipocytes, enhancing our understanding of its therapeutic potential in managing weight and insulin sensitivity in migraine patients.

Alpha-Linolenic Acid Ameliorates Cognitive Impairment and Liver Damage Caused by Obesity.

Obesity is a growing global problem that causes various complications such as diabetes, cognitive dysfunction, cardiovascular diseases, and hepatobiliary disease. Alpha-linolenic acid (ALA) has been reported to exhibit multiple pharmaceutical effects. This study aimed to explore the effects of ALA on obesity-induced adipose tissue accumulation, cognitive impairment, inflammation, and colonic mucosal barrier integrity. Mice were fed with high-fat diet (HFD) and were treated with ALA (60 or 100 mg/kg). Body weight, adipose tissue, serum glucose and lipid levels, glucose resistance, and insulin resistance were measured. Cognitive ability was analyzed using the behavior tests. PTP1B and IRS/p-AKT/p-GSK3β/p-Tau signaling were examined to evaluate inflammation and synaptogenesis. Colon mucosal barrier integrity was examined by Alcian blue staining and expression of the tight junction proteins. The production of pro-inflammatory cytokines and liver damages were evaluated. 3T3-L1 cells were used for in vitro experiments. Cell viability, migration and invasion were detected. The levels of ROS, iron, and ferrous ions were measured to assess ferroptosis. Metabolomic analysis of adipose tissues was performed. ALA treatment prevented HFD-induced adipose tissue accumulation, improved glucose and lipid homeostasis and metabolism. Administration of ALA repressed the HFD-induced increase in insulin levels and insulin resistance index. Serum and colon levels of pro-inflammatory cytokines were decreased after ALA treatment. ALA elevated mitochondrial content in brown adipose tissues. ALA ameliorated obesity-induced cognitive impairment and hippocampal inflammation, enhanced colon mucosa integrity. ALA treatment ameliorated HFD-induced liver damage and lipid accumulation and inhibited differentiation of preadipocyte 3T3-L1 cells into mature adipocytes and induces ferroptosis. Metabolomic analysis suggested that ALA may target the glycerolipid metabolism pathway to ameliorate obesity. Knockdown of AGPAT2 abolished the protective effects of ALA. ALA treatment suppressed adipose accumulation in adipocytes, improved cognitive ability and colon integrity, and alleviated liver damage by modulating the 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2).

Sex and age affect depot expression of Ca2+ channels in rat white fat adipocytes.

White adipose tissue (WAT) requires extracellular Ca2+ influx for lipolysis, differentiation, and expansion. This partly occurs via plasma membrane Ca2+ voltage-dependent channels (CaVs). However, WFA exists in different depots whose function varies with age, sex, and location. To explore whether their CaV expression profiles also differ we used RNAseq and qPCR on gonadal, mesenteric, retroperitoneal, and inguinal subcutaneous fat depots from rats of different ages and sex. CaV expression was found dependent on age, sex, and WFA location. In the gonadal depots of both sexes a significantly lower expression of CaV1.2 and CaV1.3 was seen for adults compared to pre-pubescent juveniles. A lower level of expression was also seen for CaV3.1 in adult male but not female gonadal WFA, the latter of whose expression remained unchanged with age. Relatively little expression of CaV3.2 and 3.2 was observed. In post-pubescent inguinal subcutaneous fat, where the third and fourth mammary glands are located, CaV3.1 was decreased in males but increased in females - thus suggesting that this channel is associated with mammogenesis; however, no difference in intracellular Ca2+ levels or adipocyte size were noted. For all adult depots, CaV3.1 expression was larger in females than males - a difference not seen in pre-pubescent rats. These observations are consistent with the changes of CaV3.1 expression seen in 3T3-L1 cell differentiation and the ability of selective CaV3.1 antagonists to inhibit adipogensis. Our results show that changes in CaV expression patterns occur in fat depots related to sexual dimorphism: reproductive tracts and mammogenesis.

Phaeodactylum tricornutum as a stable platform for pilot scale production and investigation of the viability of spirulina fucoxanthin as a commercial lipolysis active novel compound

The nutraceutical market has experienced a significant growth over the last years, due to changing consumer preferences towards products of natural and organic origin. One of the main areas of interest in this field is microalgae-derived carotenoids, whose antioxidant activity protects against oxidative damage in human cells. The diatom Phaeodactylum tricornutum has been widely studied due to its high fucoxanthin content (antioxidant carotenoid). However, pilot and industrial scale experiments to realize continuous and stable production of P. tricornutum and extracted fucoxanthin are limited. For this reason, this research focuses on the study of the fucoxanthin production, going through culture, harvesting, scaling procedure and encapsulation stage. Finally, the optimal culture conditions for a fucoxanthin production of 2.5 mg/day/L were verified. Furthermore, the antioxidant capacity showed a decrease during storage, but still presented a capacity of 18 months, with improved performance when encapsulated with spirulina. The study carried out on encapsulated fucoxanthin-spirulina shows an inhibitory effect on lipogenesis at all concentrations tested in the 3T3-L1 cell line and a lipolytic efficacy at concentrations of 0.2 mg/mL and 0.1 mg/mL in the 3T3-L1 cell line.

Analyzing the Antihyperglycemic Effect of Cissus quadrangularis and Bacopa monnieri on 3T3-L1 Cell Lines.

Background Cissus quadrangularisis a perennial shrub of the grape family. Other names for it include devil's backbone, veldgrape, and pirandai (Tamil).Bacopa monnieri, a perennial plant, is native to wetlands in eastern and southern India. The 3T3-L1 cell line, which was created from 3T3 cells, was used in the scientific study. The current study's purpose is to evaluate the antihyperglycemic benefits ofB. monnieriandC. quadrangularis, which will be added to the current arsenal of efficient herbal hypoglycemic medications. Aim To analyze and compare the anti-hyperglycaemic effects of the two plant extracts,C. quadrangularisandB. monnieriusing a 3T3-L1 cell line. Materials and methods C. quadrangularisseeds were gathered, and extraction was conducted. TheB. monnieriplant was harvested, and a rotary evaporator was used to extract the flower. Adipocyte cells were obtained from NCCS, Pune. A CO2 incubator was used to incubate the cells. The MTT assay and gene expression analysis were done on the cell line samples. Results The antihyperglycemic effects of C. quadrangularis IRS mRNA levels of 0.7 and AKT mRNA levels of 0.7 are compared to B. monnieri IRS1 mRNA levels of 0.6 and AKT mRNA levels of 0.6 to build better diabetic treatments. The antihyperglycemic benefits of C. quadrangularis levels of IRS mRNA and AKT mRNA are compared to the influence of B. monnieri IRS1 mRNA and AKT mRNA on the development of better diabetic drugs. Conclusion Comparing the effects ofC. quadrangularisandB. monnierion the 3T3 cell line by gene expression of IRS mRNA and AKT mRNA suggests that the particular AKT downregulation shows that insulin suppresses gluconeogenesis andC. quadrangularisinhibits hyperglycemia in 3T3-L1 cells, while research on in vitro rats suggests thatB. monnierimay minimize the signs and symptoms of diabetes via enhancing IRS1/AKT signaling.

Effect of β-Estradiol on Adipogenesis in a 3T3-L1 Cell Model of Prelamin A Accumulation.

The accumulation of farnesylated prelamin A has been suggested as one of the mechanisms responsible for the loss of fat in type 2 familial partial lipodystrophy due to variants in the LMNA gene. In this rare disease, fat loss appears in women after puberty, affecting sex-hormone-dependent anatomical areas. This study investigated the impact of 17-β-estradiol on adipogenesis in murine preadipocytes subjected to a pharmacologically induced accumulation of farnesylated and non-farnesylated prelamin A. To induce the accumulation of non-farnesylated or farnesylated prelamin A, 3T3-L1 cells were treated with the farnesyltransferase inhibitor 277 or the methyltransferase inhibitor N-acetyl-S-farnesyl-l-cysteine methylester. Subsequently, the cells were induced to undergo adipocyte differentiation in the presence or absence of 17-β-estradiol. Prelamin A accumulation was assessed through immunofluorescence, while real-time PCR and Western blot techniques were used to quantify several adipogenic genes and evaluate protein levels, respectively. The results showed that 17-β-estradiol increased adipogenesis, although the combination of this hormone plus farnesylated prelamin A led to a reduction in the number of mature adipocytes and the expression of the different genes involved in adipogenesis. In conclusion, the influence of farnesylated prelamin A accumulation on adipogenesis manifested only in the presence of estradiol. These in vitro findings suggest a potential mechanism that could explain the characteristic phenotype in women suffering type 2 familial partial lipodystrophy.

Celastrol Stabilizes Glycolipid Metabolism in Hepatic Steatosis by Binding and Regulating the Peroxisome Proliferator-Activated Receptor γ Signaling Pathway.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been increasing. Obesity, insulin resistance, and lipid metabolic dysfunction are always accompanied by NAFLD. Celastrol modulates the Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) signaling pathways, thereby promoting lipolysis in 3T3-L1 adipocytes. In the present study, oleic-acid-induced NAFLD and differentiated 3T3-L1 preadipocytes were used as models of NAFLD and obesity to investigate the protective effect of celastrol. We investigated the impact of celastrol on hepatic steatosis caused by oleic acid (OA), as well as the associated underlying molecular pathways. To address the aforementioned questions, we used a cellular approach to analyze the signaling effects of celastrol on various aspects. These factors include the improvement in fatty liver in HepG2 cells, the differentiation of 3T3-L1 preadipocytes, glucose uptake, and the modulation of key transcriptional pathways associated with PPARγ. The administration of celastrol effectively mitigated lipid accumulation caused by OA in HepG2 cells, thereby ameliorating fatty liver conditions. Furthermore, celastrol suppressed the impacts on adipocyte differentiation in 3T3-L1 adipocytes. Additionally, celastrol exhibited the ability to bind to PPARγ and modulate its transcriptional activity. Notably, the ameliorative effects of celastrol on hepatic steatosis were reversed by rosiglitazone. According to our preliminary findings from in vitro celastrol signaling studies, PPARγ is likely to be the direct target of celastrol in regulating hepatic steatosis in HepG2 cells and adipocyte differentiation in 3T3-L1 cells.

New thiazolyl-isoxazole derivatives as potential anti-infective agents: design, synthesis, in vitro and in silico antimicrobial efficacy

Antimicrobial resistance threatens the efficacious prevention and treatment of infectious diseases caused by microorganisms. To combat microbial infections, the need for new drug candidates is essential. In this context, the design, synthesis, antimicrobial screening, and in silico study of a new series of 5-aryl-3-(2-arylthiazol-4-yl)isoxazole (9a-t) have been reported. The structure of new compounds was confirmed by spectrometric methods. Compounds 9a-t were evaluated for in vitro antitubercular and antimicrobial activity. Against M. tuberculosis H37Rv, fourteen compounds showed good to excellent antitubercular activity with MIC 2.01–9.80 µM. Compounds 9a, 9b, and 9r showed four-fold more activity than the reference drug isoniazid. Nine compounds, 9a, 9b, 9d, 9e, 9i, 9q, 9r, 9s, and 9t, showed good antibacterial activity against E. coli with MIC 7.8–15.62 µg/mL. Against A. niger, four compounds showed good activity with MIC 31.25 µg/mL. Against C. albicans, all twenty compounds reported excellent to good activity with MIC 7.8–31.25 µg/mL. Compounds 9c–e, 9g–j, and 9q–t showed comparable activity concerning the reference drug fluconazole. The compounds 9a-t were screened for cytotoxicity against 3t3l1 cell lines and found to be less or non-cytotoxic. The in silico study exposed that these compounds displayed high affinity towards the M. tuberculosis targets PanK, DprE1, DHFR, PknA, KasA, and Pks13, and C. albicans targets NMT, CYP51, and CS. The compound 9r was evaluated for structural dynamics and molecular dynamics simulations. The potent antitubercular and antimicrobial activity of 5-aryl-3-(2-arylthiazol-4-yl)isoxazole (9a–t) derivatives has recommended that these compounds could assist in treating microbial infections. Communicated by Ramaswamy H. Sarma

Sophoridine Counteracts Obesity via Src-Mediated Inhibition of VEGFR Expression and PI3K/AKT Phosphorylation.

Sophoridine (SRP) is a natural quinolizidine alkaloid found in many traditional Chinese herbs, though its effect on adipose tissue is unclear. We improved serum lipid levels by administering SRP by gavage in high-fat diet (HFD)-fed C57BL/6 mice. After 11 weeks, SRP supplementation significantly reduced body weight gain and improved glucose homeostasis, while reducing subcutaneous fat and liver weight. SRP also inhibited cell proliferation and differentiation of 3T3-L1 cells. Proteomics analysis revealed that SRP inhibits adipocyte differentiation by interacting with Src, thereby suppressing vascular endothelial growth factor receptor 2 (VEGFR2) expression and PI3K/AKT phosphorylation. This study provides an empirical basis for the treatment of obesity with small molecules.

Autophagic Regulation of Adipogenesis Through TP53INP2: Insights from In Silico and In Vitro Analysis.

Obesity is an epidemic disease associated with multimorbidity resulting in higher mortality risk. The imbalance between energy storage and expenditure is the prime factor in the prognosis of the disease. Specifically, excessive lipid storage through adipogenesis leads to obesity. Adipogenesis is the process that converts preadipocytes into mature adipocytes by regulating major transcription factors like PPARγ and C/EBPα, contributes to lipid storage in adipose tissue. On the contrary, autophagy is a self-degradative process that maintains homeostasis in adipose tissue by regulating adipogenesis and lipolysis. TP53INP2 is a key player that regulates the autophagy process, and it negatively regulates adipogenesis and lipid storage. The gene expression profile GSE93637 was retrieved from the GEO database and analyzed using an integrated bioinformatics approach. The differentially expressed genes (DEGs) were analyzed using R-Bioconductor for TP53INP2 knockdown microarray dataset of 3T3L1 cells, and the DEGs were analyzed for the functional enrichment analysis. Further, the genes involved in the potential biological and molecular functions were evaluated for pathway enrichment analysis by KEGG (Kyoto Encyclopedia of Genes and Genomes). A total of 726 DEGs were found including 391 upregulated and 335 downregulated genes. Further, the functional and pathway enrichment analysis was employed to identify the highly interacting genes, and we identified a total of 56 genes that are highly interacting through a protein-protein interaction network. The DEGs mainly regulate the Peroxisome proliferator-activated receptor (PPAR) signaling pathway, lipolysis, and autophagy. Further, we investigated the associated Hub genes for enriched pathway genes and found the involvement of two autophagic genes ATG7 and sequestosome 1 (p62). In addition, in vitro studies of qRT-PCR (Quantitative real-time polymerase chain reaction) and Western blot analysis revealed that increased autophagy resulted in reduced lipid storage through down-regulation of the adipogenic gene. Moreover, increased expression of autophagic gene TP53INP2 and ATG7 facilitates the down-regulation of p62 and PPARγ gene resulting in lipolysis in mature adipocytes through autophagy. There is no specific treatment to reduce obesity other than a caloric diet and exercise. Hence, this study provides sufficient evidence to conclude that TP53INP2 negatively regulates adipogenesis and increases the degradation of lipids in mature adipocytes which is crucial for reducing obesity. Therefore, it is plausible to consider TP53INP2 as a promising therapeutic target for managing adipogenesis and obesity. However, further studies are necessary to validate their functional and molecular pathway analysis in the regulation of adipogenesis and obesity.

Flavonoid extracts of Citrus aurantium L. var. amara Engl. Promote browning of white adipose tissue in high-fat diet-induced mice

Ethnopharmacological relevanceObesity has become a public burden worldwide due to its booming incidence and various complications, and browning of white adipose tissue (WAT) is recognized as a hopeful strategy to combat it. Blossom of Citrus aurantium L. var. amara Engl. (CAVA) is a popular folk medicine and dietary supplement used for relieving dyspepsia, which is recorded in the Chinese Materia Medica. Our previous study showed that blossom of CAVA had anti-obesity potential, while its role in browning of WAT was still unclear. Aim of the studyThis study aimed to characterize the constituents in flavonoids from blossom of CAVA (CAVAF) and to clarify the anti-obesity capacities especially the effects on browning of WAT. Materials and methodsGradient ethanol eluents from blossom of CAVA were obtained by AB-8 macroporous resin. 3T3-L1 cells and pancreatic lipase inhibition assay were employed to investigate the potential anti-obesity effects in vitro. HPLC and UPLC/MS assays were performed to characterize the chemical profiles of different eluents. Network pharmacology and molecular docking assays were used to reveal potential anti-obesity targets. Furthermore, high-fat diet (HFD)-induced mice were constructed to explore the anti-obesity actions and mechanisms in vivo. Results30% ethanol eluents with high flavonoid content and great inhibition on proliferation of 3T3-L1 preadipocytes and pancreatic lipase activity were regarded as CAVAF. 19 compounds were identified in CAVAF. Network pharmacology analysis demonstrated that AMPK and PPARα were potential targets for CAVAF in alleviating obesity. Animal studies demonstrated that CAVAF intervention significantly decreased the body weight, WAT weight, serum TG, TC and LDL-C levels in HFD-fed obese mice. HFD-induced insulin resistance and morphological changes in WAT and brown adipose tissue were also markedly attenuated by CAVAF treatment. CAVAF supplementation potently inhibited iWAT inflammation by regulating IL-6, IL-1β, TNF-α and IL-10 mRNA expression in iWAT of mice. Furthermore, the gene expression levels of thermogenic markers including Cyto C, ATP synthesis, Cidea, Cox8b and especially UCP1 in iWAT of mice were significantly up-regulated by CAVAF administration. CAVAF intervention also markedly increased the expression levels of PRDM16, PGC-1α, SIRT1, AMPK-α1, PPARα and PPARγ mRNA in iWAT of mice. ConclusionCAVAF treatment significantly promoted browning of WAT in HFD-fed mice. These results suggested that flavonoid extracts from blossom of CAVA were probably promising candidates for the treatment of obesity.

GULP1 deficiency reduces adipogenesis and glucose uptake via downregulation of PPAR signaling and disturbing of insulin/ERK signaling in 3T3-L1 cells.

Obesity and metabolic disorders caused by alterations in lipid metabolism are major health issues in developed, affluent societies. Adipose tissue is the only organ that stores lipids and prevents lipotoxicity in other organs. Mature adipocytes can affect themselves and distant metabolism-related tissues by producing various adipokines, including adiponectin and leptin. The engulfment adaptor phosphotyrosine-binding domain-containing 1 (GULP1) regulates intracellular trafficking of glycosphingolipids and cholesterol, suggesting its close association with lipid metabolism. However, the role of GULP1 in adipocytes remains unknown. Therefore, this study aimed to investigate the function of GULP1 in adipogenesis, glucose uptake, and the insulin signaling pathway in adipocytes. A 3T3-L1 cell line with Gulp1 knockdown (shGulp1) and a 3T3-L1 control group (U6) were established. Changes in shGulp1 cells due to GULP1 deficiency were examined and compared to those in U6 cells using microarray analysis. Glucose uptake was monitored via insulin stimulation in shGulp1 and U6 cells using a 2-NBDG glucose uptake assay, and the insulin signaling pathway was investigated by western blot analysis. Adipogenesis was significantly delayed, lipid metabolism was altered, and several adipogenesis-related genes were downregulated in shGulp1 cells compared to those in U6 cells. Microarray analysis revealed significant inhibition of peroxisome proliferator-activated receptor signaling in shGulp1 cells compared with U6 cells. The production and secretion of adiponectin as well as the expression of adiponectin receptor were decreased in shGulp1 cells. In particular, compared with U6 cells, glucose uptake via insulin stimulation was significantly decreased in shGulp1 cells through the disturbance of ERK1/2 phosphorylation. This is the first study to identify the role of GULP1 in adipogenesis and insulin-stimulated glucose uptake by adipocytes, thereby providing new insights into the differentiation and functions of adipocytes and the metabolism of lipids and glucose, which can help better understand metabolic diseases.

Self-Assembled Oligopeptoplex-Loaded Dissolving Microneedles for Adipocyte-Targeted Anti-Obesity Gene Therapy.

Advancements in gene delivery systems are pivotal for gene-based therapeutics in oncological, inflammatory, and infectious diseases. This study delineates the design of a self-assembled oligopeptoplex (SA-OP) optimized for shRNA delivery to adipocytes, targeting obesity and associated metabolic syndromes. Conventional systems face challenges, including instability due to electrostatic interactions between genetic materials and cationic oligopeptides. Additionally, repeated injections induce discomfort and compromise patient well-being. To circumvent these issues, a dissolvable hyaluronic acid-based, self-locking microneedle (LMN) patch is developed, with improved micro-dose efficiency, for precise SA-OP delivery. This platform offers pain-free administration and improved SA-OP storage stability. In vitro studies in 3T3-L1 cells demonstrated improvements in SA-OP preservation and gene silencing efficacy. In vivo evaluation in a mice model of diet-induced type 2 diabetes yielded significant gene silencing in adipose tissue and a 21.92 ± 2.51% reduction in body weight with minimum relapse risk at 6-weeks post-treatment, representing a superior therapeutic efficacy in a truncated timeframe relative to the GLP-1 analogues currently available on the market. Additionally, SA-OP (LMN) mitigated insulin resistance, inflammation, and hepatic steatosis. These findings establish SA-OP (LMN) as a robust, minimally invasive transdermal gene delivery platform with prolonged storage stability for treating obesity and its metabolic comorbidities.

Comparing Methods for Induction of Insulin Resistance in Mouse 3T3-L1 Cells.

Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders. 1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR). In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel. We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected. In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.

Deletion of the mitochondrial calcium uniporter in adipose tissue promotes energy expenditure and alleviates diet-induced obesity

ObjectiveStudies have shown a correlation between obesity and mitochondrial calcium homeostasis, yet it is unclear whether and how Mcu regulates adipocyte lipid deposition. This study aims to provide new potential target for the treatment of obesity and related metabolic diseases, and to explore the function of Mcu in adipose tissue. MethodsWe firstly investigated the role of mitoxantrone, an Mcu inhibitor, in the regulation of glucose and lipid metabolism in mouse adipocytes (3T3-L1 cells). Secondly, C57BL/6J mice were used as a research model to investigate the effects of Mcu inhibitors on fat accumulation and glucose metabolism in mice on a high-fat diet (HFD), and by using CRISPR/Cas9 technology, adipose tissue-specific Mcu knockdown mice (Mcufl/+ AKO) and Mcu knockout of mice (Mcufl/fl AKO) were obtained, to further investigate the direct effects of Mcu on fat deposition, glucose tolerance and insulin sensitivity in mice on a high-fat diet. ResultsWe found the Mcu inhibitor reduced adipocytes lipid accumulation and adipose tissues mass in mice fed an HFD. Both Mcufl/+ AKO mice and Mcufl/fl AKO mice were resistant to HFD-induced obesity, compared to control mice. Mice with Mcufl/fl AKO showed improved glucose tolerance and insulin sensitivity as well as reduced hepatic lipid accumulation. Mechanistically, inhibition of Mcu promoted mitochondrial biogenesis and adipocyte browning, increase energy expenditure and alleviates diet-induced obesity. ConclusionsOur study demonstrates a link between adipocyte lipid accumulation and mCa2+ levels, suggesting that adipose-specific Mcu deficiency alleviates HFD-induced obesity and ameliorates metabolic disorders such as insulin resistance and hepatic steatosis. These effects may be achieved by increasing mitochondrial biosynthesis, promoting white fat browning and enhancing energy metabolism.

Anti-obesity effects by parasitic nematode (Trichinella spiralis) total lysates.

Obesity is an inducible factor for the cause of chronic diseases and is described by an increase in the size and number of adipocytes that differentiate from precursor cells (preadipocytes). Parasitic helminths are the strongest natural trigger of type 2 immune system, and several studies have showed that helminth infections are inversely correlated with metabolic syndromes. To investigate whether helminth-derived molecules have therapeutic effects on high-fat diet (HFD)-induced obesity, we isolated total lysates from Trichinella spiralis muscle larvae. We then checked the anti-obesity effect after intraperitoneal administration and intraoral administration of total lysate from T. spiralis muscle larvae in a diet-induced obesity model. T. spiralis total lysates protect against obesity by inhibiting the proinflammatory response and/or enhancing M2 macrophages. In addition, we determined the effects of total lysates from T. spiralis muscle larvae on anti-obesity activities in 3T3-L1 preadipocytes by investigating the expression levels of key adipogenic regulators, including peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα) and adipocyte protein 2 (aP2). Oil Red O staining showed that the total lysates from T. spiralis muscle larvae decreased the differentiation of 3T3-L1 preadipocytes by decreasing the number of lipid droplets. In addition, the production levels of proinflammatory cytokines IL-1β, IL-6, IFN-γ and TNF-α were examined by enzyme-linked immunosorbent assay (ELISA). T. spiralis total lysates decreased intracellular lipid accumulation and suppressed the expression levels of PPARγ, C/EBPα and aP2. These results show that T. spiralis total lysate significantly suppresses the symptoms of obesity in a diet- induced obesity model and 3T3-L1 cell differentiation and suggest that it has potential for novel anti-obesity therapeutics.

ATP7A-dependent copper sequestration contributes to termination of β-CATENIN signaling during early adipogenesis

ObjectivesAdipocyte fate determination is tightly regulated by extrinsic signaling pathways and intrinsic metabolic and morphologic changes that maintain adipose tissue function. Copper (Cu) homeostasis is required for the normal metabolism of mature adipocytes, whereas the role of Cu in adipogenesis is unclear. MethodsTo determine the role of Cu is adipocytes differentiation, we used 3T3-L1 adipocytes, immunocytochemistry, X-ray fluorescence, mass-spectrometry, pharmacological treatments, and manipulations of copper levels. ResultsIn differentiating 3T3-L1 cells, adipogenic stimuli trigger the upregulation and trafficking of the Cu transporter Atp7a, thus causing Cu redistribution from the cytosol to vesicles. Disrupting Cu homeostasis by the deletion of Atp7a results in Cu elevation and inhibition of adipogenesis. The upregulation of C/EBPβ, an initial step of adipogenesis, is not affected in Atp7a−/− cells, whereas the subsequent upregulation of PPARγ is inhibited. Comparison of changes in the Atp7a−/− and wild type cells proteomes during early adipogenesis revealed stabilization of β-catenin, a negative regulator of adipogenesis. Cu chelation, or overexpression of the Cu transporter ATP7B in Atp7a−/− cells, restored β-catenin down-regulation and intracellular targeting. ConclusionsCu buffering during early adipogenesis contributes to termination of β-catenin signaling. Abnormal upregulation of β-catenin was also observed in vivo in the livers of Atp7b−/− mice, which accumulate Cu, suggesting a tissue-independent crosstalk between Cu homeostasis and the Wnt/β-catenin pathway. These results point to a new regulatory role of Cu in adipocytes and contribute to better understanding of human disorders of Cu misbalance.

Shared Mechanisms in Pparγ1sv and Pparγ2 Expression in 3T3-L1 Cells: Studies on Epigenetic and Positive Feedback Regulation of Pparγ during Adipogenesis.

We have previously reported the identification of a novel splicing variant of the mouse peroxisome proliferator-activated receptor-γ (Pparγ), referred to as Pparγ1sv. This variant, encoding the PPARγ1 protein, is abundantly and ubiquitously expressed, playing a crucial role in adipogenesis. Pparγ1sv possesses a unique promoter and 5' untranslated region (5'UTR), distinct from those of the canonical mouse Pparγ1 and Pparγ2 mRNAs. We observed a significant increase in DNA methylation at two CpG sites within the proximal promoter region (-733 to -76) of Pparγ1sv during adipocyte differentiation. Concurrently, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac indicated marked elevations in both methylation and acetylation of histone H3, while the repressive histone mark H3K9me2 significantly decreased, at the transcription start sites of both Pparγ1sv and Pparγ2 following differentiation. Knocking down Pparγ1sv using specific siRNA also led to a decrease in Pparγ2 mRNA and PPARγ2 protein levels; conversely, knocking down Pparγ2 resulted in reduced Pparγ1sv mRNA and PPARγ1 protein levels, suggesting synergistic transcriptional regulation of Pparγ1sv and Pparγ2 during adipogenesis. Furthermore, our experiments utilizing the CRISPR-Cas9 system identified crucial PPARγ-binding sites within the Pparγ gene locus, underscoring their significance in adipogenesis. Based on these findings, we propose a model of positive feedback regulation for Pparγ1sv and Pparγ2 expression during the adipocyte differentiation process in 3T3-L1 cells.