Differentiated 3T3-L1 Adipocytes Research Articles

Impact of the Oral Administration of Polystyrene Microplastics on Hepatic Lipid, Glucose, and Amino Acid Metabolism in C57BL/6Korl and C57BL/6-Lepem1hwl/Korl Mice.

The impact of microplastics (MPs) on the metabolic functions of the liver is currently unclear and not completely understood. To investigate the effects of the administration of MPs on the hepatic metabolism of normal and obese mice, alterations in the lipid, glucose (Glu), and amino acid regulation pathways were analyzed in the liver and adipose tissues of C57BL/6Korl (wild type, WT) or C57BL/6-Lepem1hwl/Korl mice (leptin knockout, Lep KO) orally administered polystyrene (PS) MPs for 9 weeks. Significant alterations in the lipid accumulation, adipogenesis, lipogenesis, and lipolysis pathways were detected in the liver tissue of MP-treated WT and Lep KO mice compared to the vehicle-treated group. These alterations in their liver tissues were accompanied by an upregulation of the serum lipid profile, as well as alterations in the adipogenesis, lipogenesis, and lipolysis pathways in the adipose tissues of MP-treated WT and Lep KO mice. Specifically, the level of leptin was increased in the adipose tissues of MP-treated WT mice without any change in their food intake. Also, MP-induced disruptions in the glycogenolysis, Glu transporter type 4 (GLUT4)-5' AMP-activated protein kinase (AMPK) signaling pathway, levels of lipid intermediates, and the insulin resistance of the liver tissues of WT and Lep KO mice were observed. Furthermore, the levels of seven endogenous metabolites were remarkably changed in the serum of WT and Lep KO mice after MP administrations. Finally, the impact of the MP administration observed in both types of mice was further verified in differentiated 3T3-L1 adipocytes and HepG2 cells. Thus, these results suggest that the oral administration of MPs for 9 weeks may be associated with the disruption of lipid, Glu, and amino acid metabolism in the liver tissue of obese WT and Lep KO mice.

Investigating the Anti-adipogenic Potential of Xanthium strumarium Linn. Leaves Fractions: HPTLC-MS Characterization, Enzymatic Analysis, and 3T3-L1 Adipocyte Differentiation Assay in Oxidative Stress-modulated Adipogenesis Cascade

Background: Molecular pathogenesis of obesity is initiated by cellular oxidative stress along with increased adipogenesis. For the search of better alternatives of synthetic molecules in terms of their adverse events, naturally, existing compounds are now a research focus. In this context, Xanthium strumarium ( X. strumarium) Linn. exhibited anti-diabetic activities and proved to have efficacy in neutralizing very near pathological targets to obesity. Purpose: This study is aimed toward bioactivity-guided fraction isolation of methanolic leaves extract, where bioactivity refers to anti-adipogenesis and anti-oxidant efficacy in in vitro enzymatic and three-day transfer, inoculum 3×105 cells (3T3-L1) adipocyte differentiation assay. Materials and method: Collection and drying of the raw leaves followed by cold extraction in methanol and flash chromatography for bioactive fraction isolation. 3T3-L1 cell line was utilized for adipocyte differentiation assay of those and on the other hand in vitro pancreatic lipase, α-glucosidase, and α-amylase enzymatic assays were analyzed. Based on exhibited activities, selected fractions are further characterized by high-performance thin-layer chromatography-mass spectrometry (HPTLC-MS). Results: The most active fractions from these assays underwent characterization via HPTLC-MS. Fractions 2 and 3 exhibited potent activities in all enzymatic assays (α-glucosidase IC50: 2.48 ± 0.015, 2.84 ± 0.030 µg/mL; α-amylase IC50: 1.98 ± 0.050, 1.79 ± 0.045 µg/mL; pancreatic lipase IC50: 3.16 ± 0.030, 3.18 ± 0.040 µg/mL) and displayed significant inhibition of adipocyte differentiation in 3T3-L1 cell line. These fractions further identified through HPTLC-MS, contained compounds like β-sitosterol and quercetin, affirming their potential bioactivity. Conclusion: The study underscores the potential of X. strumarium Linn. fractions as promising natural alternatives for combating obesity-related pathways, highlighting their significance in developing future anti-obesity therapeutics.

Obesity-associated inflammatory macrophage polarization is inhibited by capsaicin and phytolignans.

Obesity is often accompanied by increased adipose tissue inflammation, a process that is partially driven by adipose tissue-resident macrophages. In this study, we explored the potential for plant-derived dietary compounds to exert anti-inflammatory effects in macrophages that alleviate obesity-associated adipocyte dysfunction. Capsaicin (CAP), schisandrin A (SA), enterodiol (END), and enterolactone (ENL) treatment polarized J774 macrophages to an "M2" or anti-inflammatory phenotype and inhibited responses to stimulation with lipopolysaccharide (LPS). Furthermore, these compounds blocked inflammasome activation when administered just before ATP-induced NLRP3 activation, as evidenced by the abrogation of IL-1β release in mouse macrophages and human peripheral blood monocytes. The addition of CAP, SA, or ENL during the differentiation of bone marrow-derived macrophages was also sufficient to inhibit LPS-induced IL-6 and TNFα production. Finally, CAP, END, and ENL treatment during differentiation of 3T3-L1 adipocytes induced an adiponectin-high phenotype accompanied by increases in thermogenic gene expression, and conditioned media from these adipocytes inhibited LPS-induced production of IL-1β, IL-6, and TNFα from J774 macrophages. These polarizing effects were partially mediated by the elevated adiponectin and decreased syndecan-4 in the adipocyte-conditioned media. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.NEW & NOTEWORTHY The utility of food-based products to prevent or alleviate chronic conditions such as obesity and its associated comorbidities is an attractive approach. Capsaicin, schisandrin A, enterodiol, and enterolactone, phytochemicals present in traditional medicinal food, decreased proinflammatory cytokine production from macrophages that, in turn, reduced obesity-associated adipocyte dysfunction. These results implicate the contribution of plant-derived dietary components to the modulation of macrophages and adipocytes in obesity.

Narirutin-Rich Celluclast Extract from Mandarin (Citrus unshiu) Peel Alleviates High-Fat Diet-Induced Obesity and Promotes Energy Metabolism in C57BL/6 Mice.

Mandarin peel, a main by-product from the processing of citrus juice, has been highlighted for its various bioactivities and functional ingredients. Our previous study proved the inhibitory effects of Celluclast extract from mandarin peel (MPCE) on lipid accumulation and differentiation in 3T3-L1 adipocytes. Therefore, the current study aimed to evaluate the anti-obesity effect of MPCE in high-fat diet (HFD)-induced obese mice. The high-performance liquid chromatography (HPLC) analysis exhibited that narirutin and hesperidin are the main active components of MPCE. Our current results showed that MPCE supplementation decreased adiposity by reducing body and organ weights in HFD-induced obese mice. MPCE also reduced triglyceride (TG), alanine transaminase (ALT), aspartate transaminase (AST), and leptin contents in the serum of HFD-fed mice. Moreover, MPCE significantly inhibited hepatic lipid accumulation by regulating the expression levels of proteins associated with lipid metabolism, including sterol regulatory element-binding protein (SREBP1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Furthermore, MPCE administration significantly inhibited both adipogenesis and lipogenesis, with modulation of energy metabolism by activating 5' adenosine monophosphate-activated protein kinase (AMPK) and lipolytic enzymes such as hormone-sensitive lipase (HSL) in the white adipose tissue (WAT). Altogether, our findings indicate that MPCE improves HFD-induced obesity and can be used as a curative agent in pharmaceuticals and nutraceuticals to alleviate obesity and related disorders.

Ursane-type Triterpene Glycosides from Rubus cochinchinensis Exhibited Insulin-Mimetic Activities in Differentiated 3T3-L1 Adipocytes

Ursane-type Triterpene Glycosides from Rubus cochinchinensis Exhibited Insulin-Mimetic Activities in Differentiated 3T3-L1 Adipocytes

Citrus sunki Peel Extract, Containing Nobiletin and Tangeretin, Enhances Proliferation and Differentiation in 3T3-L1 Adipocytes

In this experiment, effects of Citrus sunki peel extract (CPE) on proliferation and differentiation of 3T3-L1 cells were analyzed. Citrus sunki peel was extracted with ethanol to obtain CPE. Results of measuring DPPH and hydrogen peroxide radical scavenging activity revealed that CPE had an antioxidant ability. The 3T3-L1 cells were cultured in the basal medium (C) or with 0.05% dimethyl-sulfoxide (CDMSO) or with 50, 100, 200, 300, and 400 μg/mL CPE (CPE50, CPE100, CPE200, CPE300, and CPE400, respectively). As a result of cell counting and MTS assay, CPE significantly enhanced proliferation capacity of 3T3-L1 cells. To analyze the effect of CPE on the differentiation capacity of 3T3-L1 cells, relative gene and protein expression levels of Cebpb, Cebpa, and FASN related to adipogenesis were measured by RT-qPCR and Western blot. CPE increased adipogenesis-related gene and protein expression in 3T3-L1 cells, with CPE300 being especially effective. In Oil Red O staining, adipogenesis was significantly greater in CPE200, CPE300, and CPE400 than in C. This was confirmed image wise through Nile Red staining. Through HPLC analysis, it was confirmed that nobiletin and tangeretin were most abundant in CPE. To analyze effects of nobiletin and tangeretin on 3T3-L1 cells, the cells were cultured with various concentrations of nobiletin, tangeretin, and both. As a result of cell counting and MTS assay, nobiletin and tangeretin significantly decreased the proliferation capacity of 3T3-L1 cells. In RT-qPCR, western blot, Oil Red O staining, and Nile Red staining to analyze differentiation capacity, nobiletin and tangeretin significantly enhanced the expression of genes and proteins related to adipogenic differentiation compared to the control group. In conclusion, CPE, which had high antioxidant capacity, enhanced proliferation and differentiation in 3T3-L1 adipocytes. This improvement in differentiation is due to the influence of nobiletin and tangeretin contained in CPE.

The Effects of Body Fat Reduction through the Metabolic Control of Steam-Processed Ginger Extract in High-Fat-Diet-Fed Mice.

The prevalence of metabolic syndrome is increasing globally due to behavioral and environmental changes. There are many therapeutic agents available for the treatment of chronic metabolic diseases, such as obesity and diabetes, but the data on their efficacy and safety are lacking. Through a pilot study by our group, Zingiber officinale rhizomes used as a spice and functional food were selected as an anti-obesity candidate. In this study, steam-processed ginger extract (GGE) was used and we compared its efficacy at alleviating metabolic syndrome-related symptoms with that of conventional ginger extract (GE). Compared with GE, GGE (25-100 μg/mL) had an increased antioxidant capacity and α-glucosidase inhibitory activity in vitro. GGE was better at suppressing the differentiation of 3T3-L1 adipocytes and lipid accumulation in HepG2 cells and promoting glucose utilization in C2C12 cells than GE. In 16-week high-fat-diet (HFD)-fed mice, GGE (100 and 200 mg/kg) improved biochemical profiles, including lipid status and liver function, to a greater extent than GE (200 mg/kg). The supplementation of HFD-fed mice with GGE (200 mg/kg) resulted in the downregulation of SREBP-1c and FAS gene expression in the liver. Collectively, our results indicate that GGE is a promising therapeutic for the treatment of obesity and metabolic syndrome.

Sipyimigwanjung-tang, a traditional herbal medication, alleviates weight gain in a high-fat diet-induced obese mice model

Obesity leads to the development of metabolic syndrome and comorbidities. Overweight and obesity continue to be a relentless global issue. Sipyimigwanjung-tang (SGT), a traditional herbal medication, was first mentioned in Dongui Sasang Shinpyun and has been used to treat edema, meteorism, and jaundice, which are common findings associated with obesity. The main physiological feature of obesity is expanded adipose tissue, which causes several impairments in liver metabolism. Therefore, this study aimed to investigate the anti-obesity effects of SGT in the epididymal white adipose tissue (eWAT) and livers of high-fat diet (HFD)-induced obese mice. SGT significantly blocked HFD-induced weight gain in C57BL/6N mice. In addition, SGT effectively reduced the increased weight and adipocyte size in eWAT of HFD-induced obese C57BL/6 N mice. Moreover, SGT significantly decreased the elevated gene expression of Peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, and Sterol regulatory element-binding protein 1 in the eWAT of HFD-induced obese mice. Furthermore, SGT significantly decreased lipid accumulation in the livers of HFD-induced obese mice and differentiated 3T3-L1 adipocytes. Hence, the present study provides substantial evidence that SGT has potential therapeutic effects on obesity.

Methyl Cinnamate (MC) Alleviates Free Fatty Acids (FFAs) Induced Lipid Accumulation Through the AMPK Pathway in HepG2 Cells.

AMP-activated protein kinase (AMPK) plays a critical role in energy metabolism. Its activation leads to the phosphorylation of downstream proteins such as acetyl-CoA carboxylase (ACC) and sterol regulatory element-binding protein-1 (SREBP1), subsequently inhibiting de novo fatty acid synthesis, thereby reducing intracellular triglyceride accumulation. MC is a compound found in extracts from Zanthoxylum armatum DC plants. Research has shown that MC can inhibit the differentiation of 3T3-L1 adipocytes through the CAMKK2-AMPK pathway. However, the biological effect of MC in HepG2 cells remains unknown. In this study, we utilized HepG2 cells to establish a model of MAFLD through FFAs stimulation. We investigated the biological effects of MC on HepG2 cells and studied its impact on lipid metabolism. Small interfering RNA was employed to explore the mechanism by which MC activates AMPK. Finally, molecular docking was conducted, establishing a model of the interaction between AMPK and MC. We observed that MC can alleviate triglyceride accumulation in HepG2 cells. We observed the elevated p-AMPK/AMPK, P-ACC/ ACC, and elevated CPT1a after treatment of MC in HepG2 cells. The interference of CAMKK2 mRNA did not impact the ability of MC to phosphorylate AMPK. Compound C attenuates the ability of MC to increase p-AMPK. Molecular docking results led us to hypothesize that MC directly interacts with AMPK, resulting in AMPK phosphorylation and improved lipid accumulation in HepG2 cells.

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.

Inhibitory Effects of Sesquiterpenoids Isolated from Artemisia scoparia on Adipogenic Differentiation of 3T3-L1 Preadipocytes.

Obesity and related complications are significant health issues in modern society, largely attributed to a sedentary lifestyle and a carbohydrate-rich diet. Since anti-obesity drugs often come with severe side effects, preventative measures are being sought globally, including dietary changes and functional foods that can counteract weight gain. In this context, plant-based metabolites are extensively studied for their advantageous biological effects against obesity. Several plants within the Artemisia genus have been reported to possess anti-adipogenic properties, preventing adipocytes from maturing and accumulating lipids. The present study investigated the anti-adipogenic potential of two sesquiterpenoids, reynosin and santamarine, isolated from A. scoparia in adipose-induced 3T3-L1 preadipocytes. Differentiating 3T3-L1 adipocytes treated with these isolated compounds displayed fewer adipogenic characteristics compared to untreated mature adipocytes. The results indicated that cells treated with reynosin and santamarine accumulated 55.0% and 52.5% fewer intracellular lipids compared to untreated control adipocytes, respectively. Additionally, the mRNA expression of the key adipogenic marker, transcription factor PPARγ, was suppressed by 87.2% and 91.7% following 60 μM reynosin and santamarine treatment, respectively, in differentiated adipocytes. Protein expression was also suppressed in a similar manner, at 92.7% and 82.5% by 60 μM reynosin and santamarine treatment, respectively. Likewise, SERBP1c and C/EBPα were also downregulated at both gene and protein levels in adipocytes treated with samples during differentiation. Further analysis suggested that the anti-adipogenic effect of the compounds might be a result of AMPK activation and the subsequent suppression of MAPK phosphorylation. Overall, the present study suggested that sesquiterpenoids, reynosin, and santamarine were two potential bioactive compounds with anti-adipogenic properties. Further research is needed to explore other bioactive agents within A. scoparia and elucidate the in vivo action mechanisms of reynosin and santamarine.

The Hexane Extract of Citrus sphaerocarpa Ameliorates Visceral Adiposity by Regulating the PI3K/AKT/FoxO1 and AMPK/ACC Signaling Pathways in High-Fat-Diet-Induced Obese Mice.

Obesity is an emerging global health issue with an increasing risk of disease linked to lifestyle choices. Previously, we reported that the hexane extract of Citrus sphaerocarpa (CSHE) suppressed lipid accumulation in differentiated 3T3-L1 adipocytes. In this study, we conducted in vivo experiments to assess whether CSHE suppressed obesity in zebrafish and mouse models. We administered 10 and 20 μg/mL CSHE to obese zebrafish juveniles. CSHE significantly inhibited visceral fat accumulation compared to untreated obese fish. Moreover, the oral administration (100 μg/g body weight/day) of CSHE to high-fat-diet-induced obese mice significantly reduced their body weight, visceral fat volume, and hepatic lipid accumulation. The expression analyses of key regulatory genes involved in lipid metabolism revealed that CSHE upregulated the mRNA expression of lipolysis-related genes in the mouse liver (Pparα and Acox1) and downregulated lipogenesis-related gene (Fasn) expression in epididymal white adipose tissue (eWAT). Fluorescence immunostaining demonstrated the CSHE-mediated enhanced phosphorylation of AKT, AMPK, ACC, and FoxO1, which are crucial factors regulating adipogenesis. CSHE-treated differentiated 3T3L1 adipocytes also exhibited an increased phosphorylation of ACC. Therefore, we propose that CSHE suppresses adipogenesis and enhances lipolysis by regulating the PI3K/AKT/FoxO1 and AMPK/ACC signaling pathways. These findings suggested that CSHE is a promising novel preventive and therapeutic agent for managing obesity.

Heme oxygenase-1 mediates the inhibitory effect of ginseng (Panax ginseng) leaf extract on differentiation in 3T3-L1 adipocytes

Heme oxygenase-1 mediates the inhibitory effect of ginseng (Panax ginseng) leaf extract on differentiation in 3T3-L1 adipocytes

Anti‑adipogenic effect and underlying mechanism of lignan‑enriched nutmeg extract on 3T3‑L1 preadipocytes.

Nutmeg is the seed derived from Myristica fragrans. Nutmeg seeds contain alkylbenzene derivatives such as myristicin, which are toxic to the human organism, and lignan compounds such as nectandrin B, which possess anti-aging and anti-diabetic properties. However, the anti-adipogenic, prolipolytic and anti-inflammatory effects of lignan-enriched nutmeg extract (LNX) on preadipocytes remain unclear. In the present study, the effects of LNX on lipid accumulation, glycerol release and inflammatory cyclooxygenase-2 (COX-2) expression in differentiated 3T3-L1 preadipocytes were investigated. Oil red O staining demonstrated that treatment with LNX resulted in a concentration-dependent reduction in lipid accumulation in differentiating 3T3-L1 preadipocytes without affecting cell growth. Mechanistically, LNX treatment at 6 µg/ml led to a reduction in phosphorylation levels of signal transducer and activator of transcription 3 (STAT3), whereas it did not influence the peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT enhancer binding protein alpha (C/EBP-α) expression levels during 3T3-L1 preadipocyte differentiation. In addition, LNX treatment at 6 µg/ml led to a decrease in fatty acid synthase (FAS) expression levels on day (D) 2, but not D5 and D8, during preadipocyte differentiation. Treatment with LNX at 6 µg/ml did not affect the expression levels of perilipin A during preadipocyte differentiation. In differentiated 3T3-L1 adipocytes, LNX treatment at 6 µg/ml did not stimulate glycerol release and hormone-sensitive lipase phosphorylation, which are known lipolysis hallmarks. Furthermore, LNX treatment at the doses tested had no effect on tumor necrosis factor alpha-induced COX-2 expression in 3T3-L1 preadipocytes. Collectively, these results demonstrated that LNX has an anti-adipogenic effect on differentiating 3T3-L1 preadipocytes, which is mediated by the downregulation of STAT3 phosphorylation and FAS expression.

Roux-en-Y Gastric Bypass Improves Insulin Sensitivity in Obese Rats with Type 2 Diabetes Mellitus by Regulating the Grin3a/AMPK Signal Axis in Hypothalamic Arcuate Nucleus.

The objective of this study was to explore the effects and related mechanisms of Roux-en-Y gastric bypass (RYGB) on insulin sensitivity in obese rats with type 2 diabetes mellitus (T2DM). The obese T2DM rat model was constructed by feeding a high-fat diet and injecting streptozotocin (STZ), and treated with RYGB. Grin3a shRNA was injected into the bilateral hypothalamic arcuate nucleus (ARC) to knockdown the Grin3a expression on T2DM rats. Eight weeks after operation, the body weight, fasting blood glucose (FBG), fasting serum insulin (FSI), homeostatic model assessment of insulin resistance (HOMA-IR), and plasma triglyceride (TG) levels were assessed. Hematoxylin & eosin (H&E) staining was adopted to observe the white adipose tissue (WAT) of rats. Western blot and qRT-PCR were used to detect the expression of Grin3a, adenosine 5' monophosphate-activated protein kinase (AMPK) and p-AMPK in ARC of rats. Later, the plasmid over-expressing or knocking down Grin3a was transfected into differentiated 3T3-L1 adipocytes, and the TG level and the formation of lipid droplets in adipocyte were assessed by TG kit and oil red O staining. The expression of lipogenic transcription factors in cells was detected by qRT-PCR. RYGB reduced FBG, FSI, HOMA-IR and plasma TG levels in T2DM rats while increasing Grin3a expression and p-AMPK/AMPK ratio in ARC. Knockdown of Grin3a not only reversed the decrease of FBG, FSI, HOMA-IR and plasma TG levels in T2DM rats induced by RYGB, but also reversed the up-regulation of p-AMPK/AMPK ratio in ARC affected by RYGB. Moreover, knocking down Grin3a significantly increased the TG level, promoted the formation of lipid droplets and up-regulated the expressions of lipogenic transcription factors in adipocytes. RYGB improved the insulin sensitivity, reduced the plasma TG level and lessens the fat accumulation in obese T2DM rats by regulating the Grin3a/AMPK signal in ARC.

The Optimal Dose of the Green Tea and Coffee Extracts to Suppress the Expression of PPAR-γ and C/EBP-α on Differentiated 3T3-L1 Adipocytes

Obesity is the excess fat content in the body caused by the expansion of white adipose tissue. This condition begins with the differentiation of adipose tissue that is known to be controlled by 2 main transcription factors, such as Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and CCAAT Enhancer-Binding Protein-α (C/EBP-α). Their activation and collaboration are critical for developing functioning adipocytes and preserving metabolic balance in adipose tissue. Research on the benefits of natural bioactive components that regulate adipogenesis has recently become an exciting focus. Foodstuffs reported to affect this condition positively include green coffee and green tea. The primary substance in green coffee is chlorogenic acid (CGA), meanwhile in green tea is epigallocatechin gallate (EGCG). Based on medical benefit potential, the present study evaluated the dose that produced the best effect on PPAR-γ and C/EBP-α expression between single and combined doses compared to undifferentiated adipocytes (3T3-L1). 3T3-L1 were cultured and divided into negative (NEG) and positive (DIF) groups. The DIF group is obtained by induction of differentiation, then divided into seventeen therapeutic doses. At the end of therapy, cells were fixed to measure the expression of PPAR-γ and C/EBP-α using the immunocytochemistry method. The DIF group produced the highest expression of PPAR-γ and C/EBP-α. Among the single-dose group, the lowest PPAR-γ was found in C320 and C/EBP-α in the T320 group. Meanwhile, the dose with the lowest PPAR-γ and C/EBP-α expression was found in the combination of green tea and coffee (TC) 160/80 (p-value = 0.00). The findings of this study showed that a combination of green tea and coffee extracts at doses of 160/80 has the potential for anti-obesity by suppressing the differentiation of 3T3-L1 into adipocytes by reducing the expression of PPAR-γ and C/EBP-α. HIGHLIGHTS Obesity is the excess fat content in the body caused by the expansion of white adipose tissue. This condition begins with the differentiation of adipose tissue that is known to be controlled by Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and CCAAT Enhancer-Binding Protein-α (C/EBP-α) Natural bioactive components that regulate adipogenesis in green coffee are chlorogenic acid (CGA) and epigallocatechin gallate found in green tea (EGCG). Based on this potential, the present study evaluated the dose that produced the best effect on PPARɣ and C/EBP-α protein expression between single and combined doses compared to undifferentiated adipocytes (3T3-L1) Based on this study, the differentiated group of adipocytes (DIF) produced the highest expression of PPAR-γ and C/EBP-α. Among the single dose group, which showed the lowest average value of PPARɣ was C320, and for C/EBP-α was the T320 group. Meanwhile, the dose with the lowest PPAR-γ and C/EBP-α expression came from the combination of green tea and coffee (TC) 160/80 (p-value = 0.00) So, this research shows that green tea and green coffee extract significantly affected the expression of PPAR-γ and C/EBP-α. Green tea and coffee extracts at doses of TC 160/80 have the potential for anti-obesity by suppressing the differentiation of 3T3-L1 into adipocytes by reducing the expression of PPAR-γ and C/EBP-α GRAPHICAL ABSTRACT

Rabbit Meat Extract Induces Browning in 3T3-L1 Adipocytes via the AMP-Activated Protein Kinase Pathway.

The browning of white adipocytes may be an innovative approach to address obesity. This study investigated the effects of rabbit meat extract on 3T3-L1 adipocytes, with a specific emphasis on inducing browning. The browning effects of rabbit meat extract were evaluated by analyzing genes specifically expressed in 3T3-L1 adipocytes using quantitative PCR and immunoblotting. Rabbit meat extract increased the expression of brown adipocyte-specific markers, UCP1 and PGC1α, and mitochondrial biogenesis factors, TFAM and NRF1, without affecting cell viability in fully differentiated 3T3-L1 adipocytes. Moreover, adipocyte differentiation and the triglyceride content were decreased; hormone-sensitive lipase activity was promoted. Rabbit meat extract activated the AMPK pathway in the differentiated 3T3-L1 cells. However, in adipocytes treated with rabbit meat extract, the expression of genes related to browning was reduced by the AMP-activated protein kinase (AMPK) inhibitor, dorsomorphin dihydrochloride. To the best of our knowledge, this is the first study to demonstrate that rabbit meat extract induces the browning of white adipocytes via the activation of the AMPK pathway, thereby demonstrating its therapeutic potential in preventing obesity.

Cyclodextrin-Mediated Cholesterol Depletion Induces Adiponectin Secretion in 3T3-L1 Adipocytes.

Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.

SUMO-specific protease 2 regulates lipid droplet size through ERRα-mediated CIDEA expression in adipocytes

Lipid droplets are not only lipid storage sites but also are closely related to lipid metabolism. Lipid droplet growth increases lipid storage capacity and suppresses lipolysis via lipase associated with the lipid droplet surface. The cell death-inducing DFF45-like effector (CIDE) family of proteins mediates lipid droplet fusion, which mainly contributes to lipid droplet growth. We previously demonstrated small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) plays important roles in lipid metabolism and induction/maintenance of adipogenesis. In this study, we determined whether SENP2 regulates lipid droplet size in adipocytes. Overexpression of SENP2 increased lipid droplet size in differentiated 3T3-L1 adipocytes and facilitated CIDEA transcription. We found SENP2 increased CIDEA expression mainly through desumoylation of estrogen-related receptor α (ERRα), which acted in coordination with peroxisome proliferator-activated receptor γ-coactivator α. In addition, palmitate treatment increased SENP2 and CIDEA mRNA levels. Specific small interfering RNA-mediated knockdown of SENP2, as well as ERRα knockdown, eliminated palmitate-induced CIDEA expression. These results suggest SENP2 enhances CIDEA expression by modulating ERRα when SENP2 is upregulated, such as after palmitate treatment, to increase lipid droplet size in adipocytes.

The Circadian Nobiletin-ROR Axis Suppresses Adipogenic Differentiation and IκBα/NF-κB Signaling in Adipocytes.

Obesity is a known risk factor for metabolic diseases and is often associated with chronic inflammation in adipose tissue. We previously identified the polyethoxylated flavonoid Nobiletin (NOB) as a circadian clock modulator that directly binds to and activates the ROR receptors in the core oscillator, markedly improving metabolic fitness in obese mice. Here, we show that NOB enhanced the oscillation of core clock genes in differentiated 3T3-L1 adipocytes, including ROR target genes such as Bmal1, Cry1, Dec1, and Dec2. NOB inhibited lipid accumulation in 3T3-L1 and SVF cells, concomitant with the dysregulated circadian expression of adipogenic differentiation-related genes including Cebpb, Pparg, Lpl, Scd1, and Fas. Importantly, RORα/RORγ double knockdown in 3T3-L1 cells (Ror DKD) significantly attenuated the effects of NOB on circadian gene expression and lipid accumulation. Furthermore, whereas NOB upregulated the expression of IκBα, a target of RORs, to inhibit NF-κB activation and proinflammatory cytokine expression, Ror DKD cells exhibited a heightened activation of the NF-κB pathway, further indicating a requisite role of RORs for NOB efficacy in adipocytes. Together, these results highlight a significant regulatory function of the NOB-ROR axis in the circadian expression of clock and clock-controlled genes in adipocytes, thereby governing adipogenic differentiation, lipogenesis, and inflammation.