Strongylocentrotus intermedius Extract Suppresses Adiposity by Inhibiting Adipogenesis and Promoting Adipocyte Browning via AMPK Activation in 3T3-L1 Cells.

The current study aimed to determine whether Strongylocentrotus intermedius (S. intermedius) extract (SIE) exerts anti-obesity potentials employing 3T3-L1 cells as in vitro model. Herein we reported that treatment of SIE for 6 days reduced lipid accretion and triglyceride content whereas it increased the release of free glycerol. The inhibited lipid accumulation and induced lipolysis were evidenced by the downregulation of lipogenesis proteins, such as fatty acid synthase and lipoprotein lipase, and the upregulation of hormone-sensitive lipase expression. Furthermore, the downregulation of adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein α, and sterol regulatory element-binding protein 1, highlights that reduced lipid accumulation is supported by lowering adipocyte differentiation. Additionally, treatment activates brown adipocyte phenotype in 3T3-L1 cells by inducing expression of brown adipose tissue-specific proteins, such as uncoupling protein 1 and peroxisome proliferator-activated receptor-γ coactivator 1α. Moreover, SIE induced the phosphorylation of AMP-activated protein kinase (AMPK). The pharmacological approach using AMPK inhibitor revealed that the restraining effect of SIE on adipogenesis and promotion of adipocyte browning were blocked. In GC-MS analysis, SIE was mainly composed of cholest-5-en-3-ol (36.71%) along with saturated and unsaturated fatty acids which have favorable anti-obesity potentials. These results reveal that SIE has the possibility as a lipid-lowering agent for the intervention of obesity.

Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes.

Brown fat is a therapeutic target for the treatment of obesity-associated metabolic diseases. However, nutritional intervention strategies for increasing the mass and activity of human brown adipocytes have not yet been established. To identify vitamins required for brown adipogenesis and adipocyte browning, chemical compound-induced brown adipocytes (ciBAs) were converted from human dermal fibroblasts under serum-free and vitamin-free conditions. Choline was found to be essential for adipogenesis. Additional treatment with pantothenic acid (PA) provided choline-induced immature adipocytes with browning properties and metabolic maturation, including uncoupling protein 1 (UCP1) expression, lipolysis, and mitochondrial respiration. However, treatment with high PA concentrations attenuated these effects along with decreased glycolysis. Transcriptome analysis showed that a low PA concentration activated metabolic genes, including the futile creatine cycle-related thermogenic genes, which was reversed by a high PA concentration. Riboflavin treatment suppressed thermogenic gene expression and increased lipolysis, implying a metabolic pathway different from that of PA. Thiamine treatment slightly activated thermogenic genes along with decreased glycolysis. In summary, our results suggest that specific B vitamins and choline are uniquely involved in the regulation of adipocyte browning via cellular energy metabolism in a concentration-dependent manner.

Brown adipose tissue-derived FGF21 mediates the cardioprotection of dexmedetomidine in myocardial ischemia/reperfusion injury.

Brown adipose tissue (BAT) plays a critical role in regulating cardiovascular homeostasis through the secretion of adipokines, such as fibroblast growth factor 21 (FGF21). Dexmedetomidine (DEX) is a selective α2-adrenergic receptor agonist with a protection against myocardial ischemia/reperfusion injury (MI/RI). It remains largely unknown whether or not BAT-derived FGF21 is involved in DEX-induced cardioprotection in the context of MI/RI. Herein, we demonstrated that DEX alleviated MI/RI and improved heart function through promoting the release of FGF21 from interscapular BAT (iBAT). Surgical iBAT depletion or supplementation with a FGF21 neutralizing antibody attenuated the beneficial effects of DEX. AMPK/PGC1α signaling-induced fibroblast growth factor 21 (FGF21) release in brown adipocytes is required for DEX-mediated cardioprotection since blockade of the AMPK/PGC1α axis weakened the salutary effects of DEX. Co-culture experiments showed that DEX-induced FGF21 from brown adipocytes increased the resistance of cardiomyocytes to hypoxia/reoxygenation (H/R) injury via modulating the Keap1/Nrf2 pathway. Our results provided robust evidence that the BAT-cardiomyocyte interaction is required for DEX cardioprotection, and revealed an endocrine role of BAT in DEX-mediating protection of hearts against MIRI.

Activin E upregulates uncoupling protein 1 and fibroblast growth factor 21 in brown adipocytes.

Activin E activates brown and beige adipocytes and has been controversially implicated as a factor that induces obesity and fatty liver. Here, we sought to address this controversial issue by producing recombinant human activin E to evaluate its effects on HB2 brown adipocytes in vitro. Activin E increased uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) mRNA expression in the adipocytes. This upregulation was suppressed by SB431542, an inhibitor of activin receptor-like kinase (Alk) TGF-ß type I receptors. SB431542 also inhibited the activin E-induced phosphorylation of Smad2/3. A promoter assay using a CAGA-Luc reporter and Alk expression vectors revealed that activin E activated the TGF-ß/activin pathway via Alk7. The upregulation of Ucp1 and Fgf21 mRNA might be mediated through Alk7 and Smad2/3 phosphorylation. Activin E is a potential stimulator of energy expenditure by activating brown adipocytes and highlights its potential as a therapeutic target for treating obesity.

Itaconate alleviates diet-induced obesity via activation of brown adipocyte thermogenesis.

Despite medical advances, there remains an unmet need for better treatment of obesity. Itaconate, a product of the decarboxylation of the tricarboxylic acid cycle intermediate cis-aconitate, plays a regulatory role in both metabolism and immunity. Here, we show that itaconate, as an endogenous compound, counteracts high-fat-diet (HFD)-induced obesity through leptin-independent mechanisms in three mouse models. Specifically, itaconate reduces weight gain, reverses hyperlipidemia, and improves glucose tolerance in HFD-fed mice. Additionally, itaconate enhances energy expenditure and the thermogenic capacity of brown adipose tissue (BAT). Unbiased proteomic analysis reveals that itaconate upregulates key proteins involved in fatty acid oxidation and represses the expression of lipogenic genes. Itaconate may provoke a major metabolic reprogramming by inducing fatty acid oxidation and suppression of fatty acid synthesis in BAT. These findings highlight itaconate as a potential activator of BAT-mediated thermogenesis and a promising candidate for anti-obesity therapy.

Unveiling the Potential of Natural Compounds: A Comprehensive Review on Adipose Thermogenesis Modulation.

The process of adipocyte browning has recently emerged as a novel therapeutic target for combating obesity and obesity-related diseases. Non-shivering thermogenesis is the process of biological heat production in mammals and is primarily mediated via brown adipose tissue (BAT). The recruitment and activation of BAT can be induced through chemical drugs and nutrients, with subsequent beneficial health effects through the utilization of carbohydrates and fats to generate heat to maintain body temperature. However, since potent drugs may show adverse side effects, nutritional or natural substances could be safe and effective as potential adipocyte browning agents. This review aims to provide an extensive overview of the natural food compounds that have been shown to activate brown adipocytes in humans, animals, and in cultured cells. In addition, some key genetic and molecular targets and the mechanisms of action of these natural compounds reported to have therapeutic potential to combat obesity are discussed.

NDUFA9 and its crotonylation modification promote browning of white adipocytes by activating mitochondrial function in mice.

Protein crotonylation plays a role in regulating cellular metabolism, gene expression, and other biological processes. NDUFA9 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9) is closely associated with the activity and function of mitochondrial respiratory chain complex I. Mitochondrial function and respiratory chain are closely related to browning of white adipocytes, it's speculated that NDUFA9 and its crotonylation are associated with browning of white adipocytes. Firstly, the effect of NDUFA9 on white adipose tissue was verified in white fat browning model mice, and it was found that NDUFA9 promoted mitochondrial respiration, thermogenesis, and browning of white adipose tissue. Secondly, in cellular studies, it was discovered that NDUFA9 facilitated browning of white adipocytes by enhancing mitochondrial function, mitochondrial complex I activity, ATP synthesis, and mitochondrial respiration. Again, the level of NDUFA9 crotonylation was increased by treating cells with vorinostat (SAHA)+sodium crotonate (NaCr) and overexpressing NDUFA9, it was found that NDUFA9 crotonylation promoted browning of white adipocytes. Meanwhile, the acetylation level of NDUFA9 was increased by treating cells with SAHA+sodium acetate (NaAc) and overexpressing NDUFA9, the assay revealed that NDUFA9 acetylation inhibited white adipocytes browning. Finally, combined with the competitive relationship between acetylation and crotonylation, it was also demonstrated that NDUFA9 crotonylation promoted browning of white adipocytes. Above results indicate that NDUFA9 and its crotonylation modification promote mitochondrial function, which in turn promotes browning of white adipocytes. This study establishes a theoretical foundation for the management and intervention of obesity, which is crucial in addressing obesity and related medical conditions in the future.

The dairy-derived peptide Miltin exerts anti-obesity effects by increasing adipocyte thermogenesis.

Growing research has highlighted that the consumption of dairy products improves the metabolic health in obese individuals by functioning as regulatory modulators. However, the molecular basis of this effect remains largely unknown. Herein, we report a dairy-derived peptide, which we named Miltin, that activates the thermogenesis of brown adipocytes and increases white adipocyte browning. Previously, Miltin was merely identified for its antioxidant capacity, although it is commonly present in different dairy products. In this study, we revealed the effect of Miltin in modulating adipose thermogenesis and further explored its potential in treating obesity through in vivo and in vitro strategies. The administration of Miltin in mice fed with a high-fat diet resulted in enhanced thermogenesis, improved glucose homeostasis, and reduced body mass and lipid accumulation, indicating the anti-obesity effect of Miltin. Genomic analysis revealed that Miltin modulates thermogenesis by inducing the activation of the MAPK signaling pathway by preferentially interacting with GADD45γ to promote its stability. Together, our findings indicate that Miltin's role in initiating the thermogenesis of adipocytes makes it a potential anti-obesity therapy for future development.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

Inhibition of STAT3 enhances UCP1 expression and mitochondrial function in brown adipocytes.

Extensive studies have shown that the increasing brown adipose tissue (BAT) mass/activity possesses a strong ability to prevent obesity and its related complications. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signal pathway is known to play a role in adipocyte differentiation and development. However, its impact on thermogenic properties of mature brown adipocytes has not yet been clarified. Nifuroxazide (NFX), a potent inhibitor of STAT3, has received widespread attention due to its alternative anti-tumor and anti-inflammatory effects. Herein, we report that NFX induces lipolysis with subsequent downregulation of ACCα and FAS, while ATGL and pHSL levels are elevated in mature brown adipocytes. Furthermore, NFX treatment promotes the mitochondrial respiration of mature brown adipocytes, as evidenced by increased expression of thermogenic transcriptional factors and mitochondrial content. In addition, it also alleviates the IL-6 and TNFα inhibition on brown thermogenic programming via suppressing the STAT3/NF-κB/IL-6 signaling pathway. In general, these findings suggest that the blockade of the JAK/STAT3 pathway by NFX has a pro-thermogenic effect on mature brown adipocytes which opens new perspectives for NFX repurposing and potential therapeutic route to counteract obesity and related metabolic disorders.

ACSS3 in brown fat drives propionate catabolism and its deficiency leads to autophagy and systemic metabolic dysfunction.

Propionate is a gut microbial metabolite that has been reported to have controversial effects on metabolic health. Here we show that propionate is activated by acyl-CoA synthetase short-chain family member 3 (ACSS3), located on the mitochondrial inner membrane in brown adipocytes. Knockout of Acss3 gene (Acss3-/- ) in mice reduces brown adipose tissue (BAT) mass but increases white adipose tissue (WAT) mass, leading to glucose intolerance and insulin resistance that are exacerbated by high-fat diet (HFD). Intriguingly, Acss3-/- or HFD feeding significantly elevates propionate levels in BAT and serum, and propionate supplementation induces autophagy in cultured brown and white adipocytes. The elevated levels of propionate in Acss3-/- mice similarly drive adipocyte autophagy, and pharmacological inhibition of autophagy using hydroxychloroquine ameliorates obesity, hepatic steatosis and insulin resistance of the Acss3-/- mice. These results establish ACSS3 as the key enzyme for propionate metabolism and demonstrate that accumulation of propionate promotes obesity and Type 2 diabetes through triggering adipocyte autophagy.

Prednisone stimulates white adipocyte browning via ß3-AR/p38 MAPK/ERK signaling pathway.

AIMS: Prednisone is a corticosteroid-derived drug which is widely used for its role in immunosuppression and treatment of lung disorders. The current study reports, for the first time, the critical role of prednisone in the induction of white fat browning, thereby promoting thermogenic effect in cultured white adipocytes. MAIN METHODS: The fat-browning activity of prednisone was evaluated in 3T3-L1 cells by quantitative real-time PCR, immunoblot analysis, immunofluorescence, and molecular docking techniques. KEY FINDINGS: Exposure to prednisone stimulated browning in 3T3-L1 white adipocytes by increasing the expressions of core fat browning marker proteins (UCP1, PGC-1α and PRDM16) as well as beige-specific genes (Cd137, Cidea, Cited1, and Tbx1) via ATF2 and CREB activation mediated by p38 MAPK and ERK signaling, respectively. Prednisone exposure also resulted in the robust activation of lipolytic and fatty acid oxidation marker proteins, thereby increasing mitochondrial biogenesis. In addition, prednisone treatment resulted in reduced expression levels of adipogenic transcription factors while elevating SIRT1, as well as attenuation of lipogenesis and lipid droplets formation. Furthermore, molecular docking and mechanistic studies demonstrated the recruitment of beige fat by prednisone via the ß3-AR/p38 MAPK/ERK signaling pathway. SIGNIFICANCE: Taken together, these results indicate the unique role of prednisone as a fat-browning stimulant, and demonstrate its therapeutic potential in the treatment of obesity by enhancing thermogenesis.

ASKA technology-based pull-down method reveals a suppressive effect of ASK1 on the inflammatory NOD-RIPK2 pathway in brown adipocytes.

Recent studies have shown that adipose tissue is an immunological organ. While inflammation in energy-storing white adipose tissues has been the focus of intense research, the regulatory mechanisms of inflammation in heat-producing brown adipose tissues remain largely unknown. We previously identified apoptosis signal-regulating kinase 1 (ASK1) as a critical regulator of brown adipocyte maturation; the PKA-ASK1-p38 axis facilitates uncoupling protein 1 (UCP1) induction cell-autonomously. Here, we show that ASK1 suppresses an innate immune pathway and contributes to maintenance of brown adipocytes. We report a novel chemical pull-down method for endogenous kinases using analog sensitive kinase allele (ASKA) technology and identify an ASK1 interactor in brown adipocytes, receptor-interacting serine/threonine-protein kinase 2 (RIPK2). ASK1 disrupts the RIPK2 signaling complex and inhibits the NOD-RIPK2 pathway to downregulate the production of inflammatory cytokines. As a potential biological significance, an in vitro model for intercellular regulation suggests that ASK1 facilitates the expression of UCP1 through the suppression of inflammatory cytokine production. In parallel to our previous report on the PKA-ASK1-p38 axis, our work raises the possibility of an auxiliary role of ASK1 in brown adipocyte maintenance through neutralizing the thermogenesis-suppressive effect of the NOD-RIPK2 pathway.

Lipoxin A4 promotes adipogenic differentiation and browning of mouse embryonic fibroblasts.

Recently, it has been irrefutably discovered that brown adipocytes dissipate energy as heat and protect against obesity. Researchers make great efforts to explore approaches for its activation. Lipoxin A4 (LXA4) has been proven to reverse adipose tissue inflammation and improve insulin resistance, but its function on brown adipocyte differentiation has been poorly understood, which therefore to be investigated in the present study. Mouse embryonic fibroblasts (MEFs) were induced and differentiated to model brown adipocytes, and treated with LXA4 at 0, 1, 5, and 10 nM for 0-14 d. Afterwards, Oil Red O staining detected lipid droplets. In differentiated MEFs with or without LXA4 (10 nM) treatment, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) assessed adipocyte browning marker uncoupling protein 1 (UCP-1), and brown adipogenesis markers peroxisome proliferator-activated receptor gamma (PPARγ), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), cyclooxygenase-2 (COX-2), and positive regulation domain containing 16 (PRDM16) as well as lipogenic genes of stearoyl-CoA desaturase 1 (SCD1), fatty acid synthase (FASN), glucose transporter type 4 (GLUT4), and carbohydrate response element binding protein (ChREBP). The induced differentiation of MEFs toward brown adipocytes was successful. LXA4 promoted intracellular accumulation of lipid droplets of induced cells and increased UCP-1 expression in a dose- or time-dependent manner. Under the administration of LXA4, brown adipogenesis markers and lipogenic genes were further upregulated. LXA4 made a contribution to induce differentiation of MEFs to brown adipocytes, which could be regarded a new drug target for obesity management.

The Mechanism of Leptin on Inhibiting Fibrosis and Promoting Browning of White Fat by Reducing ITGA5 in Mice.

Leptin is a small molecule protein secreted by adipocytes, which can promote white fat browning through activating the hypothalamic nervous system and inhibiting downstream signaling pathways. Moreover, white fat browning has been proven to alleviate fat tissue fibrosis. This study explores the mechanism of leptin in regulating adipose tissue fibrosis and white fat browning. After treating mice with leptin, we screened out the recombinant integrin alpha 5 (ITGA5) through proteomics sequencing, which may play a role in adipose tissue fibrosis. Through real-time quantitative PCR (qPCR), western blotting (WB), hematoxylin-eosin (HE) staining, Masson's trichrome, immunofluorescence, immunohistochemistry, etc., the results showed that after leptin treated adipocytes, the expression of fibrosis-related genes and ITGA5 was significantly down-regulated in adipocytes. We constructed fibrosis model through transforming growth factor-ß (TGF-ß) and a high-fat diet (HFD), and treated with ITGA5 overexpression vector and interference fragments. The results indicated the expression of fibrosis-related genes were significantly down-regulated after interfering with ITGA5. After treating adipocytes with wortmannin, fibrosis-related gene expression was inhibited after overexpression of ITGA5. Moreover, after injecting mice with leptin, we also found that leptin significantly up-regulated the expression of adipose tissue browning-related genes. Overall, our research shows that leptin can inhibit the activation of phosphatidylinositol 3 kinase (PI3K)-protein kinase B (AKT) signaling pathway by reducing the expression of ITGA5, which could alleviate adipose tissue fibrosis, and further promote white fat browning. Our research provides a theoretical basis for further research on the effect of leptin in fibrosis-related adipose tissue metabolism.

A Population of M2 Macrophages Associated With Bone Formation.

We previously identified transient brown adipocyte-like cells associated with heterotopic ossification (HO). These ancillary cells support new vessel synthesis essential to bone formation. Recent studies have shown that the M2 macrophage contributes to tissue regeneration in a similar way. To further define the phenotype of these brown adipocyte-like cells they were isolated and characterized by single-cell RNAseq (scRNAseq). Analysis of the transcriptome and the presence of surface markers specific for macrophages suggest that these cells are M2 macrophages. To validate these findings, clodronate liposomes were delivered to the tissues during HO, and the results showed both a significant reduction in these macrophages as well as bone formation. These cells were isolated and shown in culture to polarize towards either M1 or M2 similar to other macrophages. To confirm that these are M2 macrophages, mice received lipopolysacheride (LPS), which induces proinflammation and M1 macrophages. The results showed a significant decrease in this specific population and bone formation, suggesting an essential role for M2 macrophages in the production of bone. To determine if these macrophages are specific to HO, we isolated these cells using fluorescence-activated cell sorting (FACS) from a bone defect model and subjected them to scRNAseq. Surprisingly, the macrophage populations overlapped between the two groups (HO-derived versus callus) suggesting that they may be essential ancillary cells for bone formation in general and not selective to HO. Of further note, their unique metabolism and lipogenic properties suggest the potential for unique cross talk between these cells and the newly forming bone.

The Effect of TLR Agonists and Myokines on Secretory Activity of Adipogenically Differentiated MSC Cultures.

We studied the effect of bacterial pathogen-associated molecular patterns and myokines on the secretion of adipokines by mesenchymal stem cells (MSC) and products of their adipogenic differentiation. The secretion of adiponectin, adipsin, leptin, and insulin by adipogenically differentiated cell cultures was quantitatively determined using multiplex ELISA. MSC obtained from the stromal vascular fraction of human subcutaneous adipose tissue were shown to secrete a known adipokine adipsin. The ability of white adipocytes to secrete significant amounts of insulin (in vitro) has been shown for the first time. Control cultures of white adipocytes secreted much higher levels of adiponectin, leptin, and insulin when compared to other adipocytes cultures. On the other hand, beige and brown adipocyte cultures secreted more adipsin than white adipocyte cultures. The influence of myokine ß-aminoisobutyric acid on the secretion of adipsin in MSC, white, beige, and brown adipocytes was also studied.

Astragalus polysaccharide regulates brown adipogenic differentiation through miR-1258-5p-modulated cut-like homeobox 1 expression.

Astragalus polysaccharide (APS) is the major natural active component of Astragalus membranaceus, which has been recognized as one of the most popular herbal medicines worldwide. Enhancing the formation and function of brown adipose tissue increases energy expenditure and hence may potentially be used against obesity and type 2 diabetes. The aim of the present study was to explore the effect and mechanism of APS on brown adipocyte formation. Mouse C3H10T 1/2 cells were subject to APS, and both proliferation and brown adipogenic differentiation were determined. The results showed that APS exhibits a decreased proliferation ability, which is accompanied by downregulated proliferating cell nuclear antigen, cyclin D1, and cyclin-dependent kinase 4. APS promotes the differentiation of C3H10T 1/2 cells into brown adipocytes and induces the expressions of key brown adipogenic transcriptional factors, including CCAAT/enhancer-binding protein ß, uncoupling protein 1, and PR domain-containing 16. Importantly, APS enables insulin sensitization in brown adipocytes, which may proceed through activation of the canonical phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and AMP-activated protein kinase (AMPK). Furthermore, the level of cut-like homeobox 1 (CUX1) is positively related to brown adipogenic differentiation, while APS regulates Cux1 expression through interaction with miR-1258-5p. Notably, the promotional effect of APS on brown adipogenic differentiation was abolished by Cux1 knockout. Collectively, our results suggest that APS enhances the differentiation of C3H10T 1/2 cells into brown adipocytes through regulating Cux1 via miR-1258-5p.

3-hydroxymorphinan enhances mitochondrial biogenesis and adipocyte browning through AMPK-dependent pathway.

3-hydroxymorphinan (3-HM), a metabolite of dextromethorphan, has previously been reported to have anti-inflammatory, anti-oxidative stress, and neuroprotective effects. However, its effect on energy metabolism in adipocytes remains unclear. Herein, we investigated 3-hydroxymorphinan (3-HM) effects on mitochondrial biogenesis, oxidative stress, and lipid accumulation in 3T3-L1 adipocytes. Further, we explored 3-HM-associated molecular mechanisms. Mouse adipocyte 3T3-L1 cells were treated with 3-HM, and various protein expression levels were determined by western blotting analysis. Mitochondria accumulation and lipid accumulation were measured by staining methods. Cell toxicity was assessed by cell viability assay. We found that treatment of 3T3-L1 adipocytes with 3-HM increased expression of brown adipocyte markers, such as uncoupling protein-1 (UCP-1) and peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1α). 3-HM promotes mitochondrial biogenesis and its-mediated gene expression. Additionally, 3-HM treatment suppressed mitochondrial ROS generation and superoxide along with improved mitochondrial complex I activity. We found that treatment of 3-HM enhanced AMPK phosphorylation. siRNA-mediated suppression of AMPK reversed all these changes in 3T3-L1 adipocytes. In sum, 3-HM promotes mitochondrial biogenesis and browning and attenuates oxidative stress and lipid accumulation in 3T3-L1 adipocytes via AMPK signaling. Thus, 3-HM-mediated AMPK activation can be considered a therapeutic approach for treating obesity and related diseases.

DHA but not EPA induces the trans-differentiation of C2C12 cells into white-like adipocytes phenotype.

Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating ß-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of lipid droplets and suppressed mitochondrial maximal respiration and spare respiratory capacity. EPA, on the other hand, reduced myogenesis regulating genes, but no significant differences were observed in the expression of adipogenesis key genes. Likewise, EPA suppressed the expression of WAT signature genes indicating that EPA and DHA have an independent role on white adipogensis. Unlike DHA treatment, EPA supplementation had no effect on the differential of C2C12 cells into brown adipocytes. In conclusion, DHA is a potent adipogenic and lipogenic factor that can change the metabolic profile of muscle cells by increasing myocellular fat.