3T3-L1 Adipocytes Research Articles

Role of SUMO-Specific Protease 2 in Leptin-Induced Fatty Acid Metabolism in White Adipocytes.

Leptin is a 16-kDa fat-derived hormone with a primary role in controlling adipose tissue levels. Leptin increases fatty acid oxidation (FAO) acutely through adenosine monophosphate-activated protein kinase (AMPK) and on delay through the SUMO-specific protease 2 (SENP2)-peroxisome proliferator-activated receptor δ/γ (PPARδ/γ) pathway in skeletal muscle. Leptin also directly increases FAO and decreases lipogenesis in adipocytes; however, the mechanism behind these effects remains unknown. Here, we investigated the role of SENP2 in the regulation of fatty acid metabolism by leptin in adipocytes and white adipose tissues. The effects of leptin mediated by SENP2 on fatty acid metabolism were tested by siRNA-mediated knockdown in 3T3-L1 adipocytes. The role of SENP2 was confirmed in vivo using adipocyte-specific Senp2 knockout (Senp2-aKO) mice. We revealed the molecular mechanism involved in the leptin-induced transcriptional regulation of carnitine palmitoyl transferase 1b (Cpt1b) and long-chain acyl-coenzyme A synthetase 1 (Acsl1) using transfection/reporter assays and chromatin immunoprecipitation. SENP2 mediated the increased expression of FAO-associated enzymes, CPT1b and ACSL1, which peaked 24 hours after leptin treatment in adipocytes. In contrast, leptin stimulated FAO through AMPK during the initial several hours after treatment. In white adipose tissues, FAO and mRNA levels of Cpt1b and Acsl1 were increased by 2-fold 24 hours after leptin injection in control mice but not in Senp2-aKO mice. Leptin increased PPARα binding to the Cpt1b and Acsl1 promoters in adipocytes through SENP2. These results suggest that the SENP2-PPARα pathway plays an important role in leptin-induced FAO in white adipocytes.

Metallothionein 3 Inhibits 3T3-L1 Adipocyte Differentiation via Reduction of Reactive Oxygen Species.

Metallothionein 3 (MT3), also known as a neuronal growth-inhibitory factor, is a member of the metallothionein family and is involved in a variety of biological functions, including protection against metal toxicity and reactive oxygen species (ROS). However, less is known about the role of MT3 in the differentiation of 3T3-L1 cells into adipocytes. In this study, we observed that MT3 levels were downregulated during 3T3-L1 adipocyte differentiation. Mt3 overexpression inhibited adipocyte differentiation and reduced the levels of the adipogenic transcription factors C/EBPα and PPARγ. Further analyses showed that MT3 also suppressed the transcriptional activity of PPARγ, and this effect was not mediated by a direct interaction between MT3 with PPARγ. In addition, Mt3 overexpression resulted in a decrease in ROS levels during early adipocyte differentiation, while treatment with antimycin A, which induces ROS generation, restored the ROS levels. Mt3 knockdown, on the other hand, elevated ROS levels, which were suppressed upon treatment with the antioxidant N-acetylcysteine. Our findings indicate a previously unknown role of MT3 in the differentiation of 3T3-L1 cells into adipocytes and provide a potential novel target that might facilitate obesity treatment.

Immature ginger reduces triglyceride accumulation by downregulating Acyl CoA carboxylase and phosphoenolpyruvate carboxykinase-1 genes in 3T3-L1 adipocytes.

Obesity is the underlying risk factor for major metabolism complications, including non-alcoholic-fatty liver disease, atherosclerosis, and cardiovascular disease. The adipose tissue is a vital endocrine organ that plays a role in the synthesis and storage of lipid and, therefore, is a contributory factor to the development and progression of obesity. A growing interest in nutraceuticals suggests that natural products can alleviate the risk factors and may be effective in mitigating obesity. The objective of this study was to examine the underlying mechanisms of immature ginger on adipocyte differentiation and lipogenesis in a 3T3-L1 cellular model. Ginger samples, extracted in 80% methanol, were dried and resuspended in DMSO at 50 μg/mL as stock solution. For analysis, the extracted samples were further diluted in media. Effects on adipogenesis were evaluated by determining lipid droplet and triglyceride accumulation, whereas effects on lipogenesis were determined by measuring triglyceride contents and fatty acid profile. The expression of key regulatory genes involved in adipogenesis and lipogenesis was also determined. Our data indicate that the intracellular lipid accumulation decreased significantly by 15 or 25% on treatment with 25 or 50 μg/mL of ginger extract. Consistent with these data, significantly reduced triglyceride levels by 30 or 50% were observed on 25 or 50 μg/mL treatment with ginger extracts, respectively. In addition, ginger treatment significantly inhibited the differentiation-induced de novo lipogenesis and Δ9 desaturase activity. Furthermore, ginger treatment reduced adipogenesis genes, C/ebpβ and C/ebpδ, expression by 47 or 64%, respectively, but significantly increased Pparγ expression by 60% and adiponectin by 75%. Ginger extracts had no effect on Fas genes but reduced lipogenesis genes, acyl CoA carboxylase (Acc) expression by two-fold, and phosphoenolpyruvate carboxy kinase 1 (Pepck1) expression by 50%. Our findings suggest immature ginger can potentially inhibit lipogenesis pathways by limiting the channeling of glucose carbon in fatty acid synthesis by inhibiting the expression of ACC and glycerol production via inhibiting the expression of PEPCK, which consequently inhibits triglyceride formation.

Efficacy of fucoxanthin extract from Sargassum horneri on 3T3-L1 pre-adipocyte differentiation.

Obesity, a chronic disease characterized by excessive body fat accumulation, is associated with significant health risks. The state of being overweight or obese leads to a number of chronic diseases, including cardiovascular disease, type 2 diabetes, cancer, and osteoarthritis. Accordingly, the regulation of adipocyte proliferation and differentiation has been the focus of many studies. The goal of the present study was to investigate the function of fucoxanthin, extracted from Sargassum horneri, in adipocyte (3T3-L1 cells) differentiation. A quantitative real-time polymerase chain reaction was conducted to investigate the mRNA expression levels of adipocyte differentiation-related genes under fucoxanthin stimulation. All adipocyte-related genes responded to PIC stimuli. Additionally, using western blotting, we confirmed that fucoxanthin reduced adipocyte differentiation. These results indicate that fucoxanthin extracted from Sargassum horneri can regulate adipogenesis. Further studies are needed to reveal the signaling pathways that lead to reduced adipocyte differentiation induced by fucoxanthin.

Advanced Oxidation Protein Products Contribute to Chronic-Kidney-Disease-Induced Adipose Inflammation through Macrophage Activation.

Fat atrophy and adipose tissue inflammation can cause the pathogenesis of metabolic symptoms in chronic kidney disease (CKD). During CKD, the serum levels of advanced oxidation protein products (AOPPs) are elevated. However, the relationship between fat atrophy/adipose tissue inflammation and AOPPs has remained unknown. The purpose of this study was to investigate the involvement of AOPPs, which are known as uremic toxins, in adipose tissue inflammation and to establish the underlying molecular mechanism. In vitro studies involved co-culturing mouse-derived adipocytes (differentiated 3T3-L1) and macrophages (RAW264.7). In vivo studies were performed using adenine-induced CKD mice and AOPP-overloaded mice. Fat atrophy, macrophage infiltration and increased AOPP activity in adipose tissue were identified in adenine-induced CKD mice. AOPPs induced MCP-1 expression in differentiated 3T3-L1 adipocytes via ROS production. However, AOPP-induced ROS production was suppressed by the presence of NADPH oxidase inhibitors and the scavengers of mitochondria-derived ROS. A co-culturing system showed AOPPs induced macrophage migration to adipocytes. AOPPs also up-regulated TNF-α expression by polarizing macrophages to an M1-type polarity, and then induced macrophage-mediated adipose inflammation. In vitro data was supported by experiments using AOPP-overloaded mice. AOPPs contribute to macrophage-mediated adipose inflammation and constitute a potential new therapeutic target for adipose inflammation associated with CKD.

Naringin promotes fat browning mediated by UCP1 activation via the AMPK signaling pathway in 3T3-L1 adipocytes.

Induction of the brown adipocyte-like phenotype in white adipocytes (fat browning) is considered a promising therapeutic strategy to treat obesity. Naringin, a citrus flavonoid, has antioxidant, anti-inflammatory, and anticancer activities. We examined the application of naringin as an anti-obesity compound based on an investigation of its induction of fat browning in 3T3-L1 adipocytes. Naringin did not induce lipid accumulation in differentiated 3T3-L1 adipocytes. Additionally, naringin reduced the expression levels of proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) involved in adipogenesis during lipid metabolism and increased the levels of PPARα and adiponectin involved in fatty acid oxidation. The expression levels of fat browning markers uncoupling protein 1 (UCP1; involved in thermogenesis) and PR domain containing 16 (PRDM16) increased. In addition, naringin treatment resulted in the activation of PPARγ coactivator 1-alpha (PGC-1α), a factor related to UCP1 transcription and mitochondrial biogenesis. Moreover, the expression of beige adipocyte-specific genes such as Cd137, Cited1, Tbx1, and Tmem26 was also induced. The small multi-lipid droplets characteristic of beige adipocytes indicated that naringin treatment increased the levels of all lipolysis markers (hormone-sensitive lipase [HSL], adipose triglyceride lipase [ATGL], perilipin [PLIN], and protein kinase A [PKA]). Adenosine monophosphate-activated protein kinase (AMPK) and UCP1 levels increased by treatment with naringin alone; this was possibly mediated by the stimulation of the AMPK signaling pathway. According to mechanistic studies, naringin activated the thermogenic protein UCP1 via the AMPK signaling pathway. In conclusion, naringin induces fat browning and is a promising therapeutic agent for metabolic disorders based on the regulation of lipid metabolism.

An Innovative Mei-Gin Formula Exerts Anti-Adipogenic and Anti-Obesity Effects in 3T3-L1 Adipocyte and High-Fat Diet-Induced Obese Rats

To investigate the potential anti-obesity properties of an innovative functional formula (called the Mei-Gin formula: MGF) consisting of bainiku-ekisu, Prunus mume (70% ethanol extract), black garlic (water extract), and Mesona procumbens Hemsl. (40% ethanol extract) for reducing lipid accumulation in 3T3-L1 adipocytes in vitro and obese rats in vivo. The prevention and regression of high-fat diet (HFD)-induced obesity by the intervention of Japan Mei-Gin, MGF-3 and -7, and positive health supplement powder were investigated in male Wistar rats. The anti-obesity effects of MGF-3 and -7 in rats with HFD-induced obesity were examined by analyzing the role of visceral and subcutaneous adipose tissue in the development of obesity. The results indicated that MGF-1-7 significantly suppressed lipid accumulation and cell differentiation through the down-regulation of GPDH activity, as a key regulator in the synthesis of triglycerides. Additionally, MGF-3 and MGF-7 exhibited a greater inhibitory effect on adipogenesis in 3T3-L1 adipocytes. The high-fat diet increased body weight, liver weight, and total body fat (visceral and subcutaneous fat) in obese rats, while these alterations were effectively improved by the administration of MGF-3 and -7, especially MGF-7. This study highlights the role of the Mei-Gin formula, particularly MGF-7, in anti-obesity action, which has the potential to be used as a therapeutic agent for the prevention or treatment of obesity.

Phenols from Potentilla anserina L. Improve Insulin Sensitivity and Inhibit Differentiation in 3T3-L1 Adipocytes in Vitro.

Potentilla anserina L., a well-known perennial herb, is widely used in traditional Tibetan medicine and used as a delicious food in humans. The present investigation reports on the activity of P. anserina phenols (PAP) in regulating glycolipid metabolism in 3T3-L1 adipocytes. Insulin sensitivity tests showed that PAP improved insulin-stimulated glucose uptake by promoting the phosphorylation of serine/threonine kinase Akt. Moreover, an assay involving the differentiation of 3T3-L1 preadipocytes demonstrated that PAP also decreased the accumulation of lipid droplets by suppressing the expression of adipokines during the differentiation process. In addition, the underlying mechanism from the aspects of energy metabolism and oxidative stress is also discussed. The improvement in energy metabolism was supported by an increase in mitochondrial membrane potential (MMP) and intracellular ATP. Amelioration of oxidative stress was supported by decreased levels of intracellular reactive oxygen species (ROS). In summary, our findings suggest that PAP can ameliorate the disorder of glycolipid metabolism in insulin resistant 3T3-L1 adipocytes by improving energy metabolism and oxidative stress and might be an attractive candidate for the treatment of diabetes.

Anti-inflammatory efficacy of brown seaweed (Padina tetrastromatica) in 3T3-L1 adipocytes and low-dose LPS induced inflammation in C57BL6 mice

Chronic systemic inflammation is a central indicator of numerous diseases. Regulation of inflammation in adipose and liver is essential in mitigating obesity. SIRT1 is known to modulate inflammation and metabolic pathways. Brown algae Padina tetrastromatica possesses anti-oxidant and anti-inflammatory properties. However, the synergistic effects of P. tetrastromatica bioactives Fucoxanthin (Fx) and Total lipids (TL) to inhibit inflammation in SIRT1 silenced 3T3-L1 adipocytes remain unknown. Furthermore, the role of anti-inflammatory food supplements in mitigating systemic inflammation stays elusive. Therefore, here we studied the effects of P.tetrastromatica bioactives on cytokine production and activation of cell signals in SIRT1 silenced and LPS-induced inflammation in 3T3-L1 adipocytes and mice models. Preliminary in silico screening of Fx and its metabolites with target inflammatory markers was performed. The effects of Fx and TL on adipogenesis and reactive oxygen species (ROS) in silenced cells were detected by Oil Red O and DCFH-DA assay. Cytokine production and protein expression of NF-κB, COX-2, IL-10 and SIRT1 were determined by ELISA and western blotting. Fx and TL significantly inhibited adipogenesis, ROS, and Nitric oxide (NO), pro-inflammatory cytokine production (IL-6, IL-1ß, MCP-1, TNF-α) in SIRT1 silenced and LPS-challenged inflamed cells. Inflammatory marker (NF-κB, COX-2) expression was downregulated in Fx and TL treated cells compared to LPS and SIRT1 silenced cells indicating their modulating mechanistic ability to mitigate inflammation. Whereas, the expression of anti-inflammatory cytokine IL-10, adiponectin and NAD+ were upregulated significantly in Fx and TL treated cells compared to silenced and LPS insulted cells. These results were further validated by feeding seaweed and barley blended anti-inflammatory food (AIF) to LPS-treated mice. Results showed that AIF supplementation inhibited ROS generation, and pro-inflammatory cytokine, COX-2 and NF-κB expression. Whereas, feeding AIF upregulated adiponectin, NAD+, IL-10, and SIRT1 levels. Results suggest potential therapeutic food applications of P. tetrastromatica to manage inflammation and obesity.

Screening, structural characterization and anti-adipogenesis effect of a water-soluble polysaccharide from Lycium barbarum L. by an activity-oriented approach

This study aimed to describe the structural features and anti-adipogenesis effect of polysaccharides obtained from Lycium barbarum L. by a bioactivity-oriented purification method. First, crude polysaccharide LICP009 was identified as the main bioactive compound with potential inhibition of intracellular lipid accumulation. Bioactivity guided fractionation was further performed using ion-exchange chromatography and Sephadex G-50 to obtain the purified component LICP009-2-1. This component was mainly composed of arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (GalA), and glucuronic acid (GlcA) in a molar ratio of 40.2:31.5:4.2:5.8:5.7 with a molecular weight of 1.35 × 104 g/mol. Linkage and NMR data indicated that the backbone of LICP009-2-1 was consisted of a repeated unit of →3,6)-β-D-Galp-(1→ residues, with three branches composed of α-L-Araf-(1→, α-L-Araf-(1 → 5)-α-L-Araf-(1 → 5)-α-L-Araf-(1 → 3)-β-D-Galp-(1→, and α-L-Araf-(1 → 2)-α-L-Araf-(1→[4)-β-D-Galp-(1]3 → . In differentiated 3T3-L1 adipocyte model, LICP009-2-1 exhibited an antiadipogenic effect by decreasing lipid accumulation, as well as downregulating the expression of key transcription factors and proteins in the adipogenesis pathway in a dose-dependent manner. In high-fat-diet-induced obese mice, oral administration of LICP009-2-1 counteracted the progression of obesity and hyperlipidemia. These results indicated that LICP009-2-1 plays anti-adipogenic and anti-obesity roles both in vitro and in vivo, and could be an anti-obesity agent utilized as medicinal, pharmacological and functional food ingredient.

Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology.

Fatty acid-binding protein 4 (FABP4) is a critical immune-metabolic modulator, mainly expressed in adipocytes and macrophages, secreted from adipocytes in association with lipolysis, and plays essential pathogenic roles in cardiovascular and metabolic diseases. We previously reported Chlamydia pneumoniae infecting murine 3T3-L1 adipocytes and causing lipolysis and FABP4 secretion invitro. However, it is still unknown whether C. pneumoniae intranasal lung infection targets white adipose tissues (WATs), induces lipolysis, and causes FABP4 secretion invivo. In this study, we demonstrate that C. pneumoniae lung infection causes robust lipolysis in WAT. Infection-induced WAT lipolysis was diminished in FABP4-/- mice or FABP4 inhibitor-pretreated wild-type mice. Infection by C. pneumoniae in wild-type but not FABP4-/- mice induces the accumulation of TNF-α- and IL-6-producing M1-like adipose tissue macrophages in WAT. Infection-induced WAT pathology is augmented by endoplasmic reticulum (ER) stress/the unfolded protein response (UPR), which is abrogated by treatment with azoramide, a modulator of the UPR. C. pneumoniae lung infection is suggested to target WAT and induce lipolysis and FABP4 secretion invivo via ER stress/UPR. FABP4 released from infected adipocytes may be taken up by other neighboring intact adipocytes or adipose tissue macrophages. This process can further induce ER stress activation and trigger lipolysis and inflammation, followed by FABP4 secretion, leading to WAT pathology. A better understanding of the role of FABP4 in C. pneumoniae infection-induced WAT pathology will provide the basis for rational intervention measures directed at C. pneumoniae infection and metabolic syndrome, such as atherosclerosis, for which robust epidemiologic evidence exists.

Cyclopia intermedia (Honeybush) Induces Uncoupling Protein 1 and Peroxisome Proliferator-Activated Receptor Alpha Expression in Obese Diabetic Female db/db Mice.

Previously, we reported that a crude polyphenol-enriched fraction of Cyclopia intermedia (CPEF), a plant consumed as the herbal tea, commonly known as honeybush, reduced lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese, diabetic female leptin receptor-deficient (db/db) mice. In the current study, the mechanisms underlying decreased body weight gain in db/db mice were further elucidated using western blot analysis and in silico approaches. CPEF induced uncoupling protein 1 (UCP1, 3.4-fold, p < 0.05) and peroxisome proliferator-activated receptor alpha (PPARα, 2.6-fold, p < 0.05) expression in brown adipose tissue. In the liver, CPEF induced PPARα expression (2.2-fold, p < 0.05), which was accompanied by a 31.9% decrease in fat droplets in Hematoxylin and Eosin (H&E)-stained liver sections (p < 0.001). Molecular docking analysis revealed that the CPEF compounds, hesperidin and neoponcirin, had the highest binding affinities for UCP1 and PPARα, respectively. This was validated with stabilising intermolecular interactions within the active sites of UCP1 and PPARα when complexed with these compounds. This study suggests that CPEF may exert its anti-obesity effects by promoting thermogenesis and fatty acid oxidation via inducing UCP1 and PPARα expression, and that hesperidin and neoponcirin may be responsible for these effects. Findings from this study could pave the way for designing target-specific anti-obesity therapeutics from C. intermedia.

Quercetin-3-O-rutinoside from Moringa oleifera Downregulates Adipogenesis and Lipid Accumulation and Improves Glucose Uptake by Activation of AMPK/Glut-4 in 3T3-L1 Cells.

Natural product-based therapeutic alternatives have drawn immense interest to deal with growing incidence of metabolic disorders. Rutin (quercetin-3-O-rutinoside) is found in a variety of fruits, vegetables, and plant beverages. In the present study, rutin was isolated from Moringa oleifera Lam., leaves and its anti-lipidemic and anti-adipogenic activity was evaluated through inhibition of key digestive enzymes and in vitro cell culture experiments using 3T3-L1 adipocytes. Rutin treatment substantially reduced α-glucosidase and pancreatic lipase activities with IC50 values of 40 and 35 μg/ml, respectively. MTT assay with 3T3-L1 cells demonstrated the non-toxic effect of rutin up to 160 μg/ml. Oil Red O-stained images of rutin-treated 3T3-L1 cells depicted that rutin considerably reduced lipid content and adipogenesis (79.9%), and enhanced glycerol release in 3T3-L1 cells when compared to untreated cells. Rutin significantly (p < 0.05) enhanced glucose uptake in 3T3-L1 adipocytes and also led to reduced levels of leptin but enhanced levels of adiponectin. Western blot analysis of rutin-treated (40 µg/ml) adipocytes showed phosphorylation of AMPK, upregulated expression of Glut-4 (1.31-fold) and UCP-1 (1.47-fold), but downregulated expression of PPAR-γ by 0.73-fold. At transcriptional level, similar trends were observed in the mRNA expression of the above genes, except AMPK. Our results demonstrate that rutin isolated from M. oleifera significantly alleviates lipid content and adipogenesis, and improves glucose uptake through regulating PPAR-γ and AMPK signaling pathways; thus, rutin can be considered as a potential therapeutic agent against adiposity and glucose intolerance.

Comparison of physicochemical properties of sorghum extract by ethanol concentration and its anti-adipogenic effect in 3T3-L1cells.

Sorghum is a vital cereal source that has various phenolic compounds and potential health-promoting benefits. This study evaluated the phenolic content, antioxidant and anti-obesity effects of sorghum extract (SE) prepared using three solvent systems: 50% (SE50), 80% (SE80), and 100% (SE100) ethanol. The results showed that SE50 exhibited the highest total polyphenol and flavonoid content among the sorghum extracts using different ethanol concentrations as extraction solvents. In addition, SE50 showed significantly higher antioxidant capacity than the other extracts. Interestingly, SE50 significantly inhibited lipid accumulation in 3T3-L1 adipocytes; however, SE80 and SE100 had no beneficial effects. Moreover, SE50 significantly downregulated the mRNA expression levels of adipogenic genes (Cebpα, Pparγ, and Fabp4) and lipogenic genes (Srebp1c, Fas, and Scd1). These results suggest that SE50 is superior to other ethanol extracts in phenolic contents, antioxidant and anti-obesity activities, and it could be used as a nutraceutical for anti-obesity.

Time of day determines postexercise metabolism in mouse adipose tissue

The circadian clock is a cell-autonomous transcription-translation feedback mechanism that anticipates and adapts physiology and behavior to different phases of the day. A variety of factors including hormones, temperature, food-intake, and exercise can act on tissue-specific peripheral clocks to alter the expression of genes that influence metabolism, all in a time-of-day dependent manner. The aim of this study was to elucidate the effects of exercise timing on adipose tissue metabolism. We performed RNA sequencing on inguinal adipose tissue of mice immediately following maximal exercise or sham treatment at the early rest or early active phase. Only during the early active phase did exercise elicit an immediate increase in serum nonesterified fatty acids. Furthermore, early active phase exercise increased expression of markers of thermogenesis and mitochondrial proliferation in inguinal adipose tissue. Invitro, synchronized 3T3-L1 adipocytes showed a timing-dependent difference in Adrb2 expression, as well as a greater lipolytic activity. Thus, the response of adipose tissue to exercise is time-of-day sensitive and may be partly driven by the circadian clock. To determine the influence of feeding state on the time-of-day response to exercise, we replicated the experiment in 10-h-fasted early rest phase mice to mimic the early active phase metabolic status. A 10-h fast led to a similar lipolytic response as observed after active phase exercise but did not replicate the transcriptomic response, suggesting that the observed changes in gene expression are not driven by feeding status. In conclusion, acute exercise elicits timing-specific effects on adipose tissue to maintain metabolic homeostasis.

Acacetin alleviates energy metabolism disorder through promoting white fat browning mediated by AC-cAMP pathway.

Acacetin (ACA), a flavone isolated from Chinese traditional medical herbs, has numerous pharmacological activities. However, little is known about the roles in white fat browning and energy metabolism. In the present study, we investigated whether and how ACA would improve energy metabolism in vivo and in vitro. ACA (20 mg/kg) was intraperitoneally injected to the mice with obesity induced by HFD for 14 consecutive days (in vivo); differentiated 3T3-L1 adipocytes were treated with ACA (20 µmol/L and 40 µmol/L) for 24 h (in vitro). The metabolic profile, lipid accumulation, fat-browning and mitochondrial contents, and so on were respectively detected. The results in vivo showed that ACA significantly reduced the body weight and visceral adipose tissue weight, alleviated the energy metabolism disorder, and enhanced the browning-related protein expressions in adipose tissue of rats. Besides, the data in vitro revealed that ACA significantly reduced the lipid accumulation, induced the expressions of the browning-related proteins and cAMP-dependent protein kinase A (PKA), and increased the mitochondrium contents, especially enhanced the energy metabolism of adipocytes; however, treatment with beta-adrenergic receptor blocker (propranolol, Pro) or adenyl cyclase (AC) inhibitor (SQ22536, SQ) abrogated the ACA-mediated effects. The data demonstrate that ACA alleviates the energy metabolism disorder through the pro-browning effects mediated by the AC-cAMP pathway. The findings would provide the experimental foundation for ACA to prevent and treat obesity and related metabolism disorders.

Deep Proteome Profiling of White Adipose Tissue Reveals Marked Conservation and Distinct Features Between Different Anatomical Depots

White adipose tissue is deposited mainly as subcutaneous adipose tissue (SAT), often associated with metabolic protection, and abdominal/visceral adipose tissue, which contributes to metabolic disease. To investigate the molecular underpinnings of these differences, we conducted comprehensive proteomics profiling of whole tissue and isolated adipocytes from these two depots across two diets from C57Bl/6J mice. The adipocyte proteomes from lean mice were highly conserved between depots, with the major depot-specific differences encoded by just 3% of the proteome. Adipocytes from SAT (SAdi) were enriched in pathways related to mitochondrial complex I and beiging, whereas visceral adipocytes (VAdi) were enriched instructural proteins and positive regulators of mTOR presumably to promote nutrient storage and cellular expansion. This indicates that SAdi are geared toward higher catabolic activity, while VAdi are more suited for lipid storage. By comparing adipocytes from mice fed chow or Western diet (WD), we define a core adaptive proteomics signature consisting of increased extracellular matrix proteins and decreased fatty acid metabolism and mitochondrial Coenzyme Qbiosynthesis. Relative to SAdi, VAdi displayed greater changes with WD including a pronounced decrease in mitochondrial proteins concomitant with upregulation of apoptotic signaling and decreased mitophagy, indicating pervasive mitochondrial stress. Furthermore, WD caused a reduction in lipid handling and glucose uptake pathwaysparticularly in VAdi, consistent with adipocyte de-differentiation. By overlaying the proteomics changes with diet in whole adipose tissue and isolated adipocytes, we uncovered concordance between adipocytes and tissue only in the visceral adipose tissue, indicating a unique tissue-specific adaptation to sustained WD in SAT. Finally, an in-depth comparison of isolated adipocytes and 3T3-L1 proteomes revealed a high degree of overlap, supporting the utility of the 3T3-L1 adipocyte model. These deep proteomes provide an invaluable resource highlighting differences between white adipose depots that may fine-tune their unique functions and adaptation to an obesogenic environment.

A transdermal treatment with MC903 ameliorates diet-induced obesity by reducing visceral fat and increasing myofiber thickness and energy consumption in mice

AimMC903 is a synthetic derivative of vitamin D3 that has been designed to diminish its impact on calcium metabolism and is clinically used as a transdermal reagent for psoriasis. Animal studies showed that an oral or intraperitoneal vitamin D3 treatment prevented the development of obesity. In contrast, the bioavailability of orally administered vitamin D3 is reported to be low in obese patients. In the current study, we aimed to investigate the impact of a transdermal treatment with MC903 in established obese mice. We further studied the underlying mechanisms of MC903-mediated metabolic improvement.Materials and methodsMale C57BL/6 J mice were fed standard chow or a 60% high-fat diet (HFD) for 7 weeks, and a transdermal treatment with MC903 on the ear auricle was initiated thereafter. The metabolic profiles of mice were analyzed during 4 weeks of treatment, and mice were dissected for histological and gene expression analyses. The direct impacts of MC903 and vitamin D3 were investigated using 3T3-L1 adipocytes and C2C12 myotubes in vitro.ResultsHFD-fed mice showed significant increases in body and epididymal white adipose tissue (eWAT) weights with enlarged adipocytes. They exhibited glucose intolerance, decreased oxygen consumption, and chronic inflammation in eWAT. The transdermal treatment with MC903 significantly ameliorated these metabolic abnormalities in HFD-fed mice without affecting food consumption. In accordance with enhanced energy metabolism, myofiber diameters and the expression of uncoupling protein 3 (UCP3) in the gastrocnemius and soleus muscle were significantly increased in MC903-treated HFD mice. In addition, vitamin D3 and MC903 both suppressed adipogenic differentiation and enhanced lipolysis in 3T3-L1 adipocytes, and increased UCP3 expression in cultured C2C12 myotubes. Furthermore, MC903 increased oxygen consumption and UCP3 knockdown significantly decreased them in C2C12 myotubes.ConclusionsA transdermal treatment with MC903 increased myofiber diameter and energy metabolism and decreased visceral fat accumulation, thereby improving obesity and glucose intolerance in mice.

In Vivo Imaging of Lipid Droplets and Oxygen Status in Hepatic Tissues of Nonalcoholic Fatty Liver Model Mice Using a Lipophilic Ir(III) Complex

Nonalcoholic fatty liver disease (NAFLD) is becoming common worldwide. In pathophysiological studies of NAFLD, an in vivo optical probe that enables visualization of lipid droplets (LDs) and imaging of oxygen status in hepatic tissues simultaneously would be very useful. Here, we present the phosphorescent Ir(III) complex BTP ((btp)2Ir(acac) (btp = benzothienylpyridine, acac = acetylacetone)) as the first probe that meets this requirement. BTP was efficiently taken up into cultured 3T3-L1 adipocytes and selectively accumulated into LDs. Quantifying oxygen levels in LDs based on the phosphorescence lifetime of BTP allowed us to track changes in cellular oxygen tension after treatment with metabolic stimulants. Phosphorescence lifetime imaging microscopy combined with intravenously administered BTP in mice enabled specific visualization of LDs in hepatic lobules and simultaneous imaging of the oxygen gradient that decreased from the portal vein (PV) to the central vein (CV). NAFL model mice were created by feeding a high-fat diet (HFD) to mice for 3 or 7 days. The mice fed an HFD showed a marked increase in the amount and size of LDs in hepatocytes compared with those fed a normal diet, leading to abnormal microvascular structures. In addition, HFD-fed mice also exhibited reduced oxygen tension in areas other than the CV. Multicolor imaging with the LD-accumulated oxygen probe BTP and vasculature-staining FITC-lectin suggested that structural distortions of the sinusoidal microvasculature caused by enlarged LDs were associated with partial hypoxia in NAFL.

7,8-Dihydroxyflavone Attenuates Inflammatory Response and Insulin Resistance Induced by the Paracrine Interaction between Adipocytes and Macrophages.

Obesity-induced inflammation and insulin resistance are mediated by macrophage infiltration into adipose tissue. We investigated the effects of 7,8-dihydroxyflavone (7,8-DHF), a flavone found in plants, on the inflammatory response and insulin resistance induced by the interaction between adipocytes and macrophages. Hypertrophied 3T3-L1 adipocytes were cocultured with RAW 264.7 macrophages and treated with 7,8-DHF (3.12, 12.5, and 50 μM). The inflammatory cytokines and free fatty acid (FFA) release were evaluated by assay kits, and signaling pathways were determined by immunoblotting. Coculture of adipocytes and macrophages increased inflammatory mediators, such as nitric oxide (NO), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) and FFA secretion but suppressed the production of anti-inflammatory adiponectin. 7,8-DHF counteracted the coculture-induced changes (p < 0.001). 7,8-DHF also inhibited c-Jun N-terminal kinase (JNK) activation and blocked nuclear factor kappa B (NF-κB) nuclear translocation in the coculture system (p < 0.01). In addition, adipocytes cocultured with macrophages did not increase glucose uptake and Akt phosphorylation in response to insulin. However, 7,8-DHF treatment recovered the impaired responsiveness to insulin (p < 0.01). These findings show that 7,8-DHF alleviates inflammation and adipocyte dysfunction in the coculture of hypertrophied 3T3-L1 adipocytes and RAW 264.7 macrophages, indicating its potential as a therapeutic agent for obesity-induced insulin resistance.