Nuclear Factor-Kappa-B Mediates the Advanced Glycation End Product-Induced Repression of Slc2a4 Gene Expression in 3T3-L1 Adipocytes.

Advanced glycated end products (AGEs) are cytotoxic compounds that are mainly increased in diabetes mellitus (DM), kidney failure, inflammation, and in response to the ingestion of AGE-rich diets. AGEs can also impair glycemic homeostasis by decreasing the expression of the Slc2a4 (solute carrier family 2 member 4) gene and its GLUT4 (solute carrier family 2, facilitated glucose transporter member 4) protein in muscle. However, the mechanisms underlying AGE's effect on adipocytes have not been demonstrated yet. This study investigated the effects of AGEs upon Slc2a4/GLUT4 expression in 3T3-L1 adipocytes, as well as the potential role of NFKB (nuclear factor NF-kappa-B) activity in the effects observed. Adipocytes were cultured in the presence of control albumin (CA) or advanced glycated albumin (GA) at concentrations of 0.4, 3.6, and 5.4 mg/mL for 24 h or 72 h. Slc2a4, Rela, and Nfkb1mRNAs were measured by RT-qPCR, GLUT4, IKKA/B, and p50/p65 NFKB subunits using Western blotting, and p50/p65 binding into the Slc2a4 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. GA at 0.4 mg/mL increased Slc2a4/GLUT4 expression after 24 h and 72 h (from 50% to 100%), but at 5.4 mg/mL, Slc2a4/GLUT4 expression decreased at 72 h (by 50%). Rela and Nfkb1 expression increased after 24 h at all concentrations, but this effect was not observed at 72 h. Furthermore, 5.4 mg/mL of GA increased the p50/p65 nuclear content and binding into Slc2a4 at 72 h. In summary, this study reveals AGE-induced and NFKB-mediated repression of Slc2a4/GLUT4 expression. This can compromise the adipocyte glucose utilization, contributing not only to the worsening of glycemic control in DM subjects but also the impairment of glycemic homeostasis in non-DM subjects under the high intake of AGE-rich foods.

Angiotensin converting enzyme inhibitory hydrolysate and peptide fractions from chicken skin collagen, as modulators of lipid accumulation in adipocytes 3 T3-L1, after in vitro gastrointestinal digestion.

Inhibitory activity against angiotensin-converting enzyme (IAACE) by chicken skin collagen hydrolysate (CSCH) and their peptide fractions before and after in-vitro gastrointestinal digestion, were evaluated; as well as their ability to modulate lipid accumulation in 3 T3-L1 adipocytes. Before digestion, peptide fraction <1 kDa (F4) showed the highest IAACE (p < 0.05) followed by CSCH. After these samples were digested, F4 presented an IAACE with IC50 similar to its digest (DF4) (188.84 and 220.03 µg/mL, respectively), which was 2-fold lower (p < 0.05) than IC50 of fraction <1 kDa from post-digested hydrolysate (FDH) (388.57 µg/mL). Nine peptides were identified as the potential ACE inhibitors in F4 and DF4. Addition of DF4 (800 µg/mL) reduced(p < 0.05) lipid accumulation by 83% within preadipocytes. A 45-60% reduction of lipid accumulation within differentiated adipocytes was obtained by adding FDH and DF4 (regardless the concentration). These results, digested CSCH and F4 with IAACE may be considered as potential adjuvants for obesity treatment.

Resveratrol butyrate esters inhibit lipid biosynthesis in 3T3-L1 cells by AMP-activated protein kinase phosphorylation.

Resveratrol butyrate esters (RBEs), which are novel resveratrol-synthesized derivatives, exhibit increased biological activity. This study elucidated the effect of RBEs on fat metabolism and their anti-obesity characteristics. Their molecular mechanism was investigated in the 3T3-L1 murine preadipocyte cells and adipocytes. RBE doses of < 2 µM did not induce a significant change in the viability of 3T3-L1 adipocytes. After RBEs treatment, intracellular lipid droplet accumulation in 3T3-L1 adipocytes was stimulated by methylisobutylxanthine, dexamethasone, and insulin-containing medium. However, a significant dose-dependent reduction in intracellular lipid levels was observed. The mRNA levels of two adipogenic transcription factors (peroxisome proliferator-activated receptor [PPAR] and CCAAT/enhancer-binding proteins [C/EBP]) and lipogenic proteins (fatty acid-binding protein 4 [FABP4] and fatty acid synthase [FAS]) were significantly attenuated by RBE treatment in both MDI-stimulated and differentiated 3T3-L1 adipocytes. Moreover, the phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK) also dramatically increased in the MDI + RBE-treated group compared to that in the MDI + vehicle-treated group. Collectively, our study provides strong evidence that RBEs inhibit adipogenesis by regulating adipogenic protein expression and increasing the p-AMPK/AMPK ratio. Future studies will be conducted on animal models to validate the application of RBEs as a functional food ingredient in improving human health. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05436-x.

D-Limonene Promotes Anti-Obesity in 3T3-L1 Adipocytes and High-Calorie Diet-Induced Obese Rats by Activating the AMPK Signaling Pathway.

D-limonene (LIM) is a common monoterpene compound, principally found in citrus essential oils. This study investigated the anti-obesity effect of LIM on the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in 3T3-L1 adipocytes and high-calorie diet-induced obese rats and confirmed the optimally effective dose of LIM. The 3T3-L1 adipocytes were treated with 0.05−0.4 mg/mL LIM for 10 days and oil red O and triglyceride (TG) content were used to determine the levels of lipid accumulation. The results showed that more than 0.05 mg/mL LIM inhibited lipid accumulation by reducing oil red O in 3T3-L1 adipocytes. Masses of 0.2 and 0.4 mg/mL LIM also decreased the TG contents in 3T3-L1 adipocytes. On the other hand, Wistar rats were given high-calorie diets, combined with LLIM (154 mg/kg) and HLIM (1000 mg/kg) treatments, for 16 weeks. The result shows that LLIM and HLIM decreased body weight, total fat tissue weight, and serum low-density lipoprotein-cholesterol (LDLc) levels. HLIM reduced serum TG and increased serum lipase and high-density lipoprotein-cholesterol (HDLc) levels. Moreover, the anti-obesity metabolic pathway showed that LIM (>0.05 mg/mL) in 3T3-L1 adipocytes and LIM (>154 mg/kg) in high-calorie diet-induced obese rats could activate the AMPK signaling pathway. The activated AMPK regulated the mRNA expression related to adipogenesis (PPARγ, C/EBPα, FABP4), lipogenesis (SREBP-1c, ACC, FAS), and lipolysis (ATGL, HSL) to inhibit obesity. This finding demonstrates that LIM has anti-obesity properties. Namely, it is seen that LIM acts by regulating the AMPK signaling pathway in 3T3-L1 adipocytes and high-calorie diet-induced obese rats. In terms of dose−response, LIM (154 mg/kg) would be an optimal effective dose for anti-obesity induced by a high-calorie diet.

The activity comparison of six dietary flavonoids identifies that luteolin inhibits 3T3-L1 adipocyte differentiation through reducing ROS generation.

Mitochondrial reactive oxygen species (ROS)generation plays an essential role in the process of adipocyte differentiation and is involved in the development of obesity and associated metabolic diseases. Various dietary flavonoids possess the substantial anti-adipogenic activity. However, it is unclear whether these flavonoids inhibit adipocyte differentiation by reducing ROS generation. In this study, the effects of six common dietary flavonoids on adipocyte differentiation were assessed in 3T3-L1 cells. The flavonoids with the same backbone of 5,7-dihydroxylflavone, including flavones apigenin, chrysin, luteolin and flavonols kaempferol, myricetin, quercetin, dose-dependently inhibited 3T3-L1 adipocyte differentiation, suggesting an associated hierarchy of inhibitory capability: luteolin > quercetin > myricetin > apigenin/kaempferol > chrysin. Meanwhile, six flavonoids were found to inhibit adipogenic gene expression and the early stage of adipocyte differentiation. Among the tested flavonoids, luteolin significantly reduced both intracellular and mitochondrial ROS generation during adipocyte differentiation. Further, luteolin treatment depressed the elevation of H2O2 concentration in the early stage of 3T3-L1 differentiation and reversed the facilitated effects of exogenous H2O2 on 3T3-L1 adipocyte differentiation and ROS generation. Altogether, the activity comparison of six dietary flavonoids identifies that luteolin inhibits 3T3-L1 adipocyte differentiation through reducing ROS generation, elucidating a new mechanism underlying the anti-adipogenic actions of flavonoids.

Cyclophilin B, a molecule chaperone, promotes adipogenesis in 3T3­L1 preadipocytes via AKT/mTOR pathway.

Cyclophilin is known to act as a molecular chaperone in the endoplasmic reticulum. Recent studies have reported that the expression of cyclophilin B (CypB) is increased in ob/ob mice and its inhibitor suppresses adipocyte differentiation. However, the mechanism of action of CypB in adipocytes remains to be elucidated. The present study investigated the role of CypB in 3T3­L1 adipocyte differentiation. It showed that the expression level of CypB was increased during 3T3­L1 adipocyte differentiation by reverse transcription­quantitative PCR and western blotting analysis. CypB knockdown using short interfering RNA delayed cell cycle progression from the G1/S to G2/M phase through the mammalian target of rapamycin (mTOR) signaling pathway and inhibited the expression levels of adipogenic transcription factors including peroxisome proliferator­activated receptor Î³ (PPARγ) and CCAAT­enhancer binding protein (C/EBP)α. Additionally, the accumulation of lipid droplets was inhibited by CypB knockdown. Conversely, overexpression of CypB promoted cell cycle progression from the G1/S to G2/M phase by the mTOR signaling pathway and enhanced the expression levels of adipogenic transcription factors including PPARγ and C/EBPα. Finally, the present study showed that CypB downregulated the expression of CHOP, a well­known negative regulator of adipogenesis. Taken together, the data suggested that CypB might serve important physiological regulatory roles in 3T3­L1 adipocyte differentiation.

Taste 2 Receptor Is Involved in Differentiation of 3T3-L1 Preadipocytes.

Expression of taste 2 receptor (T2R) genes, also known as bitter taste receptor genes, has been reported in a variety of tissues. The white adipose tissue of mice has been shown to express Tas2r108, Tas2r126, Tas2r135, Tas2r137, and Tas2r143, but the function of T2Rs in adipocytes remains unclear. Here, we show that fasting and stimulation by bitter compounds both increased Tas2r expression in mouse white adipose tissue, and serum starvation and stimulation by bitter compounds both increased the expression of Tas2r genes in 3T3-L1 adipocytes, suggesting that T2Rs have functional roles in adipocytes. RNA sequencing analysis of 3T3-L1 adipocytes stimulated by epicatechin, the ligand of Tas2r126, suggested that this receptor may play a role in the differentiation of adipocytes. Overexpression of Tas2r126 in 3T3-L1 preadipocytes decreases fat accumulation after induction of differentiation and reduces the expression of adipogenic genes. Together, these results indicate that Tas2r126 may be involved in adipocyte differentiation.

Colchicine stimulates browning via antagonism of GABA receptor B and agonism of ß3-adrenergic receptor in 3T3-L1 white adipocytes.

Colchicine has been used for therapeutic purposes and has attracted considerable attention because of its association with tubulin and the inhibition of small tubular polymerization. Although several studies have examined the possible preventive role of colchicine in metabolic diseases, its role in adipocytes is largely unknown. This study examined the novel functional role of colchicine in adipocytes demonstrating that colchicine stimulates browning in cultured white adipocytes. Colchicine stimulates browning by increasing the brown- and beige fat-specific markers in 3T3-L1 white adipocytes. Interestingly, colchicine decreased the expression of the main lipolytic proteins (ATGL, p-HSL) while it activated Ces3, suggesting a possibility for supplying essential fatty acids for inducing thermogenesis. Molecular docking analysis showed that colchicine has a strong affinity against GABA-BR and ß3-AR, and its binding activity with GABA-BR (-26.52 kJ/mol) was stronger than ß3-AR (-20.71 kJ/mol). Mechanistic studies were conducted by treating the cells separately with agonists and antagonists of GABA-BR and ß3-AR to understand the molecular mechanism underlying the browning effect of colchicine. The results showed that colchicine stimulates browning via the antagonism of GABA-BR and the agonism of ß3-AR in 3T3-L1 white adipocytes. The colchicine-mediated activation of ß3-AR stimulated the PKA/p38 MAPK signaling pathway, where consequently ATF2 acted as a positive regulator, but AFT4 was a negative regulator for the induction of browning.

Ethyl Gallate Dual-Targeting PTPN6 and PPARγ Shows Anti-Diabetic and Anti-Obese Effects.

The emergence of the high correlation between type 2 diabetes and obesity with complicated conditions has led to the coinage of the term "diabesity". AMP-activated protein kinase (AMPK) activators and peroxisome proliferator-activated receptor (PPARγ) antagonists have shown therapeutic activity for diabesity, respectively. Hence, the discovery of compounds that activate AMPK as well as antagonize PPARγ may lead to the discovery of novel therapeutic agents for diabesity. In this study, the knockdown of PTPN6 activated AMPK and suppressed adipogenesis in 3T3-L1 cells. By screening a library of 1033 natural products against PTPN6, we found ethyl gallate to be the most selective inhibitor of PTPN6 (Ki = 3.4 µM). Subsequent assay identified ethyl gallate as the best PPARγ antagonist (IC50 = 5.4 µM) among the hit compounds inhibiting PTPN6. Ethyl gallate upregulated glucose uptake and downregulated adipogenesis in 3T3-L1 cells as anticipated. These results strongly suggest that ethyl gallate, which targets both PTPN6 and PPARγ, is a potent therapeutic candidate to combat diabesity.

BGM, a Newly Synthesised Boron Compound, Induces Apoptosis and Reduces Oxidative Stress by Inhibiting Lipogenesis in 3T3-L1 Adipocytes via PPARγ and CTRP3.

Obesity is a chronic disease associated with increased morbidity and mortality. The rapidly increasing prevalence of obesity makes it a global health problem, while treatment options remain limited. Given the potential of boron in the treatment of obesity, the aim of this study is to investigate the anti-adipogenic activity of the newly synthesised boron glycine monoester compound (BGM) using 3T3-L1 adipocytes by analysing lipid accumulation, CTRP3 and PPARy gene expression, oxidative stress and apoptotic effects. 3T3-L1 fibroblast cells (ATCC® CL-173) were transformed into adipocyte cells in vitro. Fat accumulation in the 3T3-L1 adipocyte cells was detected by Oil Red O staining. Gene expression levels were determined with qPCR. Biochemical analyzes were performed using spectrophotometric method (CAT, ALP and ACP) and ELISA kit (TAS, TOS, NADP-IDH). Apoptosis studies were performed on the muse cell nalyser using the Muse Annexin V & Dead Cell Assay Kit. When BGM-treated cells were compared to control adipocyte cells, lipid accumulation decreased in a dose-dependent manner. BGM-treated adipocyte cells had higher CTRP3 expression levels and lower PPAR-γ gene expression levels compared to control adipocyte cells (p < 0.001). While BGM application increased the TAS level, it showed an antioxidant effect by regulating the activity of oxidative metabolism enzymes (p < 0.001). BGM application increased total apoptosis by 1.5-fold. These results show that BGM is a potential therapeutic agent for obesity by regulating the expression of genes related to adipogenesis and lipogenesis in adipocyte cells and by affecting the activity of enzymes of oxidative metabolism and apoptosis.

Raspberry ketone promotes FNDC5 protein expression via HO-1 upregulation in 3T3-L1 adipocytes.

Obesity is a global health problem and a risk factor for cardiovascular diseases and cancers. Exercise is an effective intervention to combat obesity. Fibronectin type III domain containing protein 5 (FNDC5)/irisin, a myokine, can stimulate the browning of white adipose tissue by increasing uncoupling protein 1 (UCP1) expression, and therefore may represent a link between the beneficial effects of exercise and improvement in metabolic diseases. Thus, upregulating the endogenous expression of FNDC5/irisin by administering medication would be a good approach for treating obesity. Herein, we evaluated the efficacy of raspberry ketone (RK) in inducing FNDC5/irisin expression and the underlying mechanisms. The expression of brown fat-specific proteins (PR domain containing 16 (PRDM16), CD137, and UCP1), heme oxygenase-1 (HO-1), FNDC5, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) in differentiated 3T3-L1 adipocyte was analyzed by western blotting or immunofluorescence. The level of irisin in the culture medium was also assayed using an enzyme-linked immunosorbent assay kit. Results showed that RK (50 µM) significantly induced the upregulation of FNDC5 protein in differentiated 3T3-L1 adipocytes; however, the irisin level in the culture media was unaffected. Moreover, RK significantly increased the levels of PGC1α, brown adipocyte markers (PRDM16, CD137, and UCP1), and HO-1. Furthermore, the upregulation of PGC1α and FNDC5 and the browning effect induced by RK were significantly reduced by SnPP or FNDC5 siRNA, respectively. In conclusion, RK can induce FNDC5 protein expression via the HO-1 signaling pathway, and this study provides new evidence for the potential use of RK in the treatment of obesity.

Chebulinic Acid Suppresses Adipogenesis in 3T3-L1 Preadipocytes by Inhibiting PPP1CB Activity.

Depletion of protein phosphatase-1 catalytic subunit beta (PPP1CB), a serine/threonine protein phosphatase and potent adipogenic activator, suppresses the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Therefore, PPP1CB is considered as a potential therapeutic target for obesity. We screened 1033 natural products for PPP1CB inhibitors and identified chebulinic acid, which is abundantly present in the seeds of Euphoria longana and fruits of Terminalia chebula. Chebulinic acid strongly inhibited the hydrolysis of 6,8-difluoro-4-methylumbelliferyl phosphate by PPP1CB (IC50 = 300 nM) and demonstrated potent antiadipogenic effects in 3T3-L1 preadipocytes in a concentration-dependent manner. Additional studies have demonstrated that chebulinic acid suppresses early differentiation by downregulating key transcription factors that control adipogenesis in 3T3-L1 cells. These results suggested that chebulinic acid may be a potential therapeutic agent for treating obesity by inhibiting PPP1CB activity.

Two new nor-lignans from Selaginella pulvinata (Hook. & Grev.) Maxim and their antihyperglycemic activities.

Two new nor-lignans, pulvin A (1) and moellenoside C (2), along with two known compounds (3-4) were isolated from the whole plant of Selaginella pulvinate (Hook. & Grev.) Maxim. The structures of the new compounds were established on the basis of spectroscopic data and acid hydrolysis. All the isolates were investigated for their antihyperglycemic activities in 3T3-L1 adipocytes. The results showed that compounds 1 and 2 promoted the glucose consumption prominently in 3T3-L1 adipocytes in a dose-response manner. Compound 1 and 2 induced 1.14-1.73 folds and 1.03-1.55 folds changes relative to the basal level, respectively, in the concentration range of 12.5 µM to 50 µM.

Anti-adipogenic and anti-oxidant effects of a standardised extract of Moro blood oranges (Citrus sinensis (L.) Osbeck) during adipocyte differentiation of 3T3-L1 preadipocytes.

Particular features in the pulp of blood oranges are marked levels of anthocyanin, a class of polyphenolic compounds well known to exert numerous health-promoting actions on human wellbeing including anti-obesity effects. In this study, we investigated in vitro, the antioxidant and anti-adipogenic activities of Morosil®, a standardised extract of Moro blood oranges. During adipocyte differentiation, 3T3-L1 cells were treated with concentrations of extract containing 2.5, 5, 10, 25 µM of anthocyanins. After seven days of treatment and differentiation, we measured reactive oxygen species production, non-proteic thiol groups content, adipokine secretion and triglyceride accumulation together with mRNA expression of adipogenic transcription factors such as PPARγ, C/EBPα, SREBP-1c. Furthermore, both ACCα, FAS protein expression and citrate synthase activity were measured. Results show that Morosil® exerts antioxidant and anti-adipogenic activities during adipocyte differentiation of 3T3-L1 pre-adipocytes.

Enhancement of the Antiobesity and Antioxidant Effect of Purple Sweet Potato Extracts and Enhancement of the Effects by Fermentation.

The browning of white adipocytes, which transforms energy-storing white adipocytes to heat-producing beige adipocytes, is considered a strategy against metabolic diseases. Several dietary compounds, such as anthocyanins, flavonoids, and phenolic acids, induce a brown adipocyte-like phenotype in white adipocytes. In this study, we demonstrated that purple sweet potato (Ipomoea batatas) extract (PSP) exhibited potent radical scavenging activity. In addition, PSP was found to contain large amounts of phenolic, flavonoid, and anthocyanin compounds; the amount of these compounds was affected by fermentation. Functionally, PSP-induced adipose browning in high-fat-diet (HFD)-induced obese mice. The administration of PSP significantly suppressed the body weight gain and abnormal expansion of white adipose tissues in the obese mice. The expression of adipose browning-related genes was higher in the inguinal white adipose tissues from the PSP-treated mice than those in the HFD-fed mice. Moreover, PSP-treated 3T3-L1 adipocytes formed multilocular lipid droplets, similar to those formed in the 3T3-L1 adipocytes treated with a browning induction cocktail. The PSP-treated cells had an increased expression level of mitochondria and lipolysis-related genes. The browning effects of PSP were enhanced by fermentation with Lactobacillus. This study, to our knowledge, is the first to identify a new mechanism to increase the antiobesity effects of PSP by inducing adipocyte browning of adipocytes.

Ferulic Acid Stimulates Adipocyte-Specific Secretory Proteins to Regulate Adipose Homeostasis in 3T3-L1 Adipocytes.

Obesity has recently emerged as a public health issue facing developing countries in the world. It is caused by the accumulation of fat in adipose, characterized by insulin resistance, excessive lipid accumulation, inflammation, and oxidative stress, leading to an increase in adipokine levels. Herein, we investigated the capacity of a bioactive polyphenolic compound (ferulic acid (FA)) to control adipocyte dysfunction in 3T3-L1 adipocytes (in vitro). Key adipocyte differentiation markers, glycerol content, lipolysis-associated mRNA, and proteins were measured in experimental adipocytes. FA-treated adipocytes exhibited downregulated key adipocyte differentiation factors peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAT enhancer binding-proteins-α (C/EBP-α) and its downstream targets in a time-dependent manner. The FA-treated 3T3-L1 adipocytes showed an increased release of glycerol content compared with non-treated adipocytes. Also, FA treatment significantly up-regulated the lipolysis-related factors, including p-HSL, and p-perilipin, and down-regulated ApoD, Sema3C, Cxcl12, Sfrp2, p-stearoyl-CoA desaturase 1 (SCD1), adiponectin, and Grk5. Also, the FA treatment showed significantly down-regulated adipokines leptin, chemerin, and irisin than the non-treated cells. The present findings indicated that FA showed significant anti-adipogenic and lipogenic activities by regulating key adipocyte factors and enzyme, enhanced lipolysis by HSL/perilipin cascade. FA is considered a potent molecule to prevent obesity and its associated metabolic changes in the future.

Alisol A 24-acetate stimulates lipolysis in 3 T3-L1 adipocytes.

BACKGROUND: Alisol A 24-acetate (AA-24-a), one of the main active triterpenes isolated from the well-known medicinal plant Alisma orientale (Sam.) Juz., exhibits multiple biological activities including hypolipidemic activity. However, its effect on lipid metabolism in adipocytes remains unclear. The present study aimed to clarify the effect of AA-24-a on adipocyte lipolysis and to determine its potential mechanism of action using 3 T3-L1 cells. METHODS: We assayed the release of glycerol into culture medium of 3 T3-L1 cells under treatment with AA-24-a. Protein and mRNA expression and phosphorylation levels of the main lipases and kinases involved in lipolysis regulation were determined by quantitative polymerase chain reaction and western blotting. Specific inhibitors of protein kinase A (PKA; H89) and extracellular signal-regulated kinase (ERK; PD98059), which are key enzymes in relevant signaling pathways, were used to examine their roles in AA-24-a-stimulated lipolysis. RESULTS: AA-24-a significantly stimulated neutral lipolysis in fully differentiated adipocytes. To determine the underlying mechanism, we assessed the changes in mRNA and protein levels of key lipolysis-related genes in the presence or absence of H89 and PD98059. Both inhibitors reduced AA-24-a-induced lipolysis. Moreover, pretreatment with H89 attenuated AA-24-a-induced phosphorylation of hormone-sensitive lipase at Ser660, while pretreatment with PD98059 attenuated AA-24-a-induced downregulation of peroxisome proliferator-activated receptor-γ and perilipin A. CONCLUSIONS: Our results indicate that AA-24-a promoted neutral lipolysis in 3 T3-L1 adipocytes by activating PKA-mediated phosphorylation of hormone-sensitive lipase and ERK- mediated downregulation of expression of perilipin A.

Antidiabetic Flavonoids from Fruits of Morus alba Promoting Insulin-Stimulated Glucose Uptake via Akt and AMP-Activated Protein Kinase Activation in 3T3-L1 Adipocytes.

Morus alba (Moraceae), known as white mulberry, has been used to treat fever, protect against liver damage, improve eyesight, and lower blood sugar levels in traditional oriental medicine. Few studies have been conducted on the antidiabetic compounds identified from M. alba and their underlying mechanisms of action. Consequently, in this study, the fruits of M. alba were investigated for potential antidiabetic natural products using 3T3-L1 adipocytes. Phytochemical analysis of the ethanolic extract of M. alba fruits, followed by high-performance liquid chromatography (HPLC), purification led to the isolation of two main compounds: rutin and quercetin-3-O-ß-d-glucoside (Q3G). Long-term use of available drugs for treating type 2 diabetes ((T2D) is often accompanied by undesirable side effects, which have generated increased interest in the development of more effective and safer antidiabetic agents. Examination of the isolated compounds, rutin and Q3G, for antidiabetic or anti-obesity properties or both in 3T3-L1 adipocytes demonstrated that they both improved glucose uptake via Akt-mediated insulin signaling pathway or AMP-activated protein kinase (AMPK) activation in 3T3-L1 adipocytes. The compounds also showed a positive effect on lipid accumulation in adipocytes, suggesting that glucose uptake occurred through activation of the Akt and AMPK signaling pathway without inducing adipogenesis. Taken together, our findings suggest that rutin and Q3G in M. alba fruits have the potential to induce fewer side effects such as weight gain, and these active compounds could be potential therapeutic candidates for the management of T2D.

A Transcriptomic Analysis Reveals Novel Patterns of Gene Expression During 3T3-L1 Adipocyte Differentiation.

BACKGROUND: Obesity is characterized by increased adipose tissue mass that results from increased fat cell size (hypertrophy) and number (hyperplasia). The molecular mechanisms that govern the regulation and differentiation of adipocytes play a critical role for better understanding of the pathological mechanism of obesity. However, the mechanism of adipocyte differentiation is still unclear. OBJECTIVE: The present study aims to compare the gene expression changes during adipocyte differentiation in the transcriptomic level, which may help to better understand the mechanism of adipocyte differentiation. METHODS: RNA sequencing (RNA-seq) technology, GO and KEGG analysis, quantitative RT-PCR, and oil red O staining methods were used in this study. RESULTS: A lot of genes were up- or down-regulated between each two differentiation stages of 3T3-L1 cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that lipid metabolism and oxidation-reduction reaction were mainly involved in the whole process of adipocyte differentiation. Decreased immune response and cell cycle adhesion occurred in the late phase of adipocyte differentiation, which was demonstrated by divergent expression pattern analysis. Moreover, quantitative RT-PCR results showed that the mRNA expression levels of Trpv4, Trpm4, Trpm5, and Trpm7 were significantly decreased in the differentiated adipocytes. On the other hand, the mRNA expression levels of Trpv1, Trpv2, Trpv6, and Trpc1 were significantly increased in the differentiated adipocytes. Besides, the mRNA expressions of TRPV2 and TRPM7 were also significantly increased in subcutaneous white adipose tissue from diet-induced mice. In addition, the activation of TRPM7, TRPV1, and TRPV2 suppressed the differentiation of adipocytes. CONCLUSION: These data present the description of transcription profile changes during adipocyte differentiation and provides an in-depth analysis of the possible mechanisms of adipocyte differentiation. These data offer new insight into the understanding of the mechanisms of adipocyte differentiation.

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine increases insulin sensitivity in palmitate-treated myotubes and induces lipolysis in adipocytes.

Obesity causes the development of insulin resistance and type 2 diabetes. Phosphatidylcholine (PPC) has been reported to increase hepatic insulin sensitivity and lipolysis in adipose tissue to resolve local obesity. In this study, we proposed 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), the main active species of PPC, as an effective substance for the treatment of obesity-mediated disorders such as impaired fat metabolism and insulin resistance. Therefore, we investigated the potential lipolytic effects of DLPC on adipocytes and insulin signaling in muscle cells. In this study, DLPC-treated 3T3-L1 adipocytes showed enhanced tumor necrosis factor α (TNF-α) release. Suppression of TNF-α by short interfering RNA (siRNA) mitigated DLPC-induced lipolysis and apoptosis. DLPC treatment increased peroxisome proliferator-activated receptor α (PPARα) expression levels in C2C12 myocytes. siRNA-mediated suppression of PPARα abrogated the suppressive effects of DLPC on palmitate-induced inflammation and insulin resistance. In conclusion, DLPC enhanced lipolysis and apoptosis via a TNFα-dependent pathway in adipocytes and attenuated palmitate-induced insulin resistance through PPARα-mediated suppression of inflammation in myocytes.