Differentiation Process Of Preadipocytes Research Articles

Membrane Protein Amuc_1100 Derived from Akkermansia muciniphila Facilitates Lipolysis and Browning via Activating the AC3/PKA/HSL Pathway.

Obesity, defined as a disorder of lipid metabolism caused by white fat accumulation, is closely related to the gut microbiota. Akkermansia muciniphila (Akk), one of the most common gut commensals, can reduce fat storage and promote the browning of white adipocytes, alleviating disorders of lipid metabolism. However, which components of Akk produce the effect remain unclear, limiting the application of Akk in the treatment of obesity. Here, we found that the membrane protein Amuc_1100 of Akk decreased formation of lipid droplets and fat accumulation during the differentiation process and stimulated browning in vivo and in vitro. Transcriptomics revealed that Amuc_1100 accelerated lipolysis through upregulation of the AC3/PKA/HSL pathway in 3T3-L1 preadipocytes. Quantitative PCR (qPCR) and Western blotting showed that Amuc_1100 intervention promotes steatolysis and browning of preadipocytes by increasing lipolysis-related genes (AC3/PKA/HSL) and brown adipocyte marker genes (PPARγ, UCP1, and PGC1α) at both the mRNA and protein levels. These findings introduce new insight into the effects of beneficial bacteria and provide new avenues for the treatment of obesity. IMPORTANCE An important intestinal bacterial strain Akkermansia muciniphila contributes to improving carbohydrate and lipid metabolism, thus alleviating obesity symptoms. Here, we find that the Akk membrane protein Amuc_1100 regulates lipid metabolism in 3T3-L1 preadipocytes. Amuc_1100 inhibits lipid adipogenesis and accumulation during the differentiation process of preadipocytes, upregulates the browning-related genes of preadipocytes, and promotes thermogenesis through activation of uncoupling protein-1 (UCP-1), including Acox1 involved in lipid oxidation. Amuc_1100 accelerates lipolysis via the AC3/PKA/HSL pathway, phosphorylating HSL at Ser 660. The experiments illustrated here identify the specific molecules and functional mechanisms of Akk. Therapeutic approaches with Amuc_1100 derived from Akk may help alleviate obesity and metabolic disorders.

Anti-Obesity Activities of Standardized Ecklonia stolonifera Extract in 3T3-L1 Preadipocytes and High-Fat-Diet-Fed ICR Mice

The purpose of this study was to prepare a clinical trial test material (ESETM, test material of Ecklonia stolonifera extract) to develop a health functional food based on its anti-obesity effect. The anti-obesity effect of ESETM was evaluated in 3T3-L1 adipocytes and obese mice fed a high-fat diet (HFD) to confirm its nonclinical trial effect before application in clinical trial. Adipogenesis is a process of preadipocyte differentiation that causes an increase in the production of reactive oxygen species (ROS) and lipid accumulation. In vitro study results indicated that ESETM outstandingly inhibits the production of ROS and lipid accumulation during adipogenesis and lipogenesis. In vivo, ESETM-treated ICR mice had reduced HFD-induced weight change, food efficiency ratio, adipose tissue weight, liver weight and showed improved serum lipid profiles. Our results show that ESETM inhibits weight change by regulating the adipogenesis, lipogenesis, lipolysis, and thermogenesis pathways.

The Role of MicroRNAs in Muscle Tissue Development in Beef Cattle.

In this review, we highlight information on microRNA (miRNA) identification and functional characterization in the beef for muscle and carcass composition traits, with an emphasis on Qinchuan beef cattle, and discuss the current challenges and future directions for the use of miRNA as a biomarker in cattle for breeding programs to improve meat quality and carcass traits. MicroRNAs are endogenous and non-coding RNA that have the function of making post-transcriptional modifications during the process of preadipocyte differentiation in mammals. Many studies claim that diverse miRNAs have an impact on adipogenesis. Furthermore, their target genes are associated with every phase of adipocyte differentiation. It has been confirmed that, during adipogenesis, several miRNAs are differentially expressed, including miR-204, miR-224, and miR-33. The development of mammalian skeletal muscle is sequentially controlled by somite commitment into progenitor cells, followed by their fusion and migration, the proliferation of myoblasts, and final modification into fast- and slow-twitch muscle fibers. It has been reported that miRNA in the bovine MEG3-DIO3 locus has a regulatory function for myoblast differentiation. Likewise, miR-224 has been associated with controlling the differentiation of bovine adipocytes by targeting lipoprotein lipase. Through the posttranscriptional downregulation of KLF6, miR-148a-3p disrupts the proliferation of bovine myoblasts and stimulates apoptosis while the miR-23a~27a~24-2 cluster represses adipogenesis. Additional to influences on muscle and fat, bta-mir-182, bta-mir-183, and bta-mir-338 represent regulators of proteolysis in muscle, which influences meat tenderness.

Placental extract suppresses differentiation of 3T3-L1 preadipocytes to mature adipocytes via accelerated activation of p38 MAPK during the early phase of adipogenesis

BackgroundAdipogenesis, the process of preadipocyte differentiation to mature adipocytes accompanied by accumulation of intracytoplasmic lipid droplets, is regulated by various genetic and environmental factors, and closely associated with the development of obesity. Numerous recent studies suggest that some bioactive peptides and proteins derived from animals, and chemical compounds isolated from plants may be useful for prevention and treatment of obesity and obesity-related chronic diseases. In the present study, we examined the broad spectrum of effects of placental extract, with a focus on the influence of placental extract on adipogenesis.MethodWe cultured 3T3-L1 cells, which are widely used as a model of white preadipocytes, under differentiation conditions in the presence of porcine placental extract (PPE) for 8 days, and then stained the lipid droplets accumulated in the cytoplasm with Oil Red O. We also analyzed the effects of PPE on the mitogen-activated protein kinases (MAPKs) signaling, mitotic clonal expansion (MCE) and gene expressions associated with 3T3-L1 differentiation.ResultsWhen we cultured 3T3-L1 cells with PPE under differentiation conditions, the accumulation of lipid droplets and expression of adipocyte differentiation marker genes (Cebpa, Pparg, Slc2a4, Fasn and Adipoq) were dramatically attenuated. The suppressive activity of PPE against adipogenesis was heat-stable and recovered in a low-molecular-weight fraction after ultrafiltration (< 3 kDa) and gel-filtration chromatography (fraction No. 9). We also found that the suppressive activity of PPE affected the early phase of adipocyte differentiation (Days 0–2) without influencing the expression levels of C/EBPβ and C/EBPδ. The PPE and fraction No. 9 obtained from gel-filtration chromatography both promoted mitotic clonal expansion of 3T3-L1 while accelerating p38 MAPK phosphorylation. In addition, SB203580, a p38 MAPK inhibitor, partially restored the accumulation of lipid droplets and the expression of adipocyte differentiation marker genes that were suppressed by fraction No. 9.ConclusionThese results indicate that PPE suppresses the differentiation of preadipocytes via accelerated activation of p38 MAPK during the early phase of adipogenesis, suggesting PPE or its functional component could be a potential therapy for treating obesity.

MiR-128-3p regulates 3T3-L1 adipogenesis and lipolysis by targeting Pparg and Sertad2.

Differentiation of adipocytes and their aggregation to adipose tissue are critical for mammalian growth and development. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play important roles in adipogenesis and lipid metabolism. miR-128-3p may contribute to adipose tissue development according to the previous studies. However, the role of miR-128-3p in the process of preadipocyte differentiation and lipid metabolism is not yet understood. The purpose of this research was to investigate the biological function and molecular mechanism of miR-128-3p in 3T3-L1 cells. In the present study, we found that miR-128-3p was downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-128-3p obstructed the expressions of adipogenic marker genes as well as the lipid droplets accumulation and triglyceride content, suggesting the importance of miR-128-3p for adipogenesis. Moreover, miR-128-3p could lead to the retardation of cell proliferation in 3T3-L1 preadipocytes. Further evidences showed that, as a negative regulator of adipogenesis, miR-128-3p could directly target peroxisome proliferator-activated receptor γ (Pparg) which resulted in the suppression of 3T3-L1 preadipocyte differentiation, and miR-128-3p could also bind with SERTA domain containing 2 (Sertad2) which drove triglyceride hydrolysis and lipolysis. In addition, inhibition of Sertad2 with siRNA displayed the same effects as overexpression of miR-128-3p. Our research demonstrated that miR-128-3p impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis. Taken together, this study offers profound insight into the mechanism of miRNA-mediated adipogenesis and lipid metabolism.

Ectopic overexpression of MCPIP1 impairs adipogenesis by modulating microRNAs

Adipogenesis is a process of preadipocyte differentiation that requires action of numerous factors. Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) possesses the N-terminus of the PilT protein (PilT N-terminus or PIN domain) that has RNase properties. This protein degrades transcripts coding for inflammation and differentiation – related proteins. Moreover, MCPIP1 is a broad suppressor of the miRNA biogenesis. We previously found that MCPIP1 degrades transcript encoding CCAAT/Enhancer Binding Protein Beta (C/EBPβ) and influences adipogenesis. Subsequently, we aimed to determine adipocyte miRNA expression profile in differentiating mouse preadipocytes, 3T3-L1, by overexpressing MCPIP1. Using Next-Generation Sequencing (NSG) we showed that MCPIP1 overexpression results in modulated levels of 58 miRNAs in adipocytes on day 2 of differentiation. Among them, 30 miRNAs showed significantly reduced levels and 28 showed increased levels in comparison to control. Approximately one third of the modulated miRNAs were not previously reported to be involved in adipocytes differentiation. Our analysis revealed that 24 down-regulated and 23 up-regulated miRNAs (at least 1.5-fold) influence 19 signaling pathways that are important for adipogenesis. Furthermore, reduced miRNA levels result in the up-regulation of their targets. By using luciferase reporter assay, we demonstrated that miR-32-5p and miR-9-3p directly target the 3′UTR region of Mapk8 and Tiam1, respectively. In addition, activation of MAP kinases pathway (JNK and p38), proposed as being regulated by down-regulated miRNAs, was higher in WTMCPIP1 than in D141NMCPIP1 or control 3T3-L1 adipocytes. Our results indicate a considerable impact of MCPIP1 on miRNAs levels and its significance in adipogenesis.

Effect of N-(D-Ribopyranosyl) Taurine Sodium Salt on the Differentiation of Human Preadipocytes and Expression of Adipokines Through Inhibition of STAT-3 Signaling in Differentiated Human Adipocytes.

We investigated whether a taurine-ribose derivative, N-(D-ribopyranosyl)taurine sodium salt, inhibits the differentiation process of preadipocytes or modulates the expression of cytokines from adipocytes as does taurine chloramine (TauCl) in vitro. To know the inhibitory effects of taurine-ribose (Tau-Ribose) on differentiation process and adipokine expression, preadipocytes were incubated with Tau-Ribose in differentiation medium for 14 days. Differentiated adipocytes were also stimulated at the concentration of IL-1β 1 ng/ml with addition of Tau-Ribose. After 7 days of incubation, the levels of adiponectin, leptin, IL-6, and IL-8 were measured from the culture supernatants. At concentrations of 10-40 mM, Tau-Ribose dose-dependently inhibited the process of adipogenesis. The treatment of Tau-Ribose decreased the expression of transcription factors, which are necessary for adipogenesis and are known as adipocyte marker. Treatment with Tau-Ribose significantly modulated the production of IL-8 and IL-6. However, it did not modulate the production of adiponectin and leptin in IL-1β-activated adipocytes. As with taurine chloramine, Tau-Ribose also inhibited STAT-3 signaling, independent of MAPK signaling. In conclusion, Tau-Ribose inhibits the signaling pathway of STAT-3 and can change adipokines production; thus, it may have a potential as an agent for treating obesity-related diseases.

Effects of Physical Loading on Adipogenic Differentiation in 3T3-L1 Preadipocytes

Adipose tissue is subjected to various types of physical loading. Previous studies show that adipocytes have mechanosensitive properties and are affected by mechanical stimulation. In this study, we investigated the effects of fluid shear stress on adipogenic differentiation using 3T3-L1 preadipocytes. The study included 3 groups: (1) No Loading, (2) Loading-Immediate harvest, and (3) Loading-Post 2 day harvest. In the loading groups, dynamic fluid shear stress of maximum 1 Pa at 1 Hz was applied to the preadipocytes for 30 mins. GAPDH, ERK, p-ERK, C/EBPβ, PPARγ proteins were assessed by Western blot analysis. Loading resulted in increased p-ERK and reduced C/EBPβ expressions compared to the No Loading group. These changes were more significant when the cells were harvested post 2 day loading. However, loading did not result in significant changes in PPARγ expression. C/EBPβ plays a major role in promoting the differentiation process of preadipocytes and also induces PPARγ which shows enhanced expressions in the terminal differentiation stage of adipocytes. These results indicate that fluid shear stress may suppress adipogenic differentiation through C/EBPβ inhibition. Greater decrease in C/EBPβ when cells were harvested post 2 day loading suggests that changes in C/EBPβ are not immediate with loading and therefore longer duration may be necessary to observe changes in PPARγ expression. Further experiments with increased harvest time as well as experiments using 3T3-L1 preadipocytes of more advanced differentiation stage are underway.

Coordinate Functional Regulation between Microsomal Prostaglandin E Synthase-1 (mPGES-1) and Peroxisome Proliferator-activated Receptor γ (PPARγ) in the Conversion of White-to-brown Adipocytes

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor and a master regulator of adipogenesis. Microsomal prostaglandin E (PGE) synthase-1 (mPGES-1) is an inducible enzyme that couples with cyclooxygenase-2 for the biosynthesis of PGE2. In this study we demonstrate the existence of a coordinate functional interaction between PPARγ and mPGES-1 in controlling the process of pre-adipocyte differentiation in white adipose tissue (WAT). Adipocyte-specific PPARγ knock-out mice carrying an aP2 promoter-driven Cre recombinase transgene showed a blunted response to the adipogenic effects of a high fat diet. Pre-adipocytes from these knock-out mice showed loss of PPARγ and were resistant to rosiglitazone-induced WAT differentiation. In parallel, WAT from these mice showed increased expression of uncoupling protein 1, a mitochondrial enzyme that dissipates chemical energy as heat. Adipose tissue from mice lacking PPARγ also showed mPGES-1 up-regulation and increased PGE2 levels. In turn, PGE2 suppressed PPARγ expression and blocked rosiglitazone-induced pre-adipocyte differentiation toward white adipocytes while directly elevating uncoupling protein 1 expression and pre-adipocyte differentiation into mature beige/brite adipocytes. Consistently, pharmacological mPGES-1 inhibition directed pre-adipocyte differentiation toward white adipocytes while suppressing differentiation into beige/brite adipocytes. This browning effect was reproduced in knockdown experiments using a siRNA directed against mPGES-1. The effects of PGE2 on pre-adipocyte differentiation were not seen in mice lacking PPARγ in adipose tissue and were not mirrored by other eicosanoids (i.e. leukotriene B4). Taken together, these findings identify PGE2 as a key regulator of white-to-brown adipogenesis and suggest the existence of a coordinate regulation of adipogenesis between PPARγ and mPGES-1.

Interaction of the Glucocorticoid and Androgen Receptors in Adipogenesis

Glucocorticoids and androgens are important regulators of adipose tissue function. A new study by Hartig et al. in this issue of Chemistry & Biology provides relevant information regarding androgen receptor activity and its link to glucocorticoid action in human adipocytes during the process of preadipocyte differentiation.

Stimulatory and Inhibitory regulation of lipolysis by the NPR-A/cGMP/PKG and NPR-C/G i pathways in rat cultured adipocytes

Objective Recent studies have suggested the abundant expression of natriuretic peptide receptor in adipose tissue. This study was designed to investigate the levels of natriuretic receptor-A (NPR-A) and NPR-C gene expression during the process of preadipocyte differentiation and its role in adipogenesis and lipid metabolism. Methods We measured mRNA levels of NPR-A and NPR-C during the process of rat preadipocyte differentiation in vitro. We also measured the effects of ANP and C-ANP, a ligand for NPR-C, on preadipocyte differentiation. In addition, we assessed the effects of ANP and C-ANP on lipolysis and the cellular mechanism. Results The mRNA levels of NPR-A and NPR-C on day 3, 6, 10 are (− 26%, + 226%), (+ 6%, + 568%), and (+ 207%, + 3232%) respectively as compared with day 1. ANP (10 − 7 M) and 8-bromo-cGMP (10 − 4 M) significantly increased Oil Red positive area and cell number of matured-adipocytes. ANP and 8-bromo-cGMP also increased the mRNA levels of adipocyte-related genes such as PPARγ, leptin, and adiponectin on day 3, whereas C-ANP did not change these parameters. ANP (10 − 9–10 − 6 M) increased intracellular cGMP levels and promoted lipolysis in adipocytes and the effects were abolished by HS-142-1, and KT5823. Conversely C-ANP (10 − 6 M) decreased intracellular cAMP levels and lipolysis and its effect was inhibited by PTX. Conclusion Results suggest that ANP may promote adipocyte differentiation and lipolysis via the NPR-A/cGMP/PKG pathway. Direct action of ANP via NPR-C in adipogenesis may be either absent or barely present, but ANP may play a counter regulatory role in lipolysis via NPR-C/Gi pathway.

Role of adrenomedullin system in lipid metabolism and its signaling mechanism in cultured adipocytes

We investigated the levels of adrenomedullin (AM) system during the process of preadipocyte differentiation and its role in lipid metabolism and cellular signaling mechanism in differentiated adipocytes. We cultured rat preadipocytes and measured the following during the process of differentiation: two molecular forms of AM in the culture medium using a specific immunoradiometric assay and gene expression of AM and its receptor component using RT-PCR analysis. In differentiated adipocytes, we measured the effects of AM on the intracellular cAMP level, lipolysis, glucose incorporation, and the protein levels. Two molecular forms of AM were secreted into the medium, and the AM-mature/AM-total ratio was increased after 6 days of differentiation. Cultured rat preadipocytes highly expressed the genes of AM and its receptor components at day 1, and they increased at day 10. Administration of AM to preadipocytes increased the number of Oil Red O-positive adipocytes and spectrophotometric absorbance of Oil Red O. AM dose dependently increased cAMP level and lipolysis, and its effect was blocked by CGRP(8-37). Isoproterenol increased lipolysis, and AM had additive effects on isoproterenol-induced lipolysis. KT5720 and U0126 significantly inhibited the AM-induced lipolysis, whereas KT5720, but not U0126, significantly inhibited the isoproterenol-induced lipolysis. AM increased glucose incorporation and its effect was blocked by wortmannin. Western blot analysis revealed that AM increased phospho PKA, ERK, and Akt. These results indicate that AM and its receptor component are highly expressed in cultured adipocytes and may play a role in lipid metabolism via a different signaling pathway.

Concomitant expression of adrenomedullin and its receptor components in rat adipose tissues.

Adrenomedullin (AM) expressed by and secreted from a variety of cells plays pluripotent roles in an autocrine/paracrine fashion. The present study was undertaken to explore the expression of AM and its receptor genes in adipose tissues, their changes during the development of obesity, and the process of preadipocyte differentiation. Both mature adipocytes and stromal vascular cells constituting adipose tissue expressed AM transcript. AM and its receptor component [calcitonin receptor-like receptor and receptor activity-modifying protein-2 (CRLR/RAMP2)] mRNAs were expressed in a variety of rat adipose tissues, including epididymal, mesenteric, retroperitoneal, and subcutaneous adipose tissue. AM mRNA levels in rat and human epididymal adipose tissue were about one-tenth of those in the kidney. Steady-state mRNA levels of AM and CRLR/RAMP2 in epididymal, mesenteric, and retroperitoneal adipose tissues in rats fed a high-fat diet for 4 wk were far greater than those in rats with normal diet accompanied by increased plasma AM levels, whereas steady-state AM mRNA levels conversely decreased in other organs, such as kidney and liver. AM mRNA expressed in a mouse preadipocyte cell line (3T3-L1) transiently decreased by day 3, returned to basal level by day 6, and then increased by day 9 during preadipocyte differentiation, which paralleled AM secretion from the cells. However, the addition of either exogenous AM or AM receptor antagonist calcitonin gene-related peptide-(8-37), to block endogenous AM did not affect lipid droplet accumulation during preadipocyte differentiation. The present study demonstrates for the first time that AM and its receptor component (CRLR/RAMP2) mRNAs were concomitantly expressed in various adipose tissues, whose tissue-specific upregulation was induced during the development of obesity. These data suggest that AM may act as a new member of adipokines, although its functional role, as well as its pathophysiological significance in obesity, remains to be determined.