The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode.

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to WT mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Laminin fragments conjugated with perlecan's growth factor-binding domain differentiate human induced pluripotent stem cells into skin-derived precursor cells.

Deriving stem cells to regenerate full-thickness human skin is important for treating skin disorders without invasive surgical procedures. Our previous protocol to differentiate human induced pluripotent stem cells (iPSCs) into skin-derived precursor cells (SKPs) as a source of dermal stem cells employs mouse fibroblasts as feeder cells and is therefore unsuitable for clinical use. Herein, we report a feeder-free method for differentiating iPSCs into SKPs by customising culture substrates. We immunohistochemically screened for laminins expressed in dermal papillae (DP) and explored the conditions for inducing the differentiation of iPSCs into SKPs on recombinant laminin E8 (LM-E8) fragments with or without conjugation to domain I of perlecan (PDI), which binds to growth factors through heparan sulphate chains. Several LM-E8 fragments, including those of LM111, 121, 332, 421, 511, and 521, supported iPSC differentiation into SKPs without PDI conjugation. However, the SKP yield was significantly enhanced on PDI-conjugated LM-E8 fragments. SKPs induced on PDI-conjugated LM111-E8 fragments retained the gene expression patterns characteristic of SKPs, as well as the ability to differentiate into adipocytes, osteocytes, and Schwann cells. Thus, PDI-conjugated LM-E8 fragments are promising agents for inducing iPSC differentiation into SKPs in clinical settings.

Senescent adipocytes as potential effectors of muscle cells dysfunction: An in vitro model.

Recently, there has been a growing body of evidence showing a negative effect of the white adipose tissue (WAT) dysfunction on the skeletal muscle function and quality. However, little is known about the effects of senescent adipocytes on muscle cells. Therefore, to explore potential mechanisms involved in age-related loss of muscle mass and function, we performed an in vitro experiment using conditioned medium obtained from cultures of mature and aged 3 T3-L1 adipocytes, as well as from cultures of dysfunctional adipocytes exposed to oxidative stress or high insulin doses, to treat C2C12 myocytes. The results from morphological measures indicated a significant decrease in diameter and fusion index of myotubes after treatment with medium of aged or stressed adipocytes. Aged and stressed adipocytes presented different morphological characteristics as well as a different gene expression profile of proinflammatory cytokines and ROS production. In myocytes treated with different adipocytes' conditioned media, we demonstrated a significant reduction of gene expression of myogenic differentiation markers as well as a significant increase of genes involved in atrophy. Finally, a significant reduction in protein synthesis as well as a significant increase of myostatin was found in muscle cells treated with medium of aged or stressed adipocytes compared to controls. In conclusion, these preliminary results suggest that aged adipocytes could influence negatively trophism, function and regenerative capacity of myocytes by a paracrine network of signaling.

Inhibition of nucleotide biosynthesis disrupts lipid accumulation and adipogenesis.

Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate, or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mechanistic target of rapamycin complex 1 is activated by purine accumulation, mechanistic target of rapamycin complex 1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation.

Kinetic networks identify TWIST2 as a key regulatory node in adipogenesis.

Adipocytes contribute to metabolic disorders such as obesity, diabetes, and atherosclerosis. Prior characterizations of the transcriptional network driving adipogenesis have overlooked transiently acting transcription factors (TFs), genes, and regulatory elements that are essential for proper differentiation. Moreover, traditional gene regulatory networks provide neither mechanistic details about individual regulatory element-gene relationships nor temporal information needed to define a regulatory hierarchy that prioritizes key regulatory factors. To address these shortcomings, we integrate kinetic chromatin accessibility (ATAC-seq) and nascent transcription (PRO-seq) data to generate temporally resolved networks that describe TF binding events and resultant effects on target gene expression. Our data indicate which TF families cooperate with and antagonize each other to regulate adipogenesis. Compartment modeling of RNA polymerase density quantifies how individual TFs mechanistically contribute to distinct steps in transcription. The glucocorticoid receptor activates transcription by inducing RNA polymerase pause release, whereas SP and AP-1 factors affect RNA polymerase initiation. We identify Twist2 as a previously unappreciated effector of adipocyte differentiation. We find that TWIST2 acts as a negative regulator of 3T3-L1 and primary preadipocyte differentiation. We confirm that Twist2 knockout mice have compromised lipid storage within subcutaneous and brown adipose tissue. Previous phenotyping of Twist2 knockout mice and Setleis syndrome Twist2 -/- patients noted deficiencies in subcutaneous adipose tissue. This network inference framework is a powerful and general approach for interpreting complex biological phenomena and can be applied to a wide range of cellular processes.

Rhododendrol, a reductive metabolite of raspberry ketone, suppresses the differentiation of 3T3­L1 cells into adipocytes.

Obesity is a serious medical condition worldwide, and a major risk factor for type 2 diabetes, metabolic syndrome, cancer and cardiovascular disease. In addition to changes in dietary habits and physical activity, consuming supplements to maintain good health and prevent obesity is important in modern society. Raspberry ketone (RK) is a natural phenolic ketone found in the European red raspberry (Rubus idaeus L.) and is hypothesized to prevent obesity when administered orally. The present study found that RK was reduced to rhododendrol (ROH) in human liver microsomes and cytosol. The present study investigated whether the metabolite ROH had anti­adipogenic effects using mouse 3T3­L1 cells. The effects of ROH or RK on lipid accumulation during differentiation of 3T3­L1 pre­adipocyte into adipocyte were determined using Oil Red O staining. CCAAT enhancer­binding protein α (C/EBPα) and peroxisome proliferator­activated receptor γ (PPARγ) mRNA and protein expression were examined using reverse transcription­quantitative PCR and western blotting analysis, respectively. The present study revealed that ROH suppressed lipid accumulation in the cells, similar to RK. In addition, ROH suppressed the mRNA expression levels of C/EBPα and PPARγ in 3T3­L1 adipocytes. Furthermore, ROH suppressed PPARγ protein expression in 3T3­L1 adipocytes. These findings suggested that ROH is an active metabolite with an anti­adipogenic effect, which may contribute to the anti­obesity effect of orally administered RK. The present study indicated that it is important to understand the biological activity of the metabolites of orally administered compounds.

Cinnamaldehyde affects lipid droplets metabolism after adipogenic differentiation of C2C12 cells.

BACKGROUND: Based on our previous research conducted on cinnamaldehyde (CA) exhibiting its ability to improve the growth performance of fattening pigs and the adipogenesis induction model of C2C12 cells constructed in our laboratory, we explored the effects of CA on the generation and development of lipid droplets (LDs) in adipogenic differentiated C2C12 cells. METHODS AND RESULTS: C2C12 cells were treated with either 0.4 mM or 0.8 mM CA. BODIPY staining and triglyceride measurements were conducted to observe the morphology of LDs, and Western blotting was used to measure the expression of their metabolism-related proteins. The results showed that the average number of LDs in the CA treatment groups was more than the control group (P < 0.05), whereas the average LD size and triglyceride content decreased (P < 0.05). Compared with the control group, the expression levels of fusion-related genes in the LDs of the CA treatment group significantly decreased, while decomposition-related genes and autophagy-related genes in the LDs in C2C12 cells significantly increased (P < 0.01). CONCLUSION: Cinnamaldehyde promoted the decomposition and autophagy of lipid droplets in C2C12 cells and inhibited the fusion of lipid droplets.

CLSTN3ß enforces adipocyte multilocularity to facilitate lipid utilization.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue1,2, but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3ß) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3ß is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3ß have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3ß is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3ß associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3ß-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

SLC3A2 and SLC7A2 Mediate the Exogenous Putrescine-Induced Adipocyte Differentiation.

Exogenous polyamines are able to induce life span and improve glucose homeostasis and insulin sensitivity. However, the effects of exogenous polyamines on adipocyte differentiation and which polyamine transporters mediate them have not been elucidated yet. Here, we identified for the first time that exogenous polyamines can clearly stimulate adipocyte differentiation through polyamine transporters, solute carrier family 3 member A2 (SLC3A2) and SLC7A1. Exogenous polyamines markedly promote 3T3-L1 adipocyte differentiation by increasing the intracellular lipid accumulation and the expression of both adipogenic and lipogenic genes in a concentration-dependent manner. In particular, exogenous putrescine mainly regulates adipocyte differentiation in the early and intermediate stages. Moreover, we have assessed the expression of polyamine transporter genes in 3T3-L1 preadipocytes and adipocytes. Interestingly, the putrescine-induced adipocyte differentiation was found to be significantly suppressed in response to a treatment with a polyamine transporter inhibitor (AMXT-1501). Furthermore, knockdown experiments using siRNA that specifically targeted SLC3A2 or SLC7A2, revealed that both SLC3A2 and SLC7A2 act as important transporters in the cellular importing of exogenous putrescine. Thus, the exogenous putrescine entering the adipocytes via cellular transporters is involved in adipogenesis through a modulation of both the mitotic clonal expansion and the expression of master transcription factors. Taken together, these results suggest that exogenous polyamines (such as putrescine) entering the adipocytes through polyamine transporters, can stimulate adipogenesis.

Systematic review on spheroids from adipose-derived stem cells: Spontaneous or artefact state?

Three-dimensional (3D) cell cultures represent the spontaneous state of stem cells with specific gene and protein molecular expression that are more alike the in vivo condition. In vitro two-dimensional (2D) cell adhesion cultures are still commonly employed for various cellular studies such as movement, proliferation and differentiation phenomena; this procedure is standardized and amply used in laboratories, however their representing the original tissue has recently been subject to questioning. Cell cultures in 2D require a support/substrate (flasks, multiwells, etc.) and use of fetal bovine serum as an adjuvant that stimulates adhesion that most likely leads to cellular aging. A 3D environment stimulates cells to grow in suspended aggregates that are defined as "spheroids." In particular, adipose stem cells (ASCs) are traditionally observed in adhesion conditions, but a recent and vast literature offers many strategies that obtain 3D cell spheroids. These cells seem to possess a greater ability in maintaining their stemness and differentiate towards all mesenchymal lineages, as demonstrated in in vitro and in vivo studies compared to adhesion cultures. To date, standardized procedures that form ASC spheroids have not yet been established. This systematic review carries out an in-depth analysis of the 76 articles produced over the past 10 years and discusses the similarities and differences in materials, techniques, and purposes to standardize the methods aimed at obtaining ASC spheroids as already described for 2D cultures.

Inhibitory Effect of Bacterial Lysates Extracted from Pediococcus acidilactici on the Differentiation of 3T3-L1 Pre-Adipocytes.

Postbiotics, including bacterial lysates, are considered alternatives to probiotics. The aim of the current study was to investigate the effect of bacterial lysates (BLs) extracted from Pediococcus acidilactici K10 (K10 BL) and P. acidilactici HW01 (HW01 BL) on the differentiation of 3T3-L1 pre-adipocytes. Both K10 and HW01 BLs significantly reduced the accumulation of lipid droplets and the amounts of cellular glycerides in 3T3-L1 cells (p < 0.05). However, another postbiotic molecule, peptidoglycan of P. acidilactici K10 and P. acidilactici HW01, moderately inhibited the accumulation of lipid droplets, whereas heat-killed P. acidilactici did not effectively inhibit the lipid accumulation. The mRNA and protein levels of the transcription factors, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, responsible for the differentiation of 3T3-L1 cells, were significantly inhibited by K10 BL and HW01 BL (p < 0.05). Both K10 and HW01 BLs decreased adipocyte-related molecules, adipocyte fatty acid-binding protein and lipoprotein lipase, at the mRNA and protein levels. Furthermore, both K10 and HW01 BLs also downregulated the mRNA expression of leptin, but not resistin. Taken together, these results suggest that P. acidilactici BLs mediate anti-adipogenic effects by inhibiting adipogenic-related transcription factors and their target molecules.

Generation of mega brown adipose tissue in adults by controlling brown adipocyte differentiation in vivo.

Brown adipose tissue (BAT) is a highly specialized adipose tissue in its immobile location and size during the entire adulthood. In response to cold exposure and other ß3-adrenoreceptor stimuli, BAT commits energy consumption by nonshivering thermogenesis (NST). However, the molecular machinery in controlling the BAT mass in adults is unknown. Here, we show our surprising findings that the BAT mass and functions can be manipulated in adult animals by controlling BAT adipocyte differentiation in vivo. Platelet-derived growth factor receptor α (PDGFα) expressed in BAT progenitor cells served a signaling function to avert adipose progenitor differentiation. Genetic and pharmacological loss-of-function of PDGFRα eliminated the differentiation barrier and permitted progenitor cell differentiation to mature and functional BAT adipocytes. Consequently, an enlarged BAT mass (megaBAT) was created by PDGFRα inhibition owing to increases of brown adipocyte numbers. Under cold exposure, a microRNA-485 (miR-485) was identified as a master suppressor of the PDGFRα signaling, and delivery of miR-485 also produced megaBAT in adult animals. Noticeably, megaBAT markedly improved global metabolism, insulin sensitivity, high-fat-diet (HFD)-induced obesity, and diabetes by enhancing NST. Together, our findings demonstrate that the adult BAT mass can be increased by blocking the previously unprecedented inhibitory signaling for BAT progenitor cell differentiation. Thus, blocking the PDGFRα for the generation of megaBAT provides an attractive strategy for treating obesity and type 2 diabetes mellitus (T2DM).

The circadian clock mediates daily bursts of cell differentiation by periodically restricting cell-differentiation commitment.

Most mammalian cells have an intrinsic circadian clock that coordinates metabolic activity with the daily rest and wake cycle. The circadian clock is known to regulate cell differentiation, but how continuous daily oscillations of the internal clock can control a much longer, multiday differentiation process is not known. Here, we simultaneously monitor circadian clock and adipocyte-differentiation progression live in single cells. Strikingly, we find a bursting behavior in the cell population whereby individual preadipocytes commit to differentiate primarily during a 12-h window each day, corresponding to the time of rest. Daily gating occurs because cells irreversibly commit to differentiate within only a few hours, which is much faster than the rest phase and the overall multiday differentiation process. The daily bursts in differentiation commitment result from a differentiation-stimulus driven variable and slow increase in expression of PPARG, the master regulator of adipogenesis, overlaid with circadian boosts in PPARG expression driven by fast, clock-driven PPARG regulators such as CEBPA. Our finding of daily bursts in cell differentiation only during the circadian cycle phase corresponding to evening in humans is broadly relevant, given that most differentiating somatic cells are regulated by the circadian clock. Having a restricted time each day when differentiation occurs may open therapeutic strategies to use timed treatment relative to the clock to promote tissue regeneration.

A monocyte-leptin-angiogenesis pathway critical for repair post-infection.

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

Adipocyte differentiation between obese and lean conditions depends on changes in miRNA expression.

Adipogenesis is the process by which precursor cells, preadipocytes (preACs), differentiate into adipocytes (ACs). Here, we investigated differentially expressed miRNAs (DEMs) between the two conditions to understand the regulatory role of miRNAs in altering adipogenesis-related mRNAs. A total of 812 and 748 DEMs were obtained in lean and obese conditions, respectively. The up- and downregulated DEMs were highly concordant with each other in both lean and obese conditions; however, DEMs related to adipogenesis in obese conditions were more strongly downregulated than DEMs related to adipogenesis in lean conditions. There were more obese-specific downregulated DEMs than lean-specific downregulated DEMs; in contrast, there were more lean-specific upregulated DEMs than obese-specific upregulated DEMs. Approximately 45% of DEMs were mapped to the list of miRNA-target mRNA pairs when DEMs were matched to the experimentally validated list of miRNA-target mRNA information of miRTarBase. Many of the target mRNAs were differentially expressed genes (DEGs) with functions in processes such as inflammatory responses and fat metabolism. In particular, a total of 25 miRNAs that target three upregulated adipogenesis-associated inflammatory genes (IL-6, TNF-α, and IL-1ß) were commonly altered during adipogenesis. Taken together, our study reveals the types of adipogenesis-related miRNAs that are altered and the degree to which they influence healthy or pathogenic adipogenesis.

Sampsonione F suppresses adipogenesis via activating p53 pathway during the mitotic clonal expansion progression of adipocyte differentiation.

The abnormal proliferation and hypertrophy of adipocytes mediate the expansion of adipose tissue and then cause obesity-related diseases. Theoretically, an approach for preventing and curing obesity is to inhibit cell proliferation during the mitotic clonal expansion (MCE) progression of adipocyte differentiation. Polycyclic polyprenylated acylphloroglucinols (PPAPs) are mainly found from Hypericum and Garcinia genus, which have been reported to have various biological activities such as anti-depressant, anti-oxidant, and anti-tumor. Previously, our group has reported that adamantane-type PPAPs exhibited blunting activity in adipogenesis. In this study, another six adamantane PPAPs were screened to investigate their anti-adipogenesis activities and then discussed the structure-activity relationship. Particularly, sampsonione F, one of the PPAPs dramatically suppressed adipogenesis dose-dependently in vitro, along with decreased expressions of C/EBPß, C/EBPα, PPARγ, FABP4, and FAS. Moreover, sampsonione F upregulated the expression of p27 by activating p53 pathway and then downregulated the expressions of key regulators during G1/S phase arrest. Our data support that sampsonione F suppressed adipogenesis by activating p53 pathway, regulating cyclins, and resulting in G1/S phase arrest during the MCE progression of adipogenesis. This work provides a new adamantane-type PPAPs in the regulation of adipogenesis and extends our knowledges on the mechanism of the type PPAPs in regulation of adipogenesis.

Hoxa5 inhibits adipocyte proliferation through transcriptional regulation of Ccne1 and blocking JAK2/STAT3 signaling pathway in mice.

The highly regulated proliferation of adipocytes plays a momentous role in fat development and obesity. Hoxa5 is an important member of the Hox family, its encoded protein is an important transcription factor related to development, and its differential expression in different adipose tissues seems to indicate that Hoxa5 may be involved in the regulation of adipocyte proliferation. To evaluate the regulatory mechanism of Hoxa5 on adipocyte proliferation, we constructed a variety of Hoxa5 expression vectors in vivo and in vitro to explore its mechanism on adipocyte proliferation and its potential impact on obesity. We observed that the overexpression of Hoxa5 strongly reduces cell counts and Hoxa5 can inhibit cell proliferation and block cell cycle progression by regulating the expression of genes such as Cyclin E, Cyclin D1, and p53. Most importantly, we demonstrated that Hoxa5 exerts its effect by regulating the signaling pathway of Janus kinase 2 (JAK2) signal transduction and transcription 3 (STAT3) activator, as well as binding to the promoter region of Ccne1 and inhibiting the transcription of Ccne1. This study provides an in-depth understanding of the potential molecular mechanism of Hoxa5 inhibiting adipocyte proliferation. Our results suggest the importance of Hoxa5 in the treatment of obesity.

Nitric Oxide Mobilizes Intracellular Zn2+ via the GC/cGMP/PKG Signaling Pathway and Stimulates Adipocyte Differentiation.

Plasma and tissue zinc ion levels are associated with the development of obesity. Previous studies have suggested that zinc ions may regulate adipocyte metabolism and that nitric oxide (NO) plays a pivotal role in the regulation of adipocyte physiology. Our previous study showed that chronic NO deficiency causes a significant decrease in adipose tissue mass in rats. Studies also suggested that zinc ions play an important modulatory role in regulating NO function. This study aims to explore the role of zinc ions in NO-regulated adipocyte differentiation. We hypothesized that NO could increase intracellular Zn2+ level and then stimulate adipocyte differentiation. ZnCl2 and the NO donor, NONOate, were used to explore the effects of Zn2+ and NO on adipocyte differentiation. Regulatory mechanisms of NO on intracellular Zn2+ mobilization were determined by detection. Then, Zn2+-selective chelator TPEN was used to clarify the role of intracellular Zn2+ on NO-regulated adipocyte differentiation. Furthermore, the relationship between adipocyte size, Zn2+ level, and NOS expression in human subcutaneous fat tissue was elucidated. Results showed that both ZnCl2 and NO stimulated adipocyte differentiation in a dose-dependent manner. NO stimulated intracellular Zn2+ mobilization in adipocytes through the guanylate cyclase (GC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway, and NO-stimulated adipocyte differentiation was Zn2+-dependent. In human subcutaneous adipose tissue, adipocyte size was negatively correlated with expression of eNOS. In conclusion, NO treatment stimulates intracellular Zn2+ mobilization through the GC/cGMP/PKG pathway, subsequently stimulating adipocyte differentiation.

An organoid model derived from human adipose stem/progenitor cells to study adipose tissue physiology.

We established a functional adipose organoid model system for human adipose stem/progenitor cells (ASCs) isolated from white adipose tissue (WAT). ASCs were forced to self-aggregate by a hanging-drop technique. Afterwards, spheroids were transferred into agar-coated cell culture dishes to avoid plastic-adherence and dis-aggregation. Adipocyte differentiation was induced by an adipogenic hormone cocktail. Morphometric analysis revealed a significant increase in organoid size in the course of adipogenesis until d 18. Whole mount staining of organoids using specific lipophilic dyes showed large multi- and unilocular fat deposits in differentiated cells indicating highly efficient differentiation of ASCs into mature adipocytes. Moreover, we found a strong induction of the expression of key adipogenesis and adipocyte markers (CCAAT/enhancer-binding protein (C/EBP) ß, peroxisome proliferator-activated receptor (PPAR) γ, fatty acid-binding protein 4 (FABP4), adiponectin) during adipose organoid formation. Secreted adiponectin was detected in the cell culture supernatant, underscoring the physiological relevance of mature adipocytes in the organoid model. Moreover, colony formation assays of collagenase-digested organoids revealed the maintenance of a significant fraction of ASCs within newly formed organoids. In conclusion, we provide a reliable and highly efficient WAT organoid model, which enables accurate analysis of cellular and molecular markers of adipogenic differentiation and adipocyte physiology.

Inhibition of the Monocarboxylate Transporter 1 (MCT1) Promotes 3T3-L1 Adipocyte Proliferation and Enhances Insulin Sensitivity.

Enlarged, hypertrophic adipocytes are less responsive to insulin and are a hallmark feature of obesity, contributing to many of the negative metabolic consequences of excess adipose tissue. Although the mechanisms remain unclear, the adipocyte size appears to be inversely correlated with insulin sensitivity and glucose tolerance, wherein smaller adipocytes are insulin-sensitive and larger adipocytes develop insulin resistance and exhibit an impaired glucose uptake. Thus, pharmacological strategies aimed at regulating adipocyte hypertrophy (increase in adipocyte size) in favor of promoting hyperplasia (increase in adipocyte number) have the potential to improve adipocyte insulin sensitivity and provide therapeutic benefits in the context of metabolic disorders. As white adipose tissue can metabolize large amounts of glucose to lactate, using transcriptomics and in vitro characterization we explore the functional consequences of inhibiting monocarboxylate transporter 1 (MCT1) activity in fully differentiated adipocytes. Our studies show that the pharmacological inhibition of MCT1, a key regulator of the cellular metabolism and proliferation, promotes the re-entry of mature adipocytes into the cell cycle. Furthermore, we demonstrate that inhibitor-treated adipocytes exhibit an enhanced insulin-stimulated glucose uptake as compared with untreated adipocytes, and that this outcome is dependent on the cyclin-dependent kinase 1 (CDK1) activity. In summary, we identify a mechanism though which MCT1 inhibition improves the insulin sensitivity of mature adipocytes by inducing cell cycle re-entry. These results provide the foundation for future studies investigating the role MCT1 plays in adipocyte hyperplasia, and its therapeutic potential as a drug target for obesity and metabolic disease.