Adipogenesis Program Research Articles

783 Deconstructing the dermal adipogenesis program during skin development and regeneration

Dermal white adipose tissue (dWAT), a unique layer which is enriched with adipocytes, preadipocytes and adipocyte progenitors, is indispensable for maintaining skin homeostasis and tissue regeneration during skin wound healing. However, the origin, developmental path and the cellular complexity and plasticity of dWAT lineage cells remain poorly understood. Here, by single-cell RNA-seq (scRNA-seq) analyses of dermal cell populations isolated from mice at various post-developmental ages or at indicated post-wounding days, we have carried out in-depth analysis to define how dermal adipocytes are differentiated from their progenitors and to identify key signaling pathways that regulate dermal adipogenesis.

Transcriptional Landscaping Identifies a Beige Adipocyte Depot in the Newborn Mouse.

The present study sought to identify gene networks that are hallmarks of the developing inguinal subcutaneous adipose tissue (iWAT) and the interscapular brown adipose tissue (BAT) in the mouse. RNA profiling revealed that the iWAT of postnatal (P) day 6 mice expressed thermogenic and lipid catabolism transcripts, along with the abundance of transcripts associated with the beige adipogenesis program. This was an unexpected finding, as thermogenic BAT was believed to be the only site of nonshivering thermogenesis in the young mouse. However, the transcriptional landscape of BAT in P6 mice suggests that it is still undergoing differentiation and maturation, and that the iWAT temporally adopts thermogenic and lipolytic potential. Moreover, P6 iWAT and adult (P56) BAT were similar in their expression of immune gene networks, but P6 iWAT was unique in the abundant expression of antimicrobial proteins and virus entry factors, including a possible receptor for SARS-CoV-2. In summary, postnatal iWAT development is associated with a metabolic shift from thermogenesis and lipolysis towards fat storage. However, transcripts of beige-inducing signal pathways including β-adrenergic receptors and interleukin-4 signaling were underrepresented in young iWAT, suggesting that the signals for thermogenic fat differentiation may be different in early postnatal life and in adulthood.

Rab8 attenuates Wnt signaling and is required for mesenchymal differentiation into adipocytes

Differentiation of mesenchymal stem cells into adipocyte requires coordination of external stimuli and depends upon the functionality of the primary cilium. The Rab8 small GTPases are regulators of intracellular transport of membrane-bound structural and signaling cargo. However, the physiological contribution of the intrinsic trafficking network controlled by Rab8 to mesenchymal tissue differentiation has not been fully defined in vivo and in primary tissue cultures. Here, we show that mouse embryonic fibroblasts (MEFs) lacking Rab8 have severely impaired adipocyte differentiation in vivo and ex vivo. Immunofluorescent localization and biochemical analyses of Rab8a-deficient, Rab8b-deficient, and Rab8a and Rab8b double-deficient MEFs revealed that Rab8 controls the Lrp6 vesicular compartment, clearance of basal signalosome, traffic of frizzled two receptor, and thereby a proper attenuation of Wnt signaling in differentiating MEFs. Upon induction of adipogenesis program, Rab8a- and Rab8b-deficient MEFs exhibited severely defective lipid-droplet formation and abnormal cilia morphology, despite overall intact cilia growth and ciliary cargo transport. Our results suggest that intracellular Rab8 traffic regulates induction of adipogenesis via proper positioning of Wnt receptors for signaling control in mesenchymal cells.

Bone marrow as a target and accomplice of vascular complications in diabetes.

Peripheral vascular complications are common in diabetic patients. While pathogenic mechanisms have received much consideration, only recently regenerative processes captured attention. There is now a consensus that the bone marrow is a source of reparative cells and that this healing mechanism is lost in people with diabetes, especially in those suffering from ischemic complications. This failure was thought to occur due to a negative impact of diabetes on the mobilization of stem/progenitor cells with angiogenic properties from the bone marrow to the circulation. Moreover, those patients showing severely reduced bone marrow cell mobilization also bared a very high risk for adverse cardiovascular events. More recently, the structural integrity of the bone marrow was recognized to be altered because of the rarefaction of local microvasculature and innervation, thus mirroring anatomical features that typically occur in peripheral tissues. Ensuing hypoxia, nutrient starvation, and creation of an acidic and oxidative environment concur in causing the depletion of stem/progenitor cells in the endosteal niche and in forcing stromal cells to activate an adipogenesis program. Moreover, stem/progenitor cells acquire a pathogenic phenotype and, once mobilized, can pass harmful signalling molecules to vascular cells of peripheral tissues thereby contributing to ischemic complications. These new pieces of evidence indicate that the bone marrow should deserve more attention in the current care of critical limb ischemia and diabetic foot. Owing to powerful reserve capacities, the bone marrow integrity could be preserved and even rescued using rehabilitation programs and pharmacological treatments with consequent benefit for local and whole-organism homeostasis.

Effect of Dietary Calcium on Adipogenesis Program and Its Role in Adipocyte Dysfunction in Male Wistar Rats

Dietary calcium influences obesity-related adipocyte dysfunction by modulating body weight. The molecular mechanism behind this weight management is under intense investigation. In the present study, the authors investigated the effect of dietary calcium on peroxisome proliferator-activated receptor-γ (PPAR-γ)-mediated adipogenesis program and its role in adipocyte dysfunction by measuring the inflammatory and oxidative markers within the adipose tissue of male albino rats. Healthy male rats were fed with a control diet based on AIN-93, and either low-calcium (0.25%) or high-calcium (1.0%) diet for 90 days. A significant inverse (p < 0.05) relationship exists between the calcium intake and anthropometric parameters like adipose tissue mass and body weight. Adipose tissue PPAR-γ activity along with its downstream enzyme activities like glyceraldehydes 3 phosphate dehydrogenase, lipoprotein lipase as well as the protein expression of fatty acid-binding protein 4 and calcium-sensing receptor were significantly increased (p < 0.05) in the low-calcium-fed rats which in turn promoted the adipogenesis program. At the same time, protein expression of inflammatory markers like tumor necrosis factor-α and interleukin-6 and oxidative stress markers like lipid peroxidation and protein carbonylation were significantly increased (p < 0.05) in low-calcium-fed rats. These deleterious effects were suppressed by the high-calcium diet by preventing the activation of the adipogenesis program. Thus, the present study showed the involvement of dietary calcium against the PPAR-γ-mediated adipogenesis program as well as its role in adipocyte dysfunction by regulating the inflammatory and oxidative stress.

Feeding a slowly digestible carbohydrate diet during pregnancy of insulin-resistant rats prevents the excess of adipogenesis in their offspring

An obesogenic environment during pregnancy has been shown to increase the risk of dysregulation on adipogenesis and insulin resistance in the offspring. Being essential for the growing fetus, glucose supply is guaranteed by a number of modifications in the mother’s metabolism, and thus, glucose control during pregnancy especially among obese or diabetic women is paramount to prevent adverse consequences in their children.Besides the election of low-glycemic-index carbohydrates, the rate of carbohydrate digestion could be relevant to keep a good glucose control. In the present study, we compared the effects of two high-fat diets with similar glycemic load but different rates of carbohydrate digestion given to pregnant insulin-resistant rats. After birth, all animals were fed a standard diet until age 14 weeks. We analyzed offspring body composition, plasma and adipocyte lipidomics, lipid metabolism in adipose tissue and insulin sensitivity. Those animals whose mothers were fed the rapid-digesting carbohydrate diet exhibited an excessive adipogenesis. Thus, these animals showed a marked lipidemia, increased lipid synthesis in the adipose tissue and reduced glucose transporter amount in the adipose. On the contrary, those animals whose mothers were fed the slow-digesting carbohydrate diet showed a profile in the measured parameters closer to that of the offspring of healthy mothers. These results support the hypothesis that not only glycemic index but the rate of carbohydrate digestion during gestation may be critical to regulate the programming of adipogenesis in the offspring.

Perinatal low-dose iron treatment influences susceptibility to diet-induced adipogenesis in early-aged male Wistar rats

The primary objective of the study was to estimate the effect of perinatal low-dose iron supplementation on diet-induced adipogenic action of a high-fat diet in the male offspring. The experimental group of pregnant dams was treated with drinking water containing 3 mg/l ferrous sulfate (FeSO4·7H2O) from the 2nd week of pregnancy till the end of lactation (the 21st day postpartum). The control group of dams obtained pure drinking water. The obtained male littermates were fed standard and high-fat diets (HFD) for 1 month. Animals' morphometric parameters as well as serum lipoprotein profile, glucose, insulin, adipokines and cytokines concentrations were estimated. Adipose tissue oxidative stress biomarkers were also measured. It is shown that HFD-fed perinatally iron treated rats had a significantly higher adipose tissue mass in comparison with HFD-control ones. The experimental iron-treated males were also characterized by increased serum glucose and insulin concentrations. Perinatally iron treated HFD-fed animals' leptin and proinflammatory cytokines concentrations exceeded the HFD-control values. Significant accumulation of free radical oxidation biomarkers is observed in adipose tissue samples. The lipoprotein spectra indicated initial atherogenic changes in the rats' serum. Taken together, the study suggests that iron takes part in the developmental programming of adipogenesis.

Developmental Programming of Offspring Obesity, Adipogenesis, and Appetite

A newly recognized primary cause of the obesity epidemic is the developmental programming effects of infants born to mothers with obesity or gestational diabetes, intrauterine growth-restricted newborns, and offspring exposed to environmental toxins including bisphenol A. The mechanisms which result in offspring obesity include the programming of the hypothalamic appetite pathway and adipogenic signals regulating lipogenesis. Processes include nutrient sensors, epigenetic modifications, and alterations in stem cell precursors of both appetite/satiety neurons and adipocytes which are modulated to potentiate offspring obesity. Future strategies for the prevention and therapy of obesity must address programming effects of the early life environment.

Cultured Preadipocytes Undergoing Stable Transfection with Cyclooxygenase-1 in the Antisense Direction Accelerate Adipogenesis During the Maturation Phase of Adipocytes

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ(12,14)-PGJ2 and Δ(12)-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

The Role of the ω‐3 Fatty Acid DHA in the Human Life Cycle

Dietary consumption of the essential fatty acids linoleic acid (LA; ω-6) and α-linolenic acid (ALA; ω-3) is necessary for human growth and development. In the past 150 years, the average Western diet has changed dramatically such that humans today consume a much higher proportion of ω-6 fatty acids relative to ω-3 fatty acids than ever before. The importance of ω-3 fatty acids in human development has been well established in fetal and neonatal development, with brain and retinal tissues highly dependent on ω-3 fatty acids, specifically docosahexaenoic acid (DHA) for membrane fluidity and signal transduction. In childhood, ω-3s have been shown to contribute to ongoing cognitive development and may be involved in metabolic programming of bone turnover and adipogenesis. ω-3s may also play important roles in adult neurophysiology and disease prevention.

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD 2 and its non-enzymatic dehydration products, PGJ 2 series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD 2 and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD 2 from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE 2 remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize Δ 12-PGJ 2 increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor γ (PPARγ) agonists including troglitazone and Δ 12-PGJ 2. Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPARγ signaling pathway without the contribution of endogenous pro-adipogenic prostanoids.

Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes

Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD2 and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD2 synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE2 was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE2 and PGF2α, while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ12,14-prostaglandin J2. These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ2 series.

15-Deoxy-Δ 12,14-prostaglandin J 2 interferes inducible synthesis of prostaglandins E 2 and F 2α that suppress subsequent adipogenesis program in cultured preadipocytes

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E 2 and PGF 2α involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ 2 interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ 12-PGJ 2 and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ 2. Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ 2 or PDTC although 15d-PGJ 2 alone has no stimulatory effect. Moreover, 15d-PGJ 2 did not block the inhibitory effects of exogenous PGE 2 and PGF 2α on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ 2 can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.

Endocrine disrupting chemicals and the developmental programming of adipogenesis and obesity.

Obesity and related disorders are a burgeoning public health epidemic, particularly in the U.S. Currently 34% of the U.S. population is clinically obese (BMI > 30) and 68% are overweight (BMI > 25), more than double the worldwide average and 10-fold higher than Japan and South Korea. Obesity occurs when energy intake exceeds energy expenditure; however, individuals vary widely in their propensity to gain weight and accrue fat mass, even at identical levels of excess caloric input. Clinical, epidemiological, and biological studies show that obesity is largely programmed during early life, including the intrauterine period. The environmental obesogen hypothesis holds that prenatal or early life exposure to certain endocrine disrupting chemicals can predispose exposed individuals to increased fat mass and obesity. Obesogen exposure can alter the epigenome of multipotent stromal stem cells, biasing them toward the adipocyte lineage at the expense of bone. Hence, humans exposed to obesogens during early life might have an altered stem cell compartment, which is preprogrammed toward an adipogenic fate. This results in a higher steady state number of adipocytes and potentially a lifelong struggle to maintain a healthy weight, which can be exacerbated by societal influences that promote poor diet and inadequate exercise. This review focuses on the developmental origins of the adipocyte, the relationship between adipocyte number and obesity, and how obesogenic chemicals may interfere with the highly efficient homeostatic mechanisms regulating adipocyte number and energy balance.

Development of enzyme-linked immunosorbent assay for Δ 12-prostaglandin J 2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D 2 can be produced in adipocytes and dehydrated to J 2 series of PGs including 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2) and Δ 12-PGJ 2, which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ 12-PGJ 2 has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ 12-PGJ 2. According to the standard curve, the amount of Δ 12-PGJ 2 can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ 2, while it only showed the cross-reaction of 28% with unstable PGJ 2. This immunological assay was applied to the determination of the endogenous formation of Δ 12-PGJ 2 in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ 12-PGJ 2 increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ 2. Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ 12-PGJ 2 in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ 2 series at various doses on adipogenesis during the maturation phase. Although Δ 12-PGJ 2 was slightly less potent than 15d-PGJ 2, each of these PGJ 2 series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ 12-PGJ 2 contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ 2 during the maturation phase of cultured adipocytes.

Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE 2 involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE 2 associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE 2 and PGF 2α, during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

Isolation and Characterization of Omental Adipose Progenitor Cells in Children: A Potential Tool to Unravel the Pathogenesis of Metabolic Syndrome

Background/Aim: Visceral adipose tissue plays a pivotal role in the pathogenesis of metabolic syndrome. Several investigations refer to the physiology of subcutaneous and visceral adipose tissue of adult obese subjects, focusing on the characterization of adipose-derived stem cells and their differentiation capacity. To date, no study has been carried out on visceral adipose tissue of children, the main limitation being the amount of fat tissue available. The aim of this investigation was to establish a method to successfully isolate adipose progenitor cells from very small specimens of omental fat obtained from children undergoing abdominal surgery. Methods: A previously described procedure employed to extract adipose progenitors from large amounts of lipoaspirate was modified. 0.5-cm³ specimens of omental fat were collected from 13 children and 2 newborns. Primary, secondary and higher passaged adipocytic progenitor cell cultures were established. Immunophenotyping of the generated lines was carried out by flow cytometry analysis. Differentiation experiments concerning the adipogenic phenotype were conducted. Results: A feasible method for isolating adipose progenitors from pediatric specimens of omental fat was established. Cells showed a fibroblast-like appearance consistent with that of mesenchymal stem cells obtained from bone marrow. The mesenchymal phenotype was confirmed by the surface antigenic profile of these cells. Cells also underwent adipogenic differentiation in vitro. Conclusion: The availability of progenitor primary cell lines from omental adipose tissue of children may allow future investigations aimed at unraveling whether fetal programming of adipogenesis is responsible for the pathogenesis of the metabolic syndrome that develops during adulthood in subjects, born with low birth weight, typically showing central fat redistribution.

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE 2 upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Δ 12,14-PGJ 2 (15d-PGJ 2) and troglitazone as peroxisome proliferator-activated receptor γ (PPARγ) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARγ and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.

Inflammation and macrophage infiltration in adipose tissue: A link between diabetes and atherosclerosis

Abstract Obesity is associated with macrophage infiltration of adipose tissue, resulting in the development of insulin resistance and cardiovascular diseases. MCP-1 is involved in monocyte recruitment to both early atherosclerotic lesion and adipose tissue. We recently described two possible mechanisms upstream MCP-1 activation. The first involved insulin dependent phosphorylation of GATA2, a transcription factor crucial for adipogenesis and inflammatory program. In the second we showed that an increased expression of several inflammation markers, including MCP-1 in metabolic and vascular tissues might due to a deregulation of TNF-alpha Converting Enzyme (TACE). GATA-2 and TACE could be new targets in the treatment of obesity related inflammation and its complications.

Mechanism of adult primitive mesenchymal ST-13 preadipocyte differentiation.

Convincing evidence supports the idea that adipogenesis occurs throughout the life of organisms. However, little is known about the adipogenesis program for adult adipocytes. We examine this issue using mouse adult primitive mesenchymal ST-13 preadipocytes that express the peroxisome proliferator-activated receptor-gamma (PPARgamma) gene while in a predifferentiated state. The gene expression of PPARgamma was sustained throughout differentiation when ST-13 preadipocytes were induced to become adipocytes by a PPARgamma ligand. However, the differentiation of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblastic cells was associated with enhanced expression of the PPARgamma gene. Immunoblotting analysis revealed that C3H10T1/2 and 3T3-L1 cells expressed low levels of PPARgamma1 from the early stage, and the amount increased during differentiation, whereas PPARgamma2 appeared at the late stage. In contrast, ST-13 preadipocytes expressed an appreciable amount of PPARgamma1 that significantly decreased on differentiation, and a small amount of PPARgamma2 appeared late in the differentiation process. Furthermore, the standard hormone cocktail containing dexamethasone, methylisobutylxanthine, and insulin induced an increase in PPARgamma1 protein only at the early stage, and a low level of PPARgamma2 protein appeared late in ST-13 cells. However, levels of both PPARgamma1 and PPARgamma2 proteins were significantly induced within 2 d in 3T3-L1 cells in this hormonal adipogenesis. Moreover, exposing ST-13 preadipocytes to dexamethasone and insulin induced differentiation, but failed to induce adipogenesis in 3T3-L1. Adipogenesis in adult rat primary preadipocytes was also induced in a similar manner to that of ST-13. Our results indicate that ST-13 cells and primary preadipocytes derived from adults possess an adipogenesis program distinct from that of 3T3-L1 and C3H10T1/2 cells, and that it may represent the adipogenesis program for adult-specific adipocytes.