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Abstract
TNF-alpha, a trimeric cytokine, was known to inhibit differention of preadipocytes to adipocytes. In the present study, we investigated signal mediators working downstream of TNF-alpha using murine 3T3-L1 cells. TNF-alpha induced activation of both c-jun NH2-terminal kinase (JNK) and nuclear transcription factor-kappaB (NF-kappaB) in 3T3-L1 cells. Blockage of these two mediators activities by specific inhibitors, SP600125 and Ad-IkappaBalpha-SR restored adipogenesis differentiation suggesting their involvement in the inhibited differentiation of 3T3-L1 cells by TNF-alpha. Consistent with previous studies, peroxisome proliferator-activated receptor gamma (PPARgamma) a key transcriptional regulator was remarkably reduced by TNF-alpha treatment. Compared with adipogenesis, however, SP600125, a chemical JNK inhibitor hardly relieved TNF-alpha effect on PPARgamma expression whereas S32A/S36A mutant of IkappaBalpha considerably recovered PPARgamma expression, indicating that two signal mediators exploit separable main routes to achieve reduced adipogenesis. These results suggest that inhibition of 3T3-L1 cells differentiation by TNF-alpha is partly implemented through NF-kappaB and one of its downstream effectors be PPARgamma.
... TAU supplementation decreased the ratio of pIκ-Bα/Iκ-Bα protein, possibly contributing to the increased Iκ-Bα content in MTAU adipose Inflammatory cytokines activate IKKB by ubiquitination of the inhibitors of kappa B (Iκ-B), which releases the nuclear factor kappa B (NF-κB) for migration to the nucleus, where it in turn activates the transcription of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 genes [9]. TNF-α is a pro-inflammatory cytokine that is primarily involved in IR in obesity, but inhibits adipogenesis [10][11][12][13]. JNK is also activated by inflammatory stimuli [14] and translocates the activator protein (AP)-1 to the nucleus, increasing the transcription of pro-inflammatory cytokines [14, 15]. ...
... However, other studies found no differences in TNF-α gene expression in the perigonadal adipose tissue of Swiss MSG mice [12], in splenic macrophages from C57Bl/6 MSG mice [4] or in the periodontal tissue of MSG rats [35]. Previous reports have also demonstrated that TNF-α decreases adipocyte differentiation via an NF-κB-dependent pathway [13]. As such, these findings, taken together with the observed reduction in the serum concentration of this pro-inflammatory cytokine, indicate that lower TNF-α in the early stages of the MSG obesity onset may contribute to adiposity. ...
Purpose:
Obesity is usually associated with low-grade inflammation, which impairs insulin action. The amino acid, taurine (TAU), regulates glucose homeostasis and lipid metabolism and presents anti-inflammatory actions. Here, we evaluated whether inflammatory markers are altered in the serum and retroperitoneal adipose tissue of monosodium glutamate (MSG) obese rats, supplemented or not with TAU.
Methods:
Male Wistar rats received subcutaneous injections of MSG (4 mg/kg body weight/day, MSG group) or hypertonic saline (CTL) during the first 5 days of life. From 21 to 120 days of age, half of each of the MSG and CTL groups received 2.5 % TAU in their drinking water (CTAU and MTAU).
Results:
At 120 days of age, MSG rats were obese and hyperinsulinemic. TAU supplementation reduced fat deposition without affecting insulinemia in MTAU rats. MSG rats presented increased pIκ-Bα/Iκ-Bα protein expression in the retroperitoneal adipose tissue. TAU supplementation decreased the ratio of pIκ-Bα/Iκ-Bα protein, possibly contributing to the increased Iκ-Bα content in MTAU adipose tissue. Furthermore, MSG obesity or supplementation did not alter TNF-α, IL-1β or IL-6 content in adipose tissue. In contrast, MSG rats presented lower serum TNF-α, IL-4 and IL-10 concentrations, and these alterations were prevented by TAU treatment.
Conclusion:
MSG obesity in rats was not associated with alterations in pro-inflammatory markers in retroperitoneal fat stores; however, reductions in the serum concentrations of anti-inflammatory cytokines and of TNF-α were observed. TAU treatment decreased adiposity, and this effect was associated with the normalization of circulating TNF-α and IL-4 concentrations in MTAU rats.
... However, this result may only be part of the mechanism. JNK and NFκB suppresses the differentiation and function of adipocytes by suppressing the expression of PPARγ 18,19 . Reports that α-tocopherol and δ-tocopherol upregulate PPARγ expression suggest that maintenance of PPARγ expression is also involved in the mechanism 8,9,20 . ...
Brown adipose tissue (BAT) functions as a radiator for thermogenesis and helps maintain body temperature and regulate metabolism. Inflammatory signals have been reported to inhibit PGC-1α activation and UCP1-mediated thermogenesis in brown adipocytes. Inflammation is mainly caused by cell hypertrophy and macrophage invasion due to obesity, and invading macrophages secrete inflammatory cytokines, including TNF-α, IL1β, and IL6, which suppress the thermogenesis in BAT. Tocopherol is a lipid-soluble vitamin with anti-inflammatory effects is expected to contribute to the suppression of inflammation in adipose tissue. In this study, we investigated the protective effect of tocopherols, α-tocopherol (α-toc) and δ-tocopherol (δ-toc), against brown adipocyte inflammation and thermogenesis dysfunction.
Inflammatory stimulation by TNF-α, a major inflammatory cytokine, significantly decreased the protein expression levels of UCP1 and PGC-1α in rat primary brown adipocytes. The pre-incubation of α-toc or δ-toc significantly suppressed the decrease in UCP1 and PGC-1α expression and lipid accumulation. Additionally, α-toc and δ-toc suppress the induction of ERK1/2 gene expression, implying that an antiinflammatory effect is involved in this protective effect. We fed mice a high-fat diet for 16 weeks and investigated the effects of α-toc and δ-toc in the diet. Intake of α-toc and δ-toc significantly suppressed weight gain and hypertrophy of brown adipocytes. Our results suggest that α-toc and δ-toc suppress the dysfunction of thermogenesis in brown adipocytes due to inflammation and contribute to the treatment of obesity and obesity-related metabolic diseases.
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... In this study, macrophage-conditioned medium induced the phosphorylation of Thr-188, suggesting a potential role of macrophages in the modulation of adipogenesis. However, TNFα secreted from macrophages is well-known to strongly inhibit adipogenesis by downregulating PPARγ expression via NF-κ B signaling 46 , which suggests that activated macrophages are involved in the inhibition of adipogenesis. Further studies are needed to reveal the molecular mechanism of C/EBPβ phosphorylation in adipocytes by the interaction with macrophages. ...
The infiltration of macrophages into adipose tissue and their interaction with adipocytes are essential for the chronic low-grade inflammation of obese adipose tissue. In this study, we identified the serum amyloid A3 (Saa3) gene as a key adipocyte-derived factor that is affected by interaction with macrophages. We showed that the Saa3 promoter in adipocytes actually responds to activated macrophages in a co-culture system. Decreasing C/EBPβ abundance in 3T3-L1 adipocytes or point mutation of C/EBPβ elements suppressed the increased promoter activity in response to activated macrophages, suggesting an essential role of C/EBPβ in Saa3 promoter activation. Bioluminescence based on Saa3 promoter activity in Saa3-luc mice was promoted in obese adipose tissue, showing that Saa3 promoter activity is most likely related to macrophage infiltration. This study suggests that the level of expression of the Saa3 gene could be utilized for the number of infiltrated macrophages in obese adipose tissue.
... However, several mech anisms may account for decreased PPARg tran scription following 10E,12ZCLA treatment. For example, NFkB, which is activated in adipose tissue treated with 10E,12ZCLA [123], interacts with and inhibits PPARg activity [136,137]. In addition, inflammatory cytokines such as IL6 increase phosphorylation of PPARg, thereby decreasing its transcriptional activity. ...
Conjugated linoleic acids (CLAs) are a series of geometric and positional isomers of linoleic acid that have been studied for their effects against diabetes, cancer and atherosclerosis, all conditions with an inflammatory component. Despite the continued interest in CLA, there are many controversies surrounding the health benefits of these fatty acids owing to isomer- and tissue-specific responses. Dietary CLA, found mainly in dairy products and ruminant meat, consists of mainly the 9cis(Z), 11trans(E) isomer, while dietary supplements consist of equal amounts of 9Z,11E- and 10E,12Z-CLA. Other CLA isomers exist, and there is increasing realization that modest alterations in fatty acid structure have profound effects on the biological response observed. In this article, the effects of CLA on inflammation will be discussed with an emphasis on the mechanisms of action and the isomer-specific effects. The primary focus is the macrophage, a classical mediator of inflammatory responses that impact atherogenesis, as well as adipocytes, cells with inflammatory capacity that influence metabolism in distant tissues. These cell types highlight the dramatically different inflammatory responses to specific isomers of CLA. By understanding the effects of the individual CLA isomers on specific cell types, in particular, the dichotomous effects of 10E,12Z-CLA in the macrophage and adipocyte, health claims for the dietary consumption or supplementation of CLA may be more adequately evaluated.
... Mutacja z utratą funkcji w TLR4, receptorze, który indukuje ścieżkę sygnałową NF-kB, zapobiega otyłości wywoływanej dietą [66]. Jednakże istnieją przeczące temu doniesienia, według których ścieżka sygnałowa NF-kB hamuje ekspresję genów swoistych dla adipocytów poprzez obniżenie ekspresji PPARg w komórkach 3T3-L1 [14] i mezenchymalnych komórkach macierzystych [26]. Możliwą przyczyną takich kontrowersji mogą być dawki leków zapalnych stosowane w badaniach prowadzonych na hodowlach komórkowych. ...
Suboptimal fetal environments due to inadequate maternal nutrition, obesity, inflammation or gestational diabetes expose the fetus to humoral cues that alter metabolism and growth parameters leading to metabolic disturbances later in life. The fetal stage is crucial for the development of skeletal muscle, a tissue playing an important role in metabolism. Maternal obesity induces inflammation in the fetus causing modifications in the development of fetal skeletal muscle. Changes in the normal course of myogenesis may arise through several mechanisms: changes in WNT/β-catenin signaling pathway, decreased AMPK activity evoked by TNF-α, increased activity of NF-κB in response to inflammation, which leads to a decrease in myogenic factor MyoD, and increased expression of TGF β1. Modification in fetal development associated with maternal obesity is attributed to epigenetic changes. Polyunsaturated fatty acids supplied in the diet did affect the development of insulin-sensitive tissues during both the fetal and postnatal period. The specific phenotype of skeletal muscle fibers may play a role in the development of obesity, i.e. fiber phenotype I (slow, oxidative) may protect against obesity and insulin resistance. Exploring the mechanisms of direct impact of maternal obesity on the development of tissues in the offspring may help to reduce the occurrence of metabolic diseases in later life.
... The eluted dye was collected, and the optical density at 540 nm was measured. 16 During the fixation of the adipocytes with formaldehyde, photographs were taken to measure cell size. The AlphaImagerÔ 2200 Series program (Alpha Innotech, San Leandro, CA, USA) was used to measure the sizes from the images of the adipocytes. ...
The purpose of the current study was to determine the anti-obesity and anti-inflammatory effects of an extract of purple sweet potatoes (PSPs) on 3T3-L1 adipocytes. For this purpose, differentiated 3T3-L1 adipocytes were treated with a PSP extract at concentrations of 1,000, 2,000, and 3,000 μg/mL for 24 hours. Then, we measured the changes in the sizes of the adipocytes, the secretion of leptin, and the mRNA/protein expression of lipogenic, inflammatory, and lipolytic factors after the treatment with the PSP extract. The PSP extract diminished leptin secretion, indicating that growth of fat droplets was suppressed. The extract also suppressed the expression of mRNAs of lipogenic and inflammatory factors and promoted lipolytic action. The antioxidative activity of the PSP extract was also measured using three different in vitro methods: 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, ferric reducing ability potential assay, and chelating activity of transition metal ions. Taken together, our study shows that PSP extract has antilipogenic, anti-inflammatory, and lipolytic effects on adipocytes and has radical scavenging and reducing activity.
... Loss-offunction mutation in TLR4, a receptor known to induce the NFKB signaling pathway, prevents diet-induced obesity [60]. However, contradictory reports also exist in which NFKB signaling inhibited the expression of adipocyte-specific genes [76] through reducing PPARG expression in 3T3-L1 cells [77] and MSCs [78, 79]. The possible reason for such controversy might be due to the inflammatory drug doses used in cell culture studies. ...
Maternal obesity coupled with Western-style high-energy diets represents a special problem that can result in poor fetal development, leading to harmful, persistent effects on offspring, including predisposition to obesity and type 2 diabetes. Mechanisms linking maternal obesity to the increased incidence of obesity and other metabolic diseases in offspring remain poorly defined. Because skeletal muscle is the principal site for glucose and fatty acid utilization and composes 40%-50% of total body mass, changes in the properties of offspring skeletal muscle and its mass resulting from maternal obesity may be responsible for the increase in type 2 diabetes and obesity. Fetal stage is crucial for skeletal muscle development because there is no net increase in the muscle fiber number after birth. Fetal skeletal muscle development involves myogenesis, adipogenesis, and fibrogenesis, which are all derived from mesenchymal stem cells (MSCs). Shifting commitment of MSCs from myogenesis to adipogenesis and fibrogenesis will result in increased intramuscular fat and connective tissue, as well as reduced numbers of muscle fiber and/or diameter, all of which have lasting negative effects on offspring muscle function and properties. Maternal obesity leads to low-grade inflammation, which changes the commitment of MSCs in fetal muscle through several possible mechanisms: 1) inflammation downregulates wingless and int (WNT) signaling, which attenuates myogenesis; 2) inflammation inhibits AMP-activated protein kinase, which promotes adipogenesis; and 3) inflammation may induce epigenetic modification through polycomb group proteins. More studies are needed to further explore the underlying mechanisms associated with maternal obesity, inflammation, and the commitment of MSCs.
... However, this does not preclude the involvement of additional signaling pathways in Stim1-mediated inhibition of differentiation since cyclosporin A and FK506, at the concentrations used here, did not completely rescue the effects of STIM1 overexpression. In some cells, Ca 2+ is known to enhance the activation of transcription factors such as NFκB (Crabtree, 2001), also an inhibitor of adipocyte differentiation (Chae and Kwak, 2003). It remains to be tested whether modulation of Stim1 levels directly affects additional signaling pathways in 3T3-L1 cells. ...
Ca(2+) plays a complex role in the differentiation of committed pre-adipocytes into mature, fat laden adipocytes. Stim1 is a single pass transmembrane protein that has an essential role in regulating the influx of Ca(2+) ions through specific plasma membrane store-operated Ca(2+) channels. Stim1 is a sensor of endoplasmic reticulum Ca(2+) store content and when these stores are depleted ER-localized Stim1 interacts with molecular components of store-operated Ca(2+) channels in the plasma membrane to activate these channels and induce Ca(2+) influx. To investigate the potential role of Stim1 in Ca(2+)-mediated adipogenesis, we investigated the expression of Stim1 during adipocyte differentiation and the effects of altering Stim1 expression on the differentiation process. Western blotting revealed that Stim1 was expressed at low levels in 3T3-L1 pre-adipocytes and was upregulated 4 days following induction of differentiation. However, overexpression of Stim1 potently inhibited their ability to differentiate and accumulate lipid, and reduced the expression of C/EBP alpha and adiponectin. Stim1-mediated differentiation was shown to be dependent on store-operated Ca(2+) entry, which was increased upon overexpression of Stim1. Overexpression of Stim1 did not disrupt cell proliferation, mitotic clonal expansion or subsequent growth arrest. siRNA-mediated knockdown of endogenous Stim1 had the opposite effect, with increased 3T3-L1 differentiation and increased expression of C/EBP alpha and adiponectin. We thus demonstrate for the first time the presence of store-operated Ca(2+) entry in 3T3-L1 adipocytes, and that Stim1-mediated Ca(2+) entry negatively regulates adipocyte differentiation. We suggest that increased expression of Stim1 during 3T3-L1 differentiation may act, through its ability to modify the level of Ca(2+) influx through store-operated channels, to balance the level of differentiation in these cells in vitro.
... Heterozygous double mutants (Twist1 -/+ , Twist2 -/+ ) exhibit loss of subcutaneous adipose tissue and severe fat deficiency in internal organs [52]. Twist1 is a downstream target of nuclear factor-κB and can repress transcription of tumor necrosis factor-α, which is a potent repressor of adipogenesis [52,53]. The differential expression during adipogenesis of Tcf19 was also confirmed in the present study. ...
Large-scale transcription profiling of cell models and model organisms can identify novel molecular components involved in fat cell development. Detailed characterization of the sequences of identified gene products has not been done and global mechanisms have not been investigated. We evaluated the extent to which molecular processes can be revealed by expression profiling and functional annotation of genes that are differentially expressed during fat cell development.
Mouse microarrays with more than 27,000 elements were developed, and transcriptional profiles of 3T3-L1 cells (pre-adipocyte cells) were monitored during differentiation. In total, 780 differentially expressed expressed sequence tags (ESTs) were subjected to in-depth bioinformatics analyses. The analysis of 3'-untranslated region sequences from 395 ESTs showed that 71% of the differentially expressed genes could be regulated by microRNAs. A molecular atlas of fat cell development was then constructed by de novo functional annotation on a sequence segment/domain-wise basis of 659 protein sequences, and subsequent mapping onto known pathways, possible cellular roles, and subcellular localizations. Key enzymes in 27 out of 36 investigated metabolic pathways were regulated at the transcriptional level, typically at the rate-limiting steps in these pathways. Also, coexpressed genes rarely shared consensus transcription-factor binding sites, and were typically not clustered in adjacent chromosomal regions, but were instead widely dispersed throughout the genome.
Large-scale transcription profiling in conjunction with sophisticated bioinformatics analyses can provide not only a list of novel players in a particular setting but also a global view on biological processes and molecular networks.
... Niektóre z cytokin np. TNF-a, hamuje różnicowanie preadypocytów oraz wzmaga wydzielanie leptyny przez adypocyty, a także wzrost jej stężenia w osoczu [14,49]. Wykazano także, iż pretranskrypcyjne białko E1A tego adenowirusa zwiększa wrażliwość komórek ssaków na TNF-a [19]. ...
This overview of the significance of viral infections in the development of human obesity is presented within context of the commonly recognized obesity risk factors, including the personal and public health consequences of obesity in various countries. In addition, the results of past and recently published studies on the recognition of six taxonomically different viruses which can undoubtedly be associated with obesity progression in some species of animals are summarized. More attention is focused on the results of preliminary epidemiological studies indicating that human infection by the avian adenovirus SMAM-1 or the human adenovirus Ad-36 can be objectively related to symptoms, prevalence, and complications of obesity in some adult men. Proposed pathogenic pathways involved in the observed cases of viral infection-dependent obesity in animals and humans are also briefly described. Urgent implementation of high-throughput diagnostics procedures is advised to extend viral infection-oriented modes of prevention, recognition, and therapy of obesity currently available in modern societies.
... They were rinsed in phosphate buffered saline (Biowhittaker) and incubated in filtered Oil-Red-O staining solution (Sigma) at 4 o C for 1 h. After the staining solution was removed, the dye retained in the cells was eluted into isopropanol and OD540 was determined (Chae and Kwak, 2003). Total protein was determined by Bradford assay. ...
Berberine (BBR), an isoquinoline alkaloid, has a wide range of pharmacological effects, yet its exact mechanism is unknown. In order to understand the anti-adipogenic effect of BBR, we studied the change of expression of several adipogenic enzymes of 3T3-L1 cells by BBR treatment. First, we measured the change of leptin and glycerol in the medium of 3T3-L1 cells treated with 1 micrometer, 5 micrometer and 10 micrometer concentrations of BBR. We also measured the changes of adipogenic and lipolytic factors of 3T3-L1. In 3T3-L1 cells, both leptin and adipogenic factors (SREBP-1c, C/EBP-alpha, PPAR-gamma, fatty acid synthase, acetyl-CoA carboxylase, acyl-CoA synthase and lipoprotein lipase) were reduced by BBR treatment. Glycerol secretion was increased, whereas expression of lipolytic enzymes (hormone-sensitive lipase and perilipin) mRNA was slightly decreased. Next, we measured the change of inflammation markers of 3T3-L1 cells by BBR treatment. This resulted in the down-regulation of mRNA level of inflammation markers such as TNF-alpha, IL-6, C- reactive protein and haptoglobin. Taken together, our data shows that BBR has both anti-adipogenic and anti-inflammatory effects on 3T3-L1 adipocytes, and the anti-adipogenic effect seems to be due to the down-regulation of adipogenic enzymes and transcription factors.
... OPG is a circulating receptor without a transmembrane domain and it inhibits osteoclast differentiation by acting as a decoy receptor to RANK that is expressed on the osteoclast precursor surface , and it interferes with RANK's interaction with RANKL (Simonet et al., 1997; Yasuda et al., 1998; Woo et al., 2002 ). Considering that NF-κB pathway is involved in the inhibitory mechanism of cytokine in adipocyte differentiation through downregulation of PPAR-γ (Chae and Kwak, 2003) and the previously mentioned mechanism of PPAR-γ agonist inhibiting osteoclast differentiation through the inhibition of NF-κB transcription for inhibiting RANKL signaling, we could assume that the inhibition of osteoclastogenesis by OPG might have direct association with PPAR-γ activation. In this study, mean serum OPG level was significantly lower and serum total ALP was significantly higher in the Pro12Ala genotype group compared with the Pro12Pro genotype group, even after adjustment for other confounding factors, and Pro12Ala was the significant determinant of serum OPG levels in the multiple regression analysis. ...
Recent evidences suggest that the activation of peroxisome proliferator-activated receptor (PPAR)-gamma, which is an important transcriptional factor in adipocyte differentiation, also plays an important role in the bone microenvironment. The objective of the study was to clarify whether Pro12Ala polymorphism was related to the serum OPG levels and bone mineral metabolism in healthy Korean women. In 239 Korean women (mean age 51 years), who participated in medical check-up program in a health promotion center, anthropometric measurements, lumbar spine and femoral neck bone mineral density (BMD), bone turnover markers, such as serum total alkaline phosphatase (ALP) levels, urine deoxypyridinoline levels, and 24-h urine calcium excretion were measured. Serum levels of OPG were measured with ELISA method. DNAs were extracted from the samples and the genotyping of the Pro12Ala polymorphism (rs1801282) in the PPAR-gamma gene was performed via an allelic discrimination assay using a TaqMan probe. In addition, we examined the haplotype analysis between two polymorphisms of PPAR-gamma gene, Pro12Ala in exon B and C161T in exon 6 (rs3856806). Allelic frequencies were 0.950 for Pro allele and 0.050 for Ala allele, which was in compliance with Hardy- Weinberg equilibrium, and there was no Ala12Ala genotype among the genotyped subjects. Mean serum OPG level was significantly lower (P=0.035), and serum total ALP was significantly higher (P=0.014) in the Pro12Ala genotype group compared with the Pro12Pro genotype group, which were consistently significant even after adjustment for weight, height, and serum follicle stimulating hormone (FSH). In multiple regression analysis with serum OPG as the dependent variable and age, weight, ALP, femoral neck BMD and Pro12Ala genotype included in the model, only Pro12Ala genotype was significant determinant of serum OPG level (b=??0.136, P=0.035). The haplotype analysis with C161T polymorphism revealed that subjects with Ala and T alleles showed significantly lower serum OPG levels compared with those with Pro12Pro/CC genotype, which were consistently significant even after adjustment for age, weight, height and FSH (P=0.010). This result suggests statistically significant association of Pro12Ala polymorphisms with serum OPG levels in Korean females.
The biological relevance of cytokines is known for more than 20 years. Evidence suggests that adipogenesis is one of the biological events involved in the regulation of cytokines, and pro-inflammatory cytokines (e.g., TNFα and IL-1β) inhibit adipogenesis through various pathways. This inhibitory effect can constrain the hyperplastic expandability of adipose tissues. Meanwhile, chronic low-grade inflammation is commonly observed in obese populations. In some individuals, the impaired ability of adipose tissues to recruit new adipocytes to adipose depots during overnutrition results in adipocyte hypertrophy, ectopic lipid accumulation, and insulin resistance. Intervention studies showed that pro-inflammatory cytokine antagonists improve metabolism in patients with metabolic syndrome. This review focuses on the cytokines currently known to regulate adipogenesis under physiological and pathophysiological circumstances. Recent studies on how inhibited adipogenesis leads to metabolic disorders were summarized. Although the interplay of cytokines and lipid metabolism is yet incompletely understood, cytokines represent a class of potential therapeutic targets in the treatment of metabolic disorders.
Tumor necrosis factor-α (TNFα) and other ligands of the TNF superfamily are potent regulators of adipose tissue metabolism and play a crucial role in the obesity-induced inflammation of adipose tissue. Adipose tissue expression levels of TRAIL (TNF-related apoptosis-inducing ligand) and its receptor were shown to be upregulated by overfeeding and decreased by fasting in mice. In the present study we aimed to elucidate the impact of TRAIL on adipogenesis. To this end, human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes as well as stromal-vascular cells isolated from human white adipose tissue were used as model systems. Human recombinant TRAIL inhibited adipogenic differentiation in a dose-dependent manner. It activated the cleavage of caspase-8 and -3, which in turn resulted in a downregulation of the key adipogenic transcription factors C/EBPα, C/EBPδ, and PPARγ. The effect was completely blocked by pharmacological or genetic inhibition of caspases. Taken together we discovered a so far unrecognized function of TRAIL in the regulation of adipogenesis. Targeting the TRAIL/TRAIL receptor system might provide a novel strategy to interfere with adipose tissue homeostasis.
Study design:
Longitudinal case control animal model.
Objective:
To investigate effects of mesenchymal stem cell (MSC) treatment on multifidus muscle remodeling after intervertebral disc (IVD) lesion.
Summary of background data:
Lesion and degeneration of IVDs causes structural remodeling of the multifidus muscle. Pro-inflammatory cytokines are thought to contribute. MSC-treatment restores IVD health after lesion but its effects on surrounding tissues remains unknown. Using an animal model of IVD degeneration, we assessed the effects of MSC-treatment of IVDs on the structural remodeling and cytokine expression within the multifidus muscle.
Methods:
An anterolateral lesion was performed on the L1-2, L3-4 and L5-6 IVDs in sheep. At either 4 (early treatment) or 12 (late treatment) weeks after IVD lesion, MSCs were injected into the lesioned IVD. Multifidus muscle was harvested from L2 (gene expression analysis) and L4 (histological analysis) at 3 or 6 months after IVD lesion and naïve controls for histological analysis of muscle, adipose and connective tissue cross sectional areas (CSA), and immunohistochemistry to study muscle fiber types. Real-time polymerase chain reactions quantified expression of TNF, IL-1β and TGF-β1.
Results:
MSC-treatment of IVD lesion prevented the increased adipose and connective tissue CSA expected after IVD lesion. MSC-treatment did not prevent slow-to-fast muscle fiber type transformation. Gene expression of pro-inflammatory cytokines within the muscle was altered by the MSC-treatment of IVD. Increased IL-1β expression was prevented in the early treatment group and TNF and TGF-β1 expression was upregulated at 6 months.
Conclusion:
Results show that although MSC-treatment prevents fatty infiltration and fibrosis of the multifidus muscle after IVD lesion, it cannot prevent a muscle inflammatory response and muscle fiber transformation. These findings highlight the potential role of MSC therapy after IVD injury, but reveals that other interventions may also be necessary to optimize recovery of muscle.
Level of evidence:
4.
Introduction: The multifidus muscle undergoes structural and behavioral changes with back pain and injury. After intervertebral disc (IVD) lesion in animals the multifidus muscle undergoes a transformation of muscle fiber types from slow-to-fast, and extensive structural remodelling with increased adipose and connective tissue. Increased expression of pro-inflammatory cytokines (tumor necrosis factor (TNF) and interleukin 1β (ΙΛ−1β)) parallel these changes and may be responsible. Treatment of IVD lesions with mesenchymal stem cells (MSC) prevents or restores loss of IVD height and proteoglycans in the nucleus pulposus, depending on the timing of application. Whether the resolution of IVD changes by MCS treatment prevents or restores changes in the multifidus muscle structure and muscle fiber composition is unknown. This study aimed to investigate whether muscle changes are prevented or restored by early or late MSC treatment of the IVD lesion, respectively, and whether this is related to modification of inflammatory cytokine expression. Material and Methods: The L1–2 and L3–4 IVDs of 18 sheep received left anterolateral partial thickness annular lesions. Six control sheep underwent no surgery. At four (3-month (n = 6) and 6-month acute (n = 6) treatment groups) or twelve (established (n = 6) treatment group) weeks after initial surgery, animals received MSC injections (0.2ml) to the operated IVD. Three (3-month acute treatment group) and six (6-month acute and established treatment groups) months after initial surgery the L4 multifidus muscle was harvested for muscle fiber type analysis using immunohistochemistry and evaluation of cross sectional area (CSA) of muscle, adipose and connective tissue using standard histology. L2 muscle was harvested for quantitative PCR measures of pro-inflammatory cytokine gene expression (TNF, ΙΛ−1β). Results: Unlike the response to IVD lesion without MSC treatment (increased connective tissue and adipose CSA), acute MSC treatment prevented increase in adipose (acute treatment=control at 3- and 6-months; p = 0.28 and p = 0.07) and connective tissue CSA (acute treatment 0.60). The effect of MSC on muscle CSA depended on the treatment timing. At 6 months, acute treatment animals had larger whole muscle CSA than controls (p = 0.002). Control and established treatment animals did not differ (p = 0.73). Despite optimistic data for tissue CSA, muscle harvested at 6 months showed reduced proportion of slow muscle fibers and increased intermediate fibers throughout the multifidus muscle (acute and established treatment group versus control; p < 0.05). TNF expression was greater at 6 months in acute and established treatment groups than control (p < 0.001). Conclusion: These results indicate that MSC treatment of the IVD lesion prevents and restores muscle structural changes, but is unable to prevent changes to multifidus muscle fiber type. This is likely to have functional relevance for neuromuscular control of the healed IVD. MSC appear to have and anti-inflammatory effect on muscle in the early phase when IVD is healing, but cannot influence the later elevation of TNF, which appears destructive for muscle fibers.
Objectives : The present study was designed to investigate effects and molecular mechanisms of Atractylodes macrocephala Koidzumi extracts(AMK) on the improvement of adipocyte dysfunction induced by TNF- in 3T3-L1 adipocytes. We examined whether AMK could directly influence the inflammation and insulin resistance in 3T3-L1 adipocytes. Methods : Potential roles of AMK in the lipolysis, production of inflammatory adipokines and ROS, expression and phosphorylation of ERK, JNK, and protein, and expression of and C/EBP were investigated in this study. Results : Our data demonstrated that TNF- significantly increased lipolysis, levels of MCP-1, IL-6, and ROS and phosphorylation of ERK, JNK, and protein, while TNF- reduced the expression of and C/EBP in adipocytes, suggesting that TNF- induced a condition with the occurrence of inflammation and insulin resistance. Those alterations induced by TNF- were prevented by the treatment of AMK. AMK down-regulated the phosphorylation of ERK, JNK, and protein and up-regulated the expression of and C/EBP on TNF--induced inflammation and insulin resistance. Conclusions : Thus, our results indicate that AMK can be used to prevent from the TNF--induced adipocyte dysfunction through MAPK, and pathways.
The elevation of circulating LPS has been associated with obesity and aging. However, whether and how LPS contributes to adipose tissue dysfunction is unclear. In this study, we investigated the effect of LPS on the adipogenic capacity and cellular senescence of adipocyte progenitors. Stromal-vascular cells were isolated from inguinal adipose tissue of C57BL/6 mice and treated with LPS during the different time periods of adipocyte differentiation. We found that LPS treatment for 24hr prior to the induction of differentiation led to the most profound effect on the inhibition of adipogenesis, as evidenced by the morphological changes and the decreased mRNA expression of adipocyte marker genes. In addition, LPS induced features of pre-mature senescence of SV cells, including the activation of p53, the elevation of SA-β-gal activity and increased hydrogen peroxide production, but not telomere length. Upon LPS treatment, SV cells also developed senescence-associated secretory phenotype (SASP), as demonstrated by the increased expression of TNF-α, IL-1β, IL-6, MCP1 and VEGFα. Blocking LPS-induced NF-κB activation and cytokine production by Bay 11-7082 failed to rescue the impaired adipogenesis and the reduction in PPARγ and Zfp423 expression. On the contrary, Rosiglitazone had little effect on cytokine production, but corrected the defective adipogenic potential. In conclusion, we demonstrate that LPS inhibits adipogenesis by disrupting the differentiation of adipocyte progenitors in a NF-κB-independent manner; LPS also induces pre-mature senescence of adipocyte progenitors. Our data suggest that LPS could be a potential contributor to the defective adipogenesis and senescence in adipose tissue during obesity.
Copyright © 2015, American Journal of Physiology - Endocrinology and Metabolism.
Although tumor necrosis factor-α (TNF-α) is elevated in adipose tissue in obesity and may contribute to the cardiovascular and metabolic risks associated with this condition, the mechanisms leading to elevated TNF-α remain elusive. We hypothesized that autoamplification of TNF-α contributes to the maintenance of elevated TNF-α in obesity. Treatment of 3T3-L1 adipocytes with TNF-α, or injection of TNF-α into C57BL/6J mice, up-regulated TNF-α mRNA in adipocytes and in adipose tissues, respectively. Ob/ob male but not female mice lacking TNF-α receptors showed significantly lower levels of adipose TNF-α mRNA when compared with TNF-α receptor-expressing ob/ob mice. Thus, the lack of endogenous TNF-α signaling reduced adipose TNF-α mRNA in ob/ob male mice. Additionally, hyperinsulinemia potentiated this TNF-α-mediated autoamplification response in adipose tissues and in adipocytes in a synergistic and dose-dependent manner. Studies in which TNF-α was injected into lean mice lacking individual TNF-α receptors indicated that TNF-α autoamplification in adipose tissues was mediated primarily via the p55 TNF-α receptor whereas the p75 TNF-α receptor appeared to augment this response. Finally, TNF-α autoamplification in adipocytes occurred via the protein kinase C signaling pathway and the transcription factor nuclear factor-κB. Thus, TNF-α can positively autoregulate its own biosynthesis in adipose tissue, contributing to the maintenance of elevated TNF-α in obesity.
Longitudinal case controlled animal study.
To investigate putative cellular mechanisms to explain structural changes in muscle, adipose and connective tissues of the back muscles after intervertebral disc(IVD) injury.
Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time-course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level.
Multifidus muscle was harvested from both sides of the spine at L1-2 and L3-4 IVDs in 27 castrated male sheep at 3(n = 10) or 6(n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery(3 months n = 4; 6 months n = 6). Tissue was harvested at L4 for histological analysis of cross sectional area(CSA) of muscle, adipose and connective tissue(whole muscle), plus immunohistochemistry to identify proportion and CSA of individual muscle fiber types in the deepest fascicle. Quantitative PCR measured gene expression of typical cytokines/signaling molecules at L2.
Contrary to predictions, there was no multifidus muscle atrophy(whole muscle or individual fiber). There was increased adipose and connective tissue(fibrotic proliferation) CSA and slow-to-fast muscle fiber transition at 6, but not 3 months. Within the multifidus muscle increases in the expression of several cytokines(TNFa and IL-1b) and molecules that signal trophic/atrophic processes for the three tissue types(e.g. growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifers [calcineurin, PCG-1α, and myostatin]) were present.
This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions.
The zinc-finger protein A20 is a key player in the negative feedback regulation of the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) pathway in response to multiple stimuli. Tumor necrosis factor alpha (TNFα), a cytokine with pleiotropic effects on cellular proliferation and differentiation, dramatically increases A20 expression in all tissues. As TNFα inhibits adipocyte differentiation, we have determined the contribution of A20 to the adipogenic capacity of human mesenchymal stromal cells (MSCs). Here we show that A20 is constitutively expressed in MSCs, which previously has been observed only in cells that are either tumor or immune cells (T/B lymphocytes). TNFα stimulation induced a rapid degradation of A20 protein mediated exclusively by the proteasome in MSCs and not by caspases. This degradation is concomitant to the induction of its own mRNA, which suggests that a tight regulation of NF-κB signaling in MSCs is fundamental. On one hand, we demonstrate that the knockdown of A20-mediated transcript dramatically decreases the adipogenic capacity of MSCs, which correlates with the phenotype observed in the presence of TNFα. On the other hand, A20 overexpression blocks NF-κB activation and drives to increased adipogenesis, even in the presence of TNFα treatment. In conclusion, our data demonstrate that the presence of A20 allows MSCs to differentiate into adipocytes by maintaining NF-κB signaling at a basal state.
Objective:
Persistent inflammation and impaired adipogenesis are frequent features of obesity and underlie the development of its complications. However, the factors behind adipose tissue dysfunction are not completely understood. Previously it was shown that histone demethylase KDM1A is required for adipogenesis.
Design and methods:
Kdm1a expression was knocked down in 3T3-L1 preadipocytes by siRNA transfection and whole-genome expression profiling was performed by microarray hybridization. The role of NF-κβ and C/EBPβ was analyzed by incubation with the inhibitor parthenolide and by cebpb knockdown, respectively.
Results:
Knockdown of kdm1a or rcor2 in 3T3-L1 preadipocytes results in impaired differentiation and induction of inflammatory gene expression. Enhanced expression of il6 in kdm1a knocked down preadipocytes is associated with increased recruitment of C/EBPβ and the NF-κβ subunit RelA to the il6 promoter. Cebpb knockdown attenuates the induction of il6 expression in kdm1a knocked down cells, whereas simultaneous cebpb knockdown and NF-κβ inhibition abrogates it. Dietary-induced and genetic mouse models of obesity display decreased KDM1A in adipose tissue, and this correlates with increased expression of proinflammatory genes and C/EBPβ.
Conclusion:
KDM1A represses the expression of inflammatory genes in preadipocytes. Dysregulated kdm1a expression in preadipocytes may thus participate in the development of obesity-associated inflammation.
Toll-like receptors (TLRs) sense microorganism components and are critical host mediators of inflammation during infection.
Recently, TLRs have been reported to be involved in cell proliferation and differentiation. We previously reported that TLR
agonists might affect proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs). In
this study, we sought to determine whether TLR signaling is dependent on MyD88 in hASCs. The hASCs were downregulated using
LV-GFP-miR-MyD88, a lentiviral construct inserted siRNA against human MyD88 that significantly inhibited cell proliferation.
MyD88 downregulation reduced NF-κB activation and enhancement of osteogenic differentiation induced by peptidoglycan (PGN)
more significantly than that induced by lipopolysaccharide (LPS). Although LPS- and PGN-induced cytokine secretions were decreased
greatly by MyD88 downregulation, IFN-gamma-induced protein-10 (IP10) and IFNβ expression were enhanced by LPS irrespective
of the downregulation of MyD88. These results suggest that TLR signaling is mediated via MyD88-independent pathways as well
as MyD88-dependent pathways in hASCs and that MyD88 contributes to the regulation of cell proliferation and differentiation
in hASCs.
The aim of this study was to examine the expression of G protein-coupled receptor 81 (GPR81) in mouse adipose tissue in response to inflammatory stimuli. GPR81 is activated by lactate resulting in the inhibition of lipolysis.
Mice were injected with saline, lipopolysaccharide (LPS), zymosan, or turpentine, N = 5 per group. 3T3-L1 adipocytes were treated with tumor necrosis factor alpha, interleukin (IL)-l beta, IL-6, or interferon gamma.
GPR81 expression levels were measured by real-time PCR and statistical significance was determined by Student's t test.
LPS resulted in a marked decrease in GPR81 mRNA level in mouse adipose tissue in C57BL/6 and OuJ mice, an effect that was not observed in HeJ mice, which have a mutation in TLR4. Zymosan and turpentine also decreased adipose tissue GPR81 expression. Cytokine treatment of 3T3-L1 adipocytes had no effect on GPR81 expression. GPR81 expression was decreased in ob/ob mice, an animal model of type 2 diabetes that is characterized by inflammation.
Inflammation decreases the expression of GPR81 in adipose tissue.
Although tumor necrosis factor-alpha (TNF-alpha) is elevated in adipose tissue in obesity and may contribute to the cardiovascular and metabolic risks associated with this condition, the mechanisms leading to elevated TNF-alpha remain elusive. We hypothesized that autoamplification of TNF-alpha contributes to the maintenance of elevated TNF-alpha in obesity. Treatment of 3T3-L1 adipocytes with TNF-alpha, or injection of TNF-alpha into C57BL/6J mice, up-regulated TNF-alpha mRNA in adipocytes and in adipose tissues, respectively. Ob/ob male but not female mice lacking TNF-alpha receptors showed significantly lower levels of adipose TNF-alpha mRNA when compared with TNF-alpha receptor-expressing ob/ob mice. Thus, the lack of endogenous TNF-alpha signaling reduced adipose TNF-alpha mRNA in ob/ob male mice. Additionally, hyperinsulinemia potentiated this TNF-alpha-mediated autoamplification response in adipose tissues and in adipocytes in a synergistic and dose-dependent manner. Studies in which TNF-alpha was injected into lean mice lacking individual TNF-alpha receptors indicated that TNF-alpha autoamplification in adipose tissues was mediated primarily via the p55 TNF-alpha receptor whereas the p75 TNF-alpha receptor appeared to augment this response. Finally, TNF-alpha autoamplification in adipocytes occurred via the protein kinase C signaling pathway and the transcription factor nuclear factor-kappaB. Thus, TNF-alpha can positively autoregulate its own biosynthesis in adipose tissue, contributing to the maintenance of elevated TNF-alpha in obesity.
Preadipocyte differentiation capacity declines between middle and old age. Expression of the adipogenic transcription factors, CCAAT/enhancer-binding protein (C/EBP) alpha and peroxisome proliferator-activated receptor gamma (PPARgamma), is lower in differentiating preadipocytes from old than young animals, although no age-related changes occur in C/EBPbeta mRNA, which is upstream of C/EBPalpha and PPARgamma. C/EBPbeta-liver-enriched inhibitory protein (C/EBPbeta-LIP), a truncated C/EBPbeta isoform that is a dominant inhibitor of differentiation, increases with aging in rat fat tissue and preadipocytes. CUG triplet repeat-binding protein-1 (CUGBP1) binds to C/EBPbeta mRNA, increasing C/EBPbeta-LIP translation. Abundance and nucleotide binding activity of CUGBP1 increased with aging in preadipocytes. CUGBP1 overexpression in preadipocytes from young animals increased C/EBPbeta-LIP and impaired adipogenesis. Decreasing CUGBP1 in preadipocytes from old rats by RNA interference reduced C/EBPbeta-LIP abundance and promoted adipogenesis. Tumor necrosis factor-alpha, levels of which are elevated in fat tissue with aging, increased CUGBP1 protein, CUGBP1 binding activity, and C/EBPbeta-LIP in preadipocytes from young rats. Thus, CUGBP1 contributes to regulation of adipogenesis in primary preadipocytes and is responsive to tumor necrosis factor-alpha. With aging, preadipocyte CUGBP1 abundance and activity increases, resulting in enhanced translation of the C/EBPbeta-LIP isoform, thereby blocking effects of adipogenic transcription factors, predisposing preadipocytes from old animals to resist adipogenesis. Altered translational processing, possibly related to changes in cytokine milieu and activation of stress responses, may contribute to changes in progenitor differentiation and tissue function with aging.
Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.
We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPdelta, GATA domain-binding protein 3 (GATA3), C/EBPbeta, peroxisome proliferator-activated receptor (PPAR) gamma2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor alpha, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARgamma coactivator 1alpha, PPARgamma coactivator 1beta, and PPARdelta] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.
Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 +/- 1.9 vs. 7.5 +/- 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 +/- 0.26 vs. 0.78 +/- 0.17 microm; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARgamma2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPbeta only tended to be lower (p = 0.07).
This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.
Mesenchymal stem cells (MSCs) are widespread in adult organisms and may be involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. Thus, it is important to discover the factors controlling MSC renewal and differentiation. Here we report that adult MSCs express functional Toll-like receptors (TLRs), confirmed by the responses of MSCs to TLR ligands. Pam3Cys, a prototypic TLR-2 ligand, augmented interleukin-6 secretion by MSC, induced nuclear factor kappa B (NF-kappaB) translocation, reduced MSC basal motility, and increased MSC proliferation. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We show herein that Pam3Cys inhibited MSC differentiation into osteogenic, adipogenic, and chondrogenic cells while sparing their immunosuppressive effect. Our study therefore shows that a TLR ligand can antagonize MSC differentiation triggered by exogenous mediators and consequently maintains the cells in an undifferentiated and proliferating state in vitro. Moreover, MSCs derived from myeloid factor 88 (MyD88)-deficient mice lacked the capacity to differentiate effectively into osteogenic and chondrogenic cells. It appears that TLRs and their ligands can serve as regulators of MSC proliferation and differentiation and might affect the maintenance of MSC multipotency.
Tumour necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is a potent negative regulator of adipocyte differentiation. However, the mechanism of TNF-alpha-mediated antiadipogenesis remains incompletely understood. In this study, we first confirm that TNF-alpha inhibits adipogenesis of 3T3-L1 preadipocytes by preventing the early induction of the adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). This suppression coincides with enhanced expression of several reported mediators of antiadipogenesis that are also targets of the Wnt/beta-catenin/T-cell factor 4 (TCF4) pathway. Indeed, we found that TNF-alpha enhanced TCF4-dependent transcriptional activity during early antiadipogenesis, and promoted the stabilisation of beta-catenin throughout antiadipogenesis. We analysed the effect of TNF-alpha on adipogenesis in 3T3-L1 cells in which beta-catenin/TCF signalling was impaired, either via stable knockdown of beta-catenin, or by overexpression of dominant-negative TCF4 (dnTCF4). The knockdown of beta-catenin enhanced the adipogenic potential of 3T3-L1 preadipocytes and attenuated TNF-alpha-induced antiadipogenesis. However, beta-catenin knockdown also promoted TNF-alpha-induced apoptosis in these cells. In contrast, overexpression of dnTCF4 prevented TNF-alpha-induced antiadipogenesis but showed no apparent effect on cell survival. Finally, we show that TNF-alpha-induced antiadipogenesis and stabilisation of beta-catenin requires a functional death domain of TNF-alpha receptor 1 (TNFR1). Taken together these data suggest that TNFR1-mediated death domain signals can inhibit adipogenesis via a beta-catenin/TCF4-dependent pathway.
Fat depot sizes peak in middle age but decrease by advanced old age. This phenomenon is associated with ectopic fat deposition, decreased adipocyte size, impaired differentiation of preadipocytes into fat cells, decreased adipogenic transcription factor expression, and increased fat tissue inflammatory cytokine generation. To define the mechanisms contributing to impaired adipogenesis with aging, we examined the release of TNFalpha, which inhibits adipogenesis, and the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which blocks activity of adipogenic C/EBP family members, in preadipocytes cultured from young, middle-aged, and old rats. Medium conditioned by fat tissue, as well as preadipocytes, from old rats impeded lipid accumulation by preadipocytes from young animals. More TNFalpha was released by preadipocytes from old than young rats. Differences in TNFalpha-converting enzyme, TNFalpha degradation, or the presence of macrophages in cultures were not responsible. TNFalpha induced rat preadipocyte CHOP expression. CHOP was higher in undifferentiated preadipocytes from old than younger animals. Overexpression of CHOP in young rat preadipocytes inhibited lipid accumulation. TNFalpha short interference RNA reduced CHOP and partially restored lipid accumulation in old rat preadipocytes. CHOP normally increases during late differentiation, potentially modulating the process. This late increase in CHOP was not affected substantially by aging: CHOP was similar in differentiating preadipocytes and fat tissue from old and young animals. Hypoglycemia, which normally causes an adaptive increase in CHOP, was less effective in inducing CHOP in preadipocytes from old than younger animals. Thus increased TNFalpha release by undifferentiated preadipocytes with elevated basal CHOP contributes to impaired adipogenesis with aging.
Skeletal muscle and adipose tissue development often has a reciprocal relationship in vivo, particularly in myodystrophic states. We have investigated whether determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPAR gamma and C/EBP alpha. When cultured under optimal conditions for muscle differentiation, murine G8 myoblasts expressing PPAR gamma and C/EBP alpha show markedly reduced levels of the myogenic basic helix-loop-helix proteins MyoD, myogenin, MRF4, and myf5 and are completely unable to differentiate into myotubes. Under conditions permissive for adipogenesis including a PPAR activator, these cells differentiate into mature adipocytes that express molecular markers characteristic of this lineage. Our results demonstrate that a developmental switch between these two related but highly specialized cell types can be controlled by the expression of key adipogenic transcription factors. These factors have an ability to inhibit myogenesis that is temporally and functionally separate from their ability to stimulate adipogenesis.
PPARγ is a member of the PPAR subfamily of nuclear receptors. In this work, the structure of the human PPARγ cDNA and gene was determined, and its promoters and tissue-specific expression were functionally characterized. Similar to the mouse, two PPAR isoforms, PPARγ1 and PPARγ2, were detected in man. The relative expression of human PPARγ was studied by a newly developed and sensitive reverse transcriptase-competitive polymerase chain reaction method, which allowed us to distinguish between PPARγ1 and γ2 mRNA. In all tissues analyzed, PPARγ2 was much less abundant than PPARγ1. Adipose tissue and large intestine have the highest levels of PPARγ mRNA; kidney, liver, and small intestine have intermediate levels; whereas PPARγ is barely detectable in muscle. This high level expression of PPARγ in colon warrants further study in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPARγs, the human PPARγs are activated by thiazolidinediones and prostaglandin J and bind with high affinity to a PPRE. The human PPARγ gene has nine exons and extends over more than 100 kilobases of genomic DNA. Alternate transcription start sites and alternate splicing generate the PPARγ1 and PPARγ2 mRNAs, which differ at their 5'-ends. PPARγ1 is encoded by eight exons, and PPARγ2 is encoded by seven exons. The 5'-untranslated sequence of PPARγ1 is comprised of exons A1 and A2, whereas that of PPARγ2 plus the additional PPARγ2-specific N-terminal amine acids are encoded by exon B, located between exons A2 and A1. The remaining six exons, termed 1 to 6, are common to the PPARγ1 and γ2. Knowledge of the gene structure will allow screening for PPARγ mutations in humans with metabolic disorders, whereas knowledge of its expression pattern and factors regulating its expression could be of major importance in understanding its biology.
Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with multiple biological activities and exists in two forms as follows: a 17-kDa soluble form that is a cleaved product of the 26-kDa transmembrane form (mTNFalpha). It has been suggested that the transmembrane form of TNFalpha is mainly responsible for localized responses via cell-cell contact. Here, we have examined the activities of transmembrane TNFalpha in cultured adipocytes. A non-cleavable transmembrane form of TNFalpha (mTNFDelta1-9K11E) was expressed in several preadipocyte cell lines using retroviral gene transfer. In wild type preadipocytes carrying both TNF receptors, expression of mTNFDelta1-9K11E resulted in inhibition of the differentiation program. The extent of this varied depending on the nature and strength of the adipogenic stimuli. The TNF receptor responsible for this function was determined by expressing mTNFDelta1-9K11E in preadipocyte cell lines lacking either TNF receptor 1 (TNFR1), 2 (TNFR2), or both. In order to confirm the results in the same cellular background, TNF receptors were also reconstituted in the cell lines lacking corresponding receptors. These experiments demonstrated that TNFR1 was necessary and sufficient for mediating mTNFDelta1-9K11E-induced inhibition of adipogenesis and that this action was similar to that of soluble TNFalpha. In conclusion, our results indicate that mTNFDelta1-9K11E is biologically active in cultured adipocytes and can alter the adipogenic program of these cells by selectively activating TNFR1. This may have physiological implications where local TNFalpha actions are thought to be generated at sites such as adipose tissue.
Mitogen-activated protein kinase (MAPK) is required for cell growth and cell differentiation. In adipogenesis, MAPK activation
opposes the differentiation process. The regulatory mechanisms or the cellular factors that regulate the switch between growth
and differentiation in the adipogenic lineage have been largely unelucidated. We show here that AEBP1, a transcriptional repressor
that is down-regulated during adipogenesis, complexes and protects MAPK from its specific phosphatase in mammalian cells.
We further show evidence that the modulation of MAPK activation by AEBP1 is a biologically relevant process in adipogenesis.
Our results suggest that modulation of MAPK activation by the protective effect of AEBP1 may constitute a critical part in
the determination between cell growth and differentiation in the adipogenic lineage. The proposed mode of action by which
a transcription factor regulates MAPK activation is novel.
Insulin is a potent adipogenic hormone that triggers an induction of a series of transcription factors governing differentiation of pre-adipocytes into mature adipocytes. However, the exact link between the insulin signaling cascade and the intrinsic cascade of adipogenesis remains incompletely understood. Herein we demonstrate that inhibition of prenylation of p21ras and Rho-A arrests insulin-stimulated adipogenesis. Inhibition of farnesylation of p21ras also blocked the ability of insulin to activate mitogen-activated protein (MAP) kinase and cyclic AMP response element-binding (CREB) protein. Expression of two structurally different inducible constitutively active CREB constructs rescued insulin-stimulated adipocyte differentiation from the inhibitory influence of prenylation inhibitors. Constitutively active CREB constructs induced expression of PPARgamma2, fatty acid synthase, GLUT-4, and leptin both in control and prenylation inhibitors-treated cells. It appears that insulin-stimulated prenylation of the Ras family GTPases assures normal phosphorylation and activation of CREB that, in turn, triggers the intrinsic cascade of adipogenesis.
Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine with a proposed role in obesity-related insulin resistance. This could be mediated by increased lipolysis in adipose tissue resulting in elevated free fatty acid levels. The early intracellular signals entailed in TNF-alpha-mediated lipolysis are unknown but may involve members of the mitogen-activated protein kinase (MAPK) family. We investigated the possible contribution of MAPK in TNF-alpha-induced lipolysis in human preadipocytes. TNF-alpha activated the three mammalian MAPK, p44/42, JNK, and p38, in a distinct time- and concentration-dependent manner. TNF-alpha also induced a concentration-dependent stimulation of lipolysis with a more than 3-fold increase at the maximal dose. Lipolysis was completely inhibited by blockers specific for p44/42 (PD98059) and JNK (dimetylaminopurine) but was not affected by the p38 blocker SB203580. Use of receptor-specific TNF-alpha mutants showed that activation of MAPK is entirely mediated by the TNFR1 receptor. The results in human preadipocytes differed from those obtained in murine 3T3-L1 adipocytes in which all three MAPK were constitutively active. Thus, studies of intracellular signaling pathways obtained in different cellular contexts should be interpreted with caution. In conclusion, although TNF-alpha activates all three known MAPK in human preadipocytes, only p44/42 and JNK appear to be involved in the regulation of lipolysis.
The process of adipogenesis is known to involve the interplay of several transcription factors. Activation of one of these factors, the nuclear hormone receptor PPARγ, is known to promote fat cell differentiation in vitro. Whether PPARγ is required for this process in vivo has remained an open question because a viable loss-of-function model for PPARγ has been lacking. We demonstrate here that mice chimeric for wild-type and PPARγ null cells show little or no contribution of null cells to adipose tissue, whereas most other organs examined do not require PPARγ for proper development. In vitro, the differentiation of ES cells into fat is shown to be dependent on PPARγ gene dosage. These data provide direct evidence that PPARγ is essential for the formation of fat.
The current study was done to assess if heterogeneity existed in the degree of adipogenesis in stromal cells (preadipocytes) from multiple donors. In addition to conventional lipid-based methods, we have employed a novel signal amplification technology, known as branched DNA, to monitor expression of an adipocyte specific gene product aP2. The fatty acid binding protein aP2 increases during adipocyte differentiation and is induced by thiazolidinediones and other peroxisome proliferator activated receptor γ ligands. The current work examined the adipogenic induction of aP2 mRNA levels in human adipose tissue stromal cells derived from 12 patients (mean age ± SEM, 38.9 ± 3.1) with mild to moderate obesity (mean body mass index ± SEM, 27.8 ± 2.4). Based on branched DNA technology, a rapid and sensitive measure of specific RNAs, the relative aP2 level in adipocytes increased by 679 ± 93-fold (mean ± SEM, n=12) compared to preadipocytes. Normalization of the aP2 mRNA levels to the housekeeping gene, glyceraldehyde phosphate dehydrogenase, did not significantly alter the fold induction in a subset of 4 patients (803.6 ± 197.5 vs 1118.5 ± 308.1). Independent adipocyte differentiation markers were compared between adipocytes and preadipocytes in parallel studies. Leptin secretion increased by up to three-orders of magnitude while measurements of neutral lipid accumulation by Oil Red O and Nile Red staining increased by 8.5-fold and 8.3-fold, respectively. These results indicate that preadipocytes isolated from multiple donors displayed varying degrees of differentiation in response to an optimal adipogenic stimulus in vitro. This work also demonstrates that branched DNA measurement of aP2 is a rapid and sensitive measure of adipogenesis in human stromal cells. The linear range of this assay extends up to three-orders of magnitude and correlates directly with independent measures of cellular differentiation. J. Cell. Biochem. 810:312–319, 2001. © 2001 Wiley-Liss, Inc.
When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.
Certain infections and malignancies in mammals cause the development of a condition known as cachexia in which the animal
continues to lose weight, often while consuming an adequate diet. When macrophages are stimulated with an endotoxin, they
produce a factor or factors, termed cachectin, that inhibits the activity of fat-producing (lipogenic) enzymes in cultured
adipocytes. This effect may reflect one of the physiological bases for cachexia. In the present study, clones of complementary
DNA from genes whose expression is increased during the differentiation of adipocytes were used to study the molecular basis
of cachectin's actions. In the presence of cachectin, the expression of the corresponding genes was reversibly and specifically
inhibited. Furthermore, when mature adipocytes were exposed to cachectin, the messenger RNA's of those genes diminished and
rapidly approached the levels present before differentiation.
Tumor necrosis factor-alpha (TNF-alpha) has been shown to have certain catabolic effects on fat cells and whole animals. An
induction of TNF-alpha messenger RNA expression was observed in adipose tissue from four different rodent models of obesity
and diabetes. TNF-alpha protein was also elevated locally and systemically. Neutralization of TNF-alpha in obese fa/fa rats
caused a significant increase in the peripheral uptake of glucose in response to insulin. These results indicate a role for
TNF-alpha in obesity and particularly in the insulin resistance and diabetes that often accompany obesity.
Peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2) is an adipocyte-specific nuclear hormone receptor that has recently been identified as a key regulator of two fat cell enhancers. Transcriptional activation by PPAR gamma 2 is potentiated by a variety of lipids and lipid-like compounds, including naturally occurring polyunsaturated fatty acids. We demonstrate here that retroviral expression of PPAR gamma 2 stimulates adipose differentiation of cultured fibroblasts. PPAR activators promote the differentiation of PPAR gamma 2-expressing cells in a dose-dependent manner. C/EBP alpha, a second transcription factor induced during adipocyte differentiation, can cooperate with PPAR gamma 2 to stimulate the adipocyte program dramatically. Our results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.
Recent studies indicate that a peroxisome proliferator-activated receptor, PPAR gamma, functions as an important adipocyte determination factor. In contrast, tumor necrosis factor-alpha (TNF alpha) inhibits adipogenesis, causes dedifferentiation of mature adipocytes, and reduces the expression of several adipocyte-specific genes. Here, we report that treatment of 3T3-L1 adipocytes with TNF alpha resulted in a time- and concentration-dependent decrease in PPAR gamma mRNA expression to the level detected in preadipocytes. PPAR gamma mRNA levels were reduced by 95% with 3 nM TNF alpha treatment for 24 h. Half-maximal effects were seen after 3 h treatment with 3 nM TNF alpha or with 50 pM TNF alpha (24-h exposure). Parallel reductions in PPAR gamma protein levels were also observed after treatment of 3T3-L1 adipocytes with TNF alpha. Using a ribonuclease protection assay, both alternatively spliced PPAR gamma isoforms (gamma 1 and gamma 2) were shown to be negatively regulated by TNF alpha. The down-regulation of PPAR gamma by TNF-alpha preceded the diminution in expression of other adipocyte-specific genes including CCAAT/enhancer binding protein and adipocyte fatty acid-binding protein (aP2). The effect of TNF alpha was specific for the gamma-isoform of PPARs, since the expression of PPAR delta mRNA was not affected by treatment with TNF alpha. Low level constitutive expression of PPAR gamma in 3T3-L1 adipocytes (at levels approximately 2- to 3-fold higher than in preadipocytes) partially blocked the inhibitory effect of TNF alpha on aP2 and adipsin expression. These findings support the following conclusions: 1) PPAR gamma expression is necessary for the maintenance of the adipocyte phenotype. 2) PPAR gamma, but not PPAR delta, expression is sufficient to attenuate TNF alpha-mediated effects on adipocyte phenotype. 3) Reduced PPAR gamma gene expression is likely to represent an important component of the mechanism by which TNF alpha exerts its antiadipogenic effects.
Obesity is highly associated with insulin resistance and is the biggest risk factor for non-insulin-dependent diabetes mellitus. The molecular basis of this common syndrome, however, is poorly understood. It has been suggested that tumour necrosis factor (TNF)-alpha is a candidate mediator of insulin resistance in obesity, as it is overexpressed in the adipose tissues of rodents and humans and it blocks the action of insulin in cultured cells and whole animals. To investigate the role of TNF-alpha in obesity and insulin resistance, we have generated obese mice with a targeted null mutation in the gene encoding TNF-alpha and those encoding the two receptors for TNF-alpha. The absence of TNF-alpha resulted in significantly improved insulin sensitivity in both diet-induced obesity and that resulting for the ob/ob model of obesity. The TNFalpha-deficient obese mice had lower levels of circulating free fatty acids, and were protected from the obesity-related reduction in the insulin receptor signalling in muscle and fat tissues. These results indicate that TNF-alpha is an important mediator of insulin resistance in obesity through its effects on several important sites of insulin action.
Multicellular organisms have the challenging task of coordinating the activities of many distinct cell types. This coordination is accomplished largely by cell-associated and soluble signalling molecules that act locally or distantly to alter target-cell physiology. The tumour necrosis factor family of cytokines are type II transmembrane proteins that are important regulators of homeostasis and have been implicated as mediators of disease. These molecules serve as ligands for a family of cell-surface receptors termed the tumour necrosis factor/nerve growth factor (TNF/NGF) receptor family. The receptors are type I transmembrane proteins capable of mediating a wide range of responses in vitro and in vivo. Signal transduction is mediated by several newly discovered cytoplasmic proteins that couple these receptors to downstream signalling events. The elucidation and use of spontaneously occurring mutants in TNF-related ligands and receptors in addition to gene-targeting experiments have begun to clarify the diverse biological effects mediated by this superfamily of cytokines.
The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) promotes adipocyte differentiation, exerts atherogenic
and anti-inflammatory effects in monocyte/macrophages, and is believed to mediate the insulin-sensitizing action of antidiabetic
thiazolidinedione ligands. As no complete PPARγ antagonists have been described hitherto, we have constructed a dominant-negative
mutant receptor to inhibit wild-type PPARγ action. Highly conserved hydrophobic and charged residues (Leu468 and Glu471) in helix 12 of the ligand-binding domain were mutated to alanine. This compound PPARγ mutant retains ligand and DNA binding,
but exhibits markedly reduced transactivation due to impaired coactivator (cAMP-response element-binding protein-binding protein
and steroid receptor coactivator-1) recruitment. Unexpectedly, the mutant receptor silences basal gene transcription, recruits
corepressors (the silencing mediator of retinoid and thyroid receptors and the nuclear corepressor) more avidly than wild-type
PPARγ, and exhibits delayed ligand-dependent corepressor release. It is a powerful dominant-negative inhibitor of cotransfected
wild-type receptor action. Furthermore, when expressed in primary human preadipocytes using a recombinant adenovirus, this
PPARγ mutant blocks thiazolidinedione-induced differentiation, providing direct evidence that PPARγ mediates adipogenesis.
Our observations suggest that, as in other mutant nuclear receptor contexts (acute promyelocytic leukemia, resistance to thyroid
hormone), dominant-negative inhibition by PPARγ is linked to aberrant corepressor interaction. Adenoviral expression of this
mutant receptor is a valuable means to antagonize PPARγ signaling.
Tumor necrosis factor alpha (TNF-alpha) has well-described effects on lipid metabolism in the context of acute inflammation, as in sepsis. Recently, increased TNF-alpha production has been observed in adipose tissue derived from obese rodents or human subjects and TNF-alpha has been implicated as a causative factor in obesity-associated insulin resistance and the pathogenesis of type 2 diabetes. Thus, current evidence suggests that administration of exogenous TNF-alpha to animals can induce insulin resistance, whereas neutralization of TNF-alpha can improve insulin sensitivity. Importantly, results from knockout mice deficient in TNF-alpha or its receptors have suggested that TNF-alpha has a role in regulating in vivo insulin sensitivity. However, the absence of TNF-alpha action might only partially protect against obesity-induced insulin resistance in mice. Multiple mechanisms have been suggested to account for these metabolic effects of TNF-alpha. These include the downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated free fatty acids via stimulation of lipolysis, and negative regulation of PPAR gamma, an important insulin-sensitizing nuclear receptor. Although current evidence suggests that neutralizing TNF-alpha in type 2 diabetic subjects is not sufficient to cause metabolic improvement, it is still probable that TNF-alpha is a contributing factor in common metabolic disturbances such as insulin resistance and dyslipidemia.
We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.