Fat Cell Precursors Research Articles

A compound directed against S6K1 hampers fat mass expansion and mitigates diet-induced hepatosteatosis

The ribosomal protein S6 kinase 1 (S6K1) is a relevant effector downstream of the mammalian target of rapamycin complex 1 (mTORC1), best known for its role in the control of lipid homeostasis. Consistent with this, mice lacking the S6k1 gene have a defect in their ability to induce the commitment of fat precursor cells to the adipogenic lineage, which contributes to a significant reduction of fat mass. Here, we assess the therapeutic blockage of S6K1 in diet-induced obese mice challenged with LY2584702 tosylate, a specific oral S6K1 inhibitor initially developed for the treatment of solid tumors. We show that diminished S6K1 activity hampers fat mass expansion and ameliorates dyslipidemia and hepatic steatosis, while modifying transcriptome-wide gene expression programs relevant for adipose and liver function. Accordingly, decreased mTORC1 signaling in fat (but increased in the liver) segregated with defective epithelial-mesenchymal transition and the impaired expression of Cd36 (coding for a fatty acid translocase) and Lgals1 (Galectin 1) in both tissues. All these factors combined align with reduced adipocyte size and improved lipidomic signatures in the liver, while hepatic steatosis and hypertriglyceridemia were improved in treatments lasting either 3 months or 6 weeks.

Effect of Wall Shear Stress Gradient on Cells: Distribution of Deformation and Rotation

The effect of wall shear stress gradient on the deformation and rotation of each cell was investigated <em>in vitro</em>. To make a Couette-type of shear flow, the culture medium fluid was sandwiched with a constant gap between parallel walls: a lower stationary culture disk, and an upper rotating disk. Mouse fat precursor cells (3T3-L1) were used in this experiment. After cultivation without flow for 24 hours for adhesion of cells on the lower plate, a shear stress of less than 2 Pa was continuously applied to cells for 24 hours in the incubator. The behavior (deformation and major axis angle) of each single cell was tracked using time-lapse images observed by an inverted phase contrast microscope placed in the incubator. For wall shear stresses of less than 1.9 Pa, each cell exhibited active behavior: migration, deformation, and rotation. Whereas, the cells transformed into spheres when wall shear stress was higher than 1.9 Pa. In addition, the cells were observed to tilt against for a wall shear stress gradient approaching 50 Pa/m.

ALDH1A1 effect on Yan Yellow Cattle preadipocyte differentiation

Aldehyde dehydrogenase 1A1 (ALDH1A1) is a cytosolic enzyme that mainly catalyzes the oxidation of acetaldehyde into acetic acid and participates in the regulation of differentiation and gene expression in fat cell growth and development. However, the physiological role of ALDH1A1 in the formation of fat cell precursors in the Yan Yellow Cattle is still not clear. Herein, we investigated the specific regulation of the gene encoding for ALDH1A1 during the differentiation process of the adipocyte cells of the Yan Yellow Cattle by interfering or overexpressing the ALDH1A1 gene. As a result, we found that the mRNA expression levels of ALDH1A1 were significantly increased during the formation of progenitor cells. In addition, the expression levels of the Lipoprotein lipase (LPL) and transcription factors (PPARγ, C/EBPα) were also significantly increased. ALDH1A1 gene overexpression and RNA interfering promoted and inhibited respectively the lipid accumulation and triglyceride production in mature adipocytes, and the expression of the LPL and transcription factors (PPARγ, C/EBPα). The changes in the protein expression levels of ALDH1A1 and adipogenic factors were in accord with the changes observed in the mRNA levels. In conclusion, our results indicate that ALDH1A1 plays an important regulatory role in the differentiation of preadipocyte cells.

RepSox, a small molecule inhibitor of the TGFβ receptor, induces brown adipogenesis and browning of white adipocytes.

Unlike white adipose tissue (WAT), brown adipose tissue (BAT) is mainly responsible for energy expenditure via thermogenesis by uncoupling the respiratory chain. Promoting the differentiation of brown fat precursor cells and the browning of white fat have become a research hotspot for the treatment of obesity and associated metabolic diseases. Several secreted factors and a number of small molecules have been found to promote brown adipogenesis. Here we report that a single small-molecule compound, RepSox, is sufficient to induce adipogenesis from mouse embryonic fibroblasts (MEFs) in fibroblast culture medium. RepSox is an inhibitor of the transforming growth factor-beta receptor I (TGF-β-RI), other inhibitors of TGF-β pathway such as SB431542, LY2157299, A83-01, and Tranilast are also effective in inducing adipogenesis from MEFs. These adipocytes express brown adipocyte-specific transcription factors and thermogenesis genes, and contain a large number of mitochondria and have a high level of mitochondrial respiratory activity. More interestingly, RepSox has also been found to promote the differentiation of the brown fat precursor cells and induce browning of the white fat precursor cells. These findings suggest that inhibitors of TGF-β signaling pathway might be developed as new therapeutics for obesity and type 2 diabetes.

Heterogeneity in the perirenal region of humans suggests presence of dormant brown adipose tissue that contains brown fat precursor cells

ObjectiveIncreasing the amounts of functionally competent brown adipose tissue (BAT) in adult humans has the potential to restore dysfunctional metabolism and counteract obesity. In this study, we aimed to characterize the human perirenal fat depot, and we hypothesized that there would be regional, within-depot differences in the adipose signature depending on local sympathetic activity. MethodsWe characterized fat specimens from four different perirenal regions of adult kidney donors, through a combination of qPCR mapping, immunohistochemical staining, RNA-sequencing, and pre-adipocyte isolation. Candidate gene signatures, separated by adipocyte morphology, were recapitulated in a murine model of unilocular brown fat induced by thermoneutrality and high fat diet. ResultsWe identified widespread amounts of dormant brown adipose tissue throughout the perirenal depot, which was contrasted by multilocular BAT, primarily found near the adrenal gland. Dormant BAT was characterized by a unilocular morphology and a distinct gene expression profile, which partly overlapped with that of subcutaneous white adipose tissue (WAT). Brown fat precursor cells, which differentiated into functional brown adipocytes were present in the entire perirenal fat depot, regardless of state. We identified SPARC as a candidate adipokine contributing to a dormant BAT state, and CLSTN3 as a novel marker for multilocular BAT. ConclusionsWe propose that perirenal adipose tissue in adult humans consists mainly of dormant BAT and provide a data set for future research on factors which can reactivate dormant BAT into active BAT, a potential strategy for combatting obesity and metabolic disease.

A delayed proinflammatory response of human preadipocytes to PCB126 is dependent on the aryl hydrocarbon receptor.

Inflammation in adipose tissue is recognized as a causative factor in the development of type II diabetes. Adipocyte hypertrophy as well as bacterial and environmental factors have been implicated in causing inflammation in mature adipocytes. Exposure to persistent organic pollutants such as polychlorinated biphenyls (PCBs) has been associated with the development of type II diabetes. We show here that PCB126, a dioxin-like PCB, activates a robust proinflammatory state in fat cell precursors (preadipocytes). The response was found to be dependent on aryl hydrocarbon receptor (AhR) activation, although induction of the response was delayed compared to upregulation of CYP1A1, a classic AhR-responsive gene. Treatment of preadipocytes with a nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) inhibitor partially attenuated the PCB126-induced inflammatory response and partly, but not completely, ameliorated disruption of adipogenesis caused by PCB126. Our results indicate a role for PCB126 in mediating an inflammatory response through AhR in preadipocytes that interferes with adipogenesis.

Effect of adrenocorticotropic hormone on UCP1 gene expression in brown adipocytes.

Like other tissues, adrenocorticotropic hormone (ACTH) can produce its effect on brown adipose tissue (BAT). This study was taken to understand the direct effect of ACTH action on thermogenin gene expression and possible relation with α receptors and caffeine with this hormone. Brown fat precursor cells were isolated from interscapular BAT of young mice and grown in culture. The cells were exposed to norepinephrine (NE) and other agents. Total RNA was isolated after harvesting the cells, and northern blot analysis was performed. Hybridization was performed with nick translated cDNA probes. Filters were exposed to film, and results were evaluated by scanning. Cyclic adenosine monophosphate (cAMP) was measured by using Amersham assay kit. ACTH stimulates thermogenin gene expression in brown adipocytes. Initiation and maximum stimulations are observed with 0.01 μM and 10 μM (about 45%) of ACTH, respectively, in comparison to 0.1 μM of NE. Maximum response of cAMP is also observed with 10 μM of ACTH (about 64%). Studies with cirazoline and ACTH show that UCP1 mRNA expression is increased significantly with 10 μM of ACTH, whereas cAMP generation is decreased. In the presence of caffeine, ACTH increases cAMP generation and UCP1 gene expression more than twofold. ACTH stimulates thermogenin gene expression in cultured brown adipocytes. The complex interrelationship of ACTH with cirazoline indicates the possibility of relation between the activity of ACTH and α receptors in brown adipocytes. Further stimulation of cAMP generation and thermogenin gene expression is possible with ACTH in conjugation with caffeine and RO 20-1724.

Increased adipocyte differentiation may be mediated by extracellular calcium levels through effects on calreticulin and peroxisome proliferator activated receptor gamma expression in intramuscular stromal vascular cells isolated from Hanwoo beef cattle

Marbling is the intramuscular deposition of adipose tissue and is said to be directly associated with beef quality. Nutritional manipulations are a common practice among beef cattle producers in order to achieve better quality meat. The objective of the current study was to investigate the effects of increasing extracellular calcium on the differentiation of intramuscular stromal vascular cells (IM SVCs) isolated from Hanwoo beef cattle. Primary cell isolates of SVCs were differentiated for 14 days while exposed to increasing concentrations (1.8, 3.6, 7.2, 10.8 mM) of calcium in the media. The correlation between adipogenesis and calcium concentrations was determined through SVC differentiation monitored by RT-PCR, Western blot analysis for PPARγ, C/EBPα, calreticulin, FABP4 and GLUT4 expression. In addition, expression of both phospholipase C gamma (PLC-γ) and protein kinase C (PKC) were investigated since both have possible links to intracellular calcium increase and the expression of several adipogenic genes including FABP4 and GLUT4. Results of the current study provide evidence that stromal vascular cells exposed to the lower concentrations of extracellular calcium have higher rates of adipogenesis possibly due to a decrease in the expression of calreticulin, a known inhibitor of PPARγ expression, and PKC activation followed by PLC-γ activation, leading to the expression of adipogenic genes. Data derived from this study shows that in vitro, decreasing calcium levels present in the microenvironment of fat precursor cells lead to a higher percentage of adipogenesis.

Obesity: High-fat binges lead to depot-specific activation of fat cell precursors.

Obesity: High-fat binges lead to depot-specific activation of fat cell precursors.

Ebf2 is a selective marker of brown and beige adipogenic precursor cells.

Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.

Epigenetic Modifications and Canonical Wingless/int-1 Class (WNT) Signaling Enable Trans-differentiation of Nonosteogenic Cells into Osteoblasts

Mesenchymal cells alter and retain their phenotype during skeletal development through activation or suppression of signaling pathways. For example, we have shown that Wnt3a only stimulates osteoblast differentiation in cells with intrinsic osteogenic potential (e.g. MC3T3-E1 pre-osteoblasts) and not in fat cell precursors or fibroblasts (3T3-L1 pre-adipocytes or NIH3T3 fibroblasts, respectively). Wnt3a promotes osteogenesis in part by stimulating autocrine production of the osteoinductive ligand Bmp2. Here, we show that the promoter regions of the genes for Bmp2 and the osteoblast marker Alp are epigenetically locked to prevent their expression in nonosteogenic cells. Both genes have conserved CpG islands that exhibit increased CpG methylation, as well as decreased acetylation and increased methylation of histone H3 lysine 9 (H3-K9) specifically in nonosteogenic cells. Treatment of pre-adipocytes or fibroblasts with the CpG-demethylating agent 5'-aza-2'-deoxycytidine or the histone deacetylase inhibitor trichostatin-A renders Bmp2 and Alp responsive to Wnt3a. Hence, drug-induced epigenetic activation of Bmp2 gene expression contributes to Wnt3a-mediated direct trans-differentiation of pre-adipocytes or fibroblasts into osteoblasts. We propose that direct conversion of nonosteogenic cells into osteoblastic cell types without inducing pluripotency may improve prospects for novel epigenetic therapies to treat skeletal afflictions.

Developmental programming of adult obesity and cardiovascular disease in rodents by maternal nutrition imbalance

Studies on fetal undernutrition have generated the hypothesis that fetal programming corresponds to an attempt of the fetus to adapt to adverse conditions encountered in utero. These adaptations would be beneficial if these conditions prevail later in life, but they become detrimental in the case of normal or plentiful nutrition and favor the appearance of the metabolic syndrome. In this article, the discussion is limited to the developmental programming of obesity and cardiovascular disorders caused by an early mismatched nutrition, particularly intrauterine growth retardation followed by postnatal catch-up growth. Selected data in humans are reviewed before evoking some mechanisms revealed or suggested by experiments in rodents. A variety of physiologic mechanisms are implicated in obesity programming, 2 of which are detailed. In some, but not all observations, hyperphagia resulting namely from perturbed development of the hypothalamic circuitry devoted to appetite regulation may contribute to obesity. Another contribution may be the developmental changes in the population of fat cell precursors in adipose tissue. Even if the link between obesity and cardiovascular disease is well established, alteration of blood pressure regulation may appear independently of obesity. A loss of diurnal variation in heart rate and blood pressure in adulthood has resulted from maternal undernutrition followed by postnatal overnutrition. Further research should clarify the effect of mismatched early nutrition on the development of brain centers regulating energy intake, energy expenditure, and circadian rhythms.

Transcriptional Control of Brown Adipogenesis and Energy Homeostasis

Obesity is a disorder of energy balance, and can occur when food intake exceeds energy expenditure. We have been particularly interested in the transcriptional pathways controlling energy expenditure through regulation of mitochondrial function, using brown adipose cells as a rich source from which to “mine” molecules with broad roles in these processes. Most recently, we have found that the large, nuclear zinc‐finger protein PRDM16 is a “master” regulator of the determination of brown fat cell fate. This molecule can alter the differentiation pathway of fat cell precursors and hence, might represent a new pathway for the treatment of obesity. PRDM16 can alter the gene expression pattern of white fat precursor cells, so that they differentiate into brown fat cells. Expression of sh‐RNA against PRDM16 in immortalized brown fat cells shows that PRDM16 is required for essentially the entire brown fat program. Interestingly, when the same sh‐RNA against PRDM16 is expressed in primary brown preadipose cells, these cells differentiate into muscle cells, strongly suggesting a relationship between brown fat and skeletal muscle. Conversely, expression of PRDM16 alone is sufficient to convert primary brown fat precursor cells into skeletal muscle cells, replete with expression of myofibrillar proteins. Fate mapping experiments indicate that skeletal muscle and brown adipose arise from myf‐5‐expressing cells, while white fat does not come from this lineage. Finally, purification of the PRDM16 complex in brown fat cells indicates that PRDM16 binds to PPAR‐γ and C/EBPβ. The coactivation of these proteins indicates how this protein can be adipogenic in a muscle cell. These data, taken together, show that PRDM16 is a “master” regulator of the brown fat lineage and shows that brown fat and skeletal muscle arise from a similar or identical cell lineage. Thus brown and white fat represent a cellular and biochemical convergence through expression and function of PPAR‐γ but they arise from very different developmental pathways. The potential uses of the PRDM16 pathway in novel approaches to the therapy of human obesity will be discussed.

Distribution and Development of Brown Adipocytes in the Murine and Human Adipose Organ

Murine white and brown adipocytes are found together in dissectible visceral and subcutaneous fat depots supplied by specific vessels and nerves, forming a multi-depot organ with plastic properties. Many of the anatomo-physiological features of murine fat depots apply to humans.

Human Multipotent Adipose-Derived Stem Cells Differentiate into Functional Brown Adipocytes

In contrast to the earlier contention, adult humans have been shown recently to possess active brown adipose tissue with a potential of being of metabolic significance. Up to now, brown fat precursor cells have not been available for human studies. We have shown previously that human multipotent adipose-derived stem (hMADS) cells exhibit a normal karyotype and high self-renewal ability; they are known to differentiate into cells that exhibit the key properties of human white adipocytes, that is, uncoupling protein two expression, insulin-stimulated glucose uptake, lipolysis in response to beta-agonists and atrial natriuretic peptide, and release of adiponectin and leptin. Herein, we show that, upon chronic exposure to a specific PPARgamma but not to a PPARbeta/delta or a PPARalpha agonist, hMADS cell-derived white adipocytes are able to switch to a brown phenotype by expressing both uncoupling protein one (UCP1) and CIDEA mRNA. This switch is accompanied by an increase in oxygen consumption and uncoupling. The expression of UCP1 protein is associated to stimulation of respiration by beta-AR agonists, including beta3-AR agonist. Thus, hMADS cells represent an invaluable cell model to screen for drugs stimulating the formation and/or the uncoupling capacity of human brown adipocytes that could help to dissipate excess caloric intake of individuals.

Effects of dihydrotestosterone on differentiation and proliferation of human mesenchymal stem cells and preadipocytes.

The mechanisms by which androgens regulate fat mass are poorly understood. Although testosterone has been reported to increase lipolysis and inhibit lipid uptake, androgen effects on proliferation and differentiation of human mesenchymal stem cells (hMSCs) and preadipocytes have not been studied. Here, we investigated whether dihydrotestosterone (DHT) regulates proliferation, differentiation, or functional maturation of hMSCs and human preadipocytes from different fat depots. DHT (0-30 nM) dose-dependently inhibited lipid accumulation in adipocytes differentiated from hMSCs and downregulated expression of aP2, PPARgamma, leptin, and C/EBPalpha. Bicalutamide attenuated DHT's inhibitory effects on adipogenic differentiation of hMSCs. Adipocytes differentiated in presence of DHT accumulated smaller oil droplets suggesting reduced extent of maturation. DHT decreased the incorporation of labeled fatty acid into triglyceride, and downregulated acetyl CoA carboxylase and DGAT2 expression in adipocytes derived from hMSCs. DHT also inhibited lipid accumulation and downregulated aP2 and C/EBPalpha in human subcutaneous, mesenteric and omental preadipocytes. DHT stimulated forskolin-stimulated lipolysis in subcutaneous and mesenteric preadipocytes and inhibited incorporation of fatty acid into triglyceride in adipocytes differentiated from preadipocytes from all fat depots. DHT inhibits adipogenic differentiation of hMSCs and human preadipocytes through an AR-mediated pathway, but it does not affect the proliferation of either hMSCs or preadipocytes. Androgen effects on fat mass represent the combined effect of decreased differentiation of fat cell precursors, increased lipolysis, and reduced lipid accumulation.

A Pericellular Collagenase Directs the 3-Dimensional Development of White Adipose Tissue

White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development.

Gaining a solid grip on adipogenesis

Obesity is presently being combated by fitness regimens, drugs and diet. Increasing our understanding of the physiology of adipocytes, by deducing the regulatory pathways involved in lipid metabolism and all aspects of adipogenesis, will provide alternative strategies to reduce adverse problems of obesity. Research has suggested that mature fat cells may dedifferentiate to form proliferative-competent fat cell precursors. Knowledge of the dedifferentiation process will allow us to gain a solid grip on adipogenesis.

Wasting Away: Stress response keeps aging cells from fattening up (Fat cell formation)

BOSTON --Many people battle fat for years, only to find at the end of their lives that they're losing it in all the wrong places. Sunken eyes, inability to control body temperature, and susceptibility to infection plague elderly people as their fat pads shrink. New work, presented 16 February at the AAAS meeting here, fills in some molecular steppingstones that mark the path to a decrease in fat cell formation. As people age, they lose fat where it should be--under the skin, for instance--and gain it where it shouldn't be--in tissues such as bone marrow and muscle. This redistribution occurs in part because precursor cells destined to store fat lose their bearings. Over the past several years, scientists have established that two key proteins--whose concentrations decline with age--activate a set of genes required for precursor cells to mature into fat cells. Replacing the regulator proteins restores precursor cells to their destinies, indicating that the genes are still able to guide maturation if prompted. Researchers are therefore trying to figure out what regulates the regulators. James Kirkland, a cell biologist at Boston University, has been picking his way upstream of the regulators. CUG triplet repeat binding protein (CUGBP) cranks up production of another protein that in turn blocks output of both regulators. Kirkland reported at the meeting that precursor fat cells from old rats produce more CUGBP than do similar cells from young rats. Swelling CUGBP concentrations in older animals probably contribute to the decline in the two key regulators, he says. Going back one more step, Kirkland's team examined a protein called TNFα, which is produced in response to stress, accumulates in fat tissues with age, and thwarts production of the two maturation regulators--but the molecular underpinnings of these events have been unclear. Now Kirkland's team has found that treating precursor fat cells with TNFα causes their CUGBP concentrations to double. Other cellular events might similarly stunt precursor cells' maturation. When the researchers stimulated production of reactive oxygen species--destructive molecules implicated in aging--in cultured fat precursor cells from young rats, CUGBP concentrations shot up. Together, Kirkland says, the results suggest that cellular events associated with aging activate stress pathways, which operate through a string of regulator proteins to stem fat cell formation. The emerging molecular details might provide the basis for future therapies, he says: "In fat tissue, there might be points where you can intervene." Success at restoring specialized functions to old fat cells might provide a model for rejuvenating other tissues, he adds. Kirkland's approach is unusual, says Huber Warner, associate director for the Biology of Aging Program at the National Institute on Aging in Bethesda, Maryland: "He's looking at a phenomenon we're all familiar with--the changes in amounts and location of fat--but he's kind of alone in investigating the molecular basis for it." --Evelyn Strauss

Protective effects of noradrenaline against tumor necrosis factor-α-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression

To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes. Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers. Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels. Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection. These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.