The inflammatory receptor CD40 is expressed on human adipocytes: contribution to crosstalk between lymphocytes and adipocytes

Aims/hypothesis  Obesity is associated with adipose tissue inflammation. The CD40 molecule, TNF receptor superfamily member 5 (CD40)/CD40 ligand (CD40L) pathway plays a role in the onset and maintenance of the inflammatory reaction, but has not been studied in human adipose tissue. Our aim was to examine CD40 expression by human adipocytes and its participation in adipose tissue inflammation. Methods   CD40 expression was investigated in human whole adipose tissue and during adipocyte differentiation by real-time PCR, Western blot and immunohistochemistry. The CD40/CD40L pathway was studied using recombinant CD40L (rCD40L) in adipocyte culture and neutralising antibodies in lymphocyte/adipocyte co-culture. Results   CD40 mRNA levels in subcutaneous adipose tissue were higher in the adipocyte than in the stromal–vascular fraction. CD40 expression was upregulated during adipocyte differentiation. Addition of rCD40L to adipocytes induced mitogen activated protein kinase (MAPK) activation, stimulated inflammatory adipocytokine production, and decreased insulin-induced glucose transport in parallel with a downregulation of IRS1 and GLUT4 (also known as SCL2A4). rCD40L decreased the expression of lipogenic genes and increased lipolysis. CD40 mRNA levels were significantly higher in subcutaneous adipose tissue than in visceral adipose tissue of obese patients and were positively correlated with BMI, and with IL6 and leptin mRNA levels. Lymphocyte/adipocyte co-culture led to an upregulation of proinflammatory adipocytokines and a downregulation of leptin and adiponectin. Physical separation of the two cell types attenuated these effects, suggesting the involvement of a cell–cell contact. Blocking the CD40/CD40L interaction with neutralising antibodies reduced IL-6 secretion from adipocytes. Conclusions/interpretation  Adipocyte CD40 may contribute to obesity-related inflammation and insulin resistance. T lymphocytes regulate adipocytokine production through both the release of soluble factor(s) and heterotypic contact with adipocytes involving CD40.     

Effects of leptin on the differentiation and metabolism of human adipocytes.

BACKGROUND: Leptin is an adipose protein regulating food intake in the hypothalamus. Animal studies have suggested that leptin also acts in an auto-/paracrine fashion on adipose cell function. OBJECTIVE: The aim of this study was to investigate the effects of leptin on the differentiation and metabolism of cultured human adipocytes. MATERIAL: Adipose tissue from young healthy, lean women (body mass index (BMI) <27 kg/m(2)) undergoing elective mammary reduction surgery and young obese individuals (BMI>40 kg/m(2)) undergoing laparoscopic gastric banding. METHODS: Human preadipocytes in primary culture were induced to undergo differentiation by defined adipogenic factors. Mature adipocytes were isolated by collagenase digestion and kept in culture suspension. Glycero-3-phosphate dehydrogenase (GPDH) activity was used as a marker of adipose differentiation; glucose uptake, lipolysis and PAI-1 secretion were measured as parameters of fat cell function. RESULTS: Human preadipocytes and adipocytes from lean and obese subjects expressed the long leptin receptor isoform and two of the three short forms as assessed by polymerase chain reaction (PCR). Leptin at a supraphysiological concentration induced a transient increase of GPDH activity, but had no effect on glucose uptake and PAI-1 secretion from human adipocytes. In addition, basal and isoproterenol-stimulated lipolysis as well as the antilipolytic action of insulin in human adipocytes was not significantly affected by leptin exposure. CONCLUSION: In contrast to animal data, the results of our experiments do not demonstrate significant effects of leptin on main metabolic functions of human adipocytes arguing against a local auto-/paracrine action of leptin in human adipose tissue.     

Hexosamines regulate leptin production in human subcutaneous adipocytes

The hexosamine biosynthetic pathway has recently been proposed as a mechanism through which cells "sense" nutrient flux to regulate leptin release. This study was undertaken to examine the regulation of leptin production by hexosamines in human adipocytes. Adipose tissue UDP-N-acetylglucosamine, an end product of hexosamine biosynthesis, was elevated 3.2-fold, and ob messenger ribonucleic acid was elevated 2-fold in the sc adipose tissue of 17 obese [body mass index (BMI), 41.3+/-12.0 kg/m2; age, 31+/-5 yr] subjects compared to 14 lean (BMI, 23.4+/-1.6 kg/m2; age, 33+/-11 yr) subjects. Serum leptin was increased 2.7-fold in the obese subjects. A significant positive relationship was found between adipose tissue UDP-N-acetylglucosamine and BMI (Spearman correlation = 0.576; P = 0.0007) and between UDP-N-acetylglucosamine and serum leptin (Spearman correlation = 0.4650; P = 0.0145). Treatment of isolated sc adipocytes with 1 mmol/L glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin release 21.4+/-17.6% (mean +/- SD) over control (P = 0.0365) and 74.5+/-82.8% over control (P = 0.0271) in adipocytes from lean (BMI, 23.2+/-1.6 kg/m2; n = 6) and obese (BMI, 55.4+/-13.0 kg/m2,; n = 9) subjects, respectively, by 48 h of culture. Inhibition of UDP-N-acetylglucosamine biosynthesis with 6-diazo-5-oxo-norleucine reduced glucose-stimulated leptin release from cultured adipocytes 21.8+/-32.4% (P = 0.0395; n = 12) and ob gene expression 19.9+/-18.9% (P = 0.0208; n = 8) by 48 h of treatment. These findings suggest that hexosamine biosynthesis regulates leptin production in human adipose tissue.     

Regulation of leptin release by insulin,glucocorticoids, G(i)-coupled receptor agonists,and pertussis toxin in adipocytes and adipose tissue explants from obese humans in primary culture

The basal release of leptin by adipocytes from massively obese human subjects incubated for 48 hours in serum-free suspension culture was comparable to that by explants of subcutaneous adipose tissue from the same obese individuals. There was no stimulation due to dexamethasone or insulin alone of leptin release by adipocytes. However, the combination of insulin and dexamethasone doubled leptin release by adipocytes. The release of leptin was also stimulated by agonists of G(i)-coupled receptors (prostaglandin E(2) [PGE(2)], brimonidine [an alpha(2) catecholamine agonist] and cyclopentyladenosine [CPA]) in the presence of dexamethasone. Leptin release by these agents was further enhanced by insulin in both adipocytes and adipose tissue. Pertussis toxin, which irreversibly inactivates G(i) heterotrimers, inhibited leptin release and abolished the stimulatory effects of G(i)-coupled receptor agonists. However, pertussis toxin did not block the stimulation of leptin release by insulin in either adipose tissue or adipocytes. These data indicate that the release of leptin by human adipocytes cultured for 48 hours in a serum-free medium is comparable to that by explants of adipose tissue except that dexamethasone stimulation of leptin release requires the presence of insulin.     

Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes

The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.     

Production and release of macrophage migration inhibitory factor from human adipocytes

BACKGROUND AND AIM OF THE STUDY: Macrophage migration inhibitory factor (MIF) has been identified as a critical mediator of inflammatory responses. Because of its potent migration inhibition activity, it regulates macrophage accumulation in tissues. We therefore analyzed whether human adipocytes produce MIF, in the search of candidate mediators of macrophage infiltration of obese adipose tissue. METHODS: Human adipose tissue samples were obtained from various depots. The precursor cells were allowed to differentiate under defined adipogenic culture conditions. MIF expression was analyzed by RT-PCR, ELISA, and immunocytochemistry. RESULTS: Human preadipocytes secreted MIF in a differentiation-dependent fashion with maximum concentrations at d 12, whereas MIF mRNA was detected in both undifferentiated and differentiated cells at relatively constant levels. Immunocytochemical analysis showed that MIF protein was present in preadipocytes and more pronounced in differentiated adipocytes. Freshly isolated mature adipocytes from sc, omental, and mammary depots released MIF at rates of up to 10,000 pg/ml.24 h. Most importantly, MIF production was positively correlated with donor body mass index. Secretion of MIF was not influenced by lipopolysaccharide, interferon-gamma, or IL-4. The rates of MIF release from sc and omental adipocytes were similar but approximately 10 times higher compared with mammary adipocytes. CONCLUSIONS: Human preadipocytes and mature adipocytes from different depots spontaneously release substantial amounts of MIF. Expression levels were positively associated with donor body mass index. Hence, MIF may be an obesity-dependent mediator of macrophage infiltration of adipose tissue.     

PRL receptor-mediated effects in female mouse adipocytes: PRL induces suppressors of cytokine signaling expression and suppresses insulin-induced leptin production in adipocytes in vitro

PRL has been reported to regulate fat metabolism in several species. We recently reported PRL receptor (PRLR) expression in mouse adipocytes and increased levels of PRLR expression in the adipose tissue of lactating and PRL-transgenic mice compared with controls. These results suggest PRLR-mediated effects in adipose tissue. However, to date most studies have been performed in vivo, and it is unclear whether PRL has direct effects on adipocytes. The PRLR belongs to the cytokine receptor family, and a family of suppressors of cytokine signaling (SOCS) was recently identified. The present study was performed to investigate whether PRL has direct effects on adipocytes. The expression of cytokine-inducible SH2-domain-containing protein (CIS), SOCS-3, and SOCS-2 mRNA and protein was analyzed using ribonuclease protection assay and immunoblotting, respectively. Ovine PRL induced CIS mRNA expression and a combination of oPRL and insulin induced SOCS-3 mRNA expression in adipocytes cultured in vitro for 0-240 min, demonstrating PRLR-mediated direct effects in these cells. Furthermore, CIS, SOCS-3, and SOCS-2 mRNA and protein were all transiently expressed in adipose tissue obtained from female mice stimulated with oPRL (1 microg/g BW) for 0-24 h. In adipose tissue of female mice with endogenously high PRL levels, PRL-transgenic mice, only SOCS-2 expression was increased. The level of SOCS-2 mRNA was also increased in adipose tissue during pregnancy and lactation compared with that in wild-type virgin female mice. A possible reason for increased SOCS-2 expression after prolonged PRL exposure during lactation and in the PRL transgenes could be to restore the sensitivity of adipose tissue to PRL. In addition, the direct effect of PRL on leptin production was investigated in adipocytes cultured in vitro for 6 h. PRL inhibited insulin-induced leptin production in vitro. However, PRL had no effect on leptin production in the absence of insulin. In contrast, serum leptin concentrations were increased in PRL-transgenic females compared with control mice. In conclusion, our results demonstrate functional PRLRs in mouse adipocytes and suggest a role for CIS, SOCS-3, and SOCS-2 in regulating PRL signal transduction in adipose tissue.     

Ceiling culture of mature human adipocytes: use in studies of adipocyte functions

Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed and the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which uses their buoyant property to allow them to adhere to a floating glass surface, where they remain viable for several weeks. Using confocal immunofluorescence microscopy we showed the cellular expression and subcellular localization of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-alpha treatment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes showing positive incorporation after 4 h labelling. Proliferation was also evident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) and was assayed by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analysed by liquid scintillation counting, was increased by forskolin (10 microM for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insulin (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured adipocytes maintain adipocyte-specific functions and that ceiling culture, which overcomes the shortcomings of adipocyte suspension culture, can be used to study adipocyte cell biology.     

Leptin secretion and negative autocrine crosstalk with insulin in brown adipocytes

Leptin is an important adipocytokine whose main regulative effects on energy metabolism are exerted via activation of signalling pathways in the central nervous system. Another important regulator of energy homeostasis is insulin. The role of direct autocrine leptin effects on adipose tissue and crosstalk with insulin, in particular in the thermogenically active brown adipose tissue, remains unclear. In the present study, we have investigated leptin secretion and interaction with insulin in highly insulin-responsive immortalised mouse brown adipocytes. Leptin was secreted in a differentiation-dependent manner, and acute leptin treatment of mature adipocytes dose- and time-dependently stimulated phosphorylation of STAT3 and MAP kinase. Interestingly, acute pretreatment of fully differentiated brown adipocytes with leptin (100 nM) significantly diminished insulin-induced glucose uptake by approximately 25%. This inhibitory effect was time-dependent and maximal after 60 min of leptin prestimulation. Furthermore, it correlated with a 35% reduction in insulin-stimulated insulin receptor kinase activity after acute leptin pretreatment. Insulin-induced insulin receptor substrate-1 tyrosine phosphorylation and binding to the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI 3-kinase) were diminished by approximately 60% and 40%, respectively. Taken together, this study has demonstrated strong differentiation-dependent leptin secretion in brown adipocytes and PI 3-kinase-mediated negative autocrine effects of this hormone on insulin action. Direct peripheral leptin-insulin crosstalk may play an important role in the regulation of energy homeostasis.     

A promising culture model for analyzing the interaction between adipose tissue and cardiomyocytes

The heart has epicardial adipose tissue that produces adipokines and mesenchymal stem cells. Systemic adipose tissue is involved in the pathophysiology of obesity-related heart diseases. However, the method for analyzing the direct interaction between adipose tissue and cardiomyocytes has not been established. Here we show the novel model, using collagen gel coculture of adipose tissue fragments (ATFs) and HL-1 cardiomyocytes, and electron microscopy, immunohistochemistry, real-time RT-PCR, and ELISA. HL-1 cells formed a stratified layer on ATF-nonembedded gel, whereas they formed almost a monolayer on ATF-embedded gel. ATFs promoted the apoptosis, lipid accumulation, and fatty acid transport protein (FATP) expression of FATP4 and CD36 in HL-1 cells, whereas ATFs inhibited the growth and mRNA expression of myosin, troponin T, and atrial natriuretic peptide. Treatment of leptin (100 ng/ml) and adiponectin (10 μg/ml) neither replicated nor abolished the ATF-induced morphology of HL-1 cells, whereas that of FATP4 and CD36 antibodies (25 μg/ml) never abolished it. HL-1 cells prohibited the development of CD44+/CD105+ mesenchymal stem cell-like cells and lipid-laden preadipocytes from ATFs. HL-1 cells increased the production of adiponectin in ATFs, whereas they decreased that of leptin. The data indicate that our model actively creates adipose tissue-HL-1 cardiomyocyte interaction, suggesting first that ATFs may be related to the lipotoxiciy of HL-1 cells via unknown factors plus FATP4 and CD36 and second that HL-1 cells may help to retain the static state of ATFs, affecting adipokine secretion. Our model will serve to study adipose tissue-cardiomyocyte interaction and mechanisms of obesity-related lipotoxicity and heart diseases.     

ACTH and alpha-MSH inhibit leptin expression and secretion in 3T3-L1 adipocytes: model for a central-peripheral melanocortin-leptin pathway

Leptin is the 167 amino-acid protein product of the Lep (obese) gene that is released predominantly from adipose tissue and circulates at levels related to the amount of fat. Leptin expression is hormonally regulated: insulin and glucocorticoids are stimulators, while inhibitors include beta-adrenergic agonists and testosterone. Recently, adenylate cyclase-coupled melanocortin receptors have been identified in murine adipose tissue, the 3T3-L1 adipocyte cell line, and in human fat tissue. These studies prompted us to evaluate the effects of pro-opiomelanocortin (POMC)-derived peptides on leptin production and expression in 3T3-L1 adipocytes in culture. 3T3-L1 pre-adipocytes differentiated by the insulin/indomethacin (I/I) method produced leptin at levels that were two times higher than those obtained in cells differentiated by the more traditional insulin/dexamethasone/isobutylmethylxanthine (I/D/M) method. By RT-PCR studies, 3T3-L1 cells expressed both the melanocortin 2 receptors (MC2-R) and melanocortin 5 receptors (MC5-R) isoforms of the melanocortin receptor at an early stage of differentiation. When I/I differentiated 3T3-L1 adipocytes were incubated with different concentrations of dibutyryl cAMP (db-cAMP) or POMC-derived peptides (ACTH and alpha-MSH), ACTH and alpha-MSH stimulated cAMP production after 30 min (2-fold increase) associated with a dose-dependent inhibition of leptin secretion (ACTHz.Gt;alpha-MSH; IC(50)=3.2+/-0.4 SE and 36+/-5 nM, respectively), maximal after 3 h of incubation (30% inhibition). In addition, 100 nM ACTH and alpha-MSH induced a 60% reduction in leptin expression by RT-PCR. Incubation of cells with 0.5 mM db-cAMP led to a more prominent inhibition of leptin expression and secretion (up to 80% at 1 and 24 h, respectively). The ACTH and alpha-MSH inhibitory effects on leptin secretion were mediated by activation of the MC2-R and MC5-R and were reversed by the MC-R antagonists ACTH(11-24) and ACTH(7-38). In summary, we have shown that POMC-peptides are potent inhibitors of leptin expression and production in 3T3-L1 adipocytes. The finding of ACTH/alpha-MSH receptor-induced inhibition of leptin production and expression in adipocytes support the possibility that there is a control mechanism for modulation of adipose tissue function via a melanocortin-leptin axis.     

Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans

The purpose of this study was to examine the source of adipokines released by the visceral and sc adipose tissues of obese humans. Human adipose tissue incubated in primary culture for 48 h released more prostaglandin E(2), IL-8, and IL-6 than adiponectin, whereas the release of plasminogen activator inhibitor 1 and hepatocyte growth factor was less than that of adiponectin but greater than that of leptin. IL-10 and TNFalpha were released in amounts less than those of leptin, whereas vascular endothelial growth factor and IL1-beta were released in much lower amounts. The accumulation of adipokines was also examined in the three fractions (adipose tissue matrix, isolated stromovascular cells, and adipocytes) obtained by collagenase digestion of adipose tissue. Over 90% of the adipokine release by adipose tissue, except for adiponectin and leptin, could be attributed to nonfat cells. Visceral adipose tissue released greater amounts of vascular endothelial growth factor, IL-6, and plasminogen activator inhibitor 1 compared with abdominal sc tissue. The greatly enhanced total release of TNFalpha, IL-8, and IL-10 by adipose tissue from individuals with a body mass index of 45 compared with 32 was due to nonfat cells. Furthermore, most of the adipokine release by the nonfat cells of adipose tissue was due to cells retained in the tissue matrix after collagenase digestion.     

Insulin enhances vascular endothelial growth factor, interleukin-8, and plasminogen activator inhibitor 1 but not interleukin-6 release by human adipocytes

The present studies were designed to investigate the hormonal regulation of vascular endothelial growth factor (VEGF) release by human subcutaneous adipose tissue explants and adipocytes incubated in primary culture for 48 hours. Vascular endothelial growth factor and IL-8 release by adipocytes were less than 10% of that by tissue explants, whereas that of leptin in adipocytes was comparable to that by tissue. Dexamethasone inhibited VEGF formation by both adipose tissue explants and isolated adipocytes, whereas insulin stimulated VEGF release only in isolated adipocytes. Insulin also enhanced the formation of IL-8 and plasminogen activation inhibitor 1 (PAI-1), but not that of IL-6 by adipocytes although having little effect on that of IL-6 or PAI-1 by adipose tissue explants. Pertussis toxin stimulated lipolysis and inhibited leptin release by human adipose tissue or adipocytes but did not affect release of IL-8 or VEGF. Isoproterenol also stimulated lipolysis by human adipocytes, but this was not accompanied by any significant changes in VEGF, IL-8, IL-6, or PAI-1 release. In contrast, insulin stimulated VEGF release by human adipocytes, and this stimulation was enhanced in the presence of isoproterenol. Insulin stimulated VEGF formation as well as that of PAI-1 by human adipocytes, but not by explants under conditions where it had little effect on that of IL-6. The ability of insulin to stimulate VEGF formation by adipocytes suggests that the elevated circulating levels of insulin in obesity promote angiogenesis in adipose tissue as well as the enhanced accumulation of fat in human adipocytes.     

Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes.

A mutation within the obese gene was recently identified as the genetic basis for obesity in the ob/ob mouse. The obese gene product, leptin, is a 16-kDa protein expressed predominantly in adipose tissue. Consistent with leptin's postulated role as an extracellular signaling protein, human embryonic kidney 293 cells transfected with the obese gene secreted leptin with minimal intracellular accumulation. Upon differentiation of 3T3-L1 preadipocytes into adipocytes, the leptin mRNA was expressed concomitant with mRNAs encoding adipocyte marker proteins. A factor(s) present in calf serum markedly activated expression of leptin by fully differentiated 3T3-L1 adipocytes. A 16-hr fast decreased (by approximately 85%) the leptin mRNA level of adipose tissue of lean (ob/+ or +/+) mice but had no effect on the approximately 4-fold higher level in obese (ob/ob) littermates. Since the mutation at the ob locus fails to produce the functional protein, yet its cognate mRNA is overproduced, it appears that leptin is necessary for its own downregulation. Leptin mRNA was also suppressed in adipose tissue of rats during a 16-hr fast and was rapidly induced during a 4-hr refeeding period. Insulin deficiency provoked by streptozotocin also markedly down-regulated leptin mRNA and this suppression was rapidly reversed by insulin. These results suggest that insulin may regulate the expression of leptin.     

Monocyte chemoattractant protein-1 is produced in isolated adipocytes, associated with adiposity and reduced after weight loss in morbid obese subjects

BACKGROUND: Obesity is associated with insulin resistance and premature atherosclerosis. The human adipose tissue produce several adipokines including monocyte chemoattractant protein (MCP)-1, associated with cardiovascular disease and found to be involved in the pathogenesis of atherosclerosis in vitro. OBJECTIVE: (1) To compare mRNA levels of MCP-1, leptin and a macrophage-specific marker (CD68) in isolated adipocytes vs stromal-vascular (SV) cells, (2) to compare mRNA levels of MCP-1 in human adipose tissue to circulating MCP-1 and adiposity (eg BMI: kg/m2) and (3) investigate the effect of weight loss in obese subjects on circulating MCP-1 and leptin. RESULTS: (1) MCP-1 and CD68 mRNA levels in isolated adipocytes vs SV cells were 17% (P<0.01) and approximately 2% (P<0.001), respectively. Leptin mRNA levels in SV cells were approximately 1% of that in isolated adipocytes (P<0.01). (2) MCP-1 mRNA levels correlated with circulating MCP-1 (P<0.05) and BMI (P<0.05). (3) A 12% weight loss (P<0.001) was associated with a 25% decrease in insulin levels (P<0.01). Circulating MCP-1 and leptin decreased by 20% (P<0.001) and by 24% (P<0.001), respectively. DISCUSSION: The findings demonstrate that MCP-1 is produced in isolated human adipocytes. In addition, the findings suggest that MCP-1 may be involved in obesity-related health complications and support the hypothesis that weight loss is beneficial by improving the low-grade inflammation observed in obesity.     

Osteopontin is an activator of human adipose tissue macrophages and directly affects adipocyte function

Osteopontin (OPN) is highly up-regulated in adipose tissue in human and murine obesity and has been recently shown to be functionally involved in the pathogenesis of obesity-induced adipose tissue inflammation and associated insulin resistance in mice. OPN is a protein with multiple functions and acts as a chemokine and an inflammatory cytokine through a variety of different receptors (CD44, integrins). It is expressed in many cell types including adipose tissue macrophages (ATM). However, the target cells of OPN action in obese adipose tissue are still elusive. Here, we investigated expression of OPN receptors and the impact of OPN on ATM, adipocytes, and other cells of human adipose tissue. We found broad expression of OPN receptors in different adipose tissue cell types including adipocytes. OPN stimulated inflammatory signaling pathways and secretion of cytokines in model macrophages as well as isolated human ATM. Moreover, OPN impaired differentiation and insulin sensitivity of primary adipocytes as determined by peroxisomal proliferator-activated receptor-γ and adiponectin gene expression and insulin-stimulated glucose uptake. Furthermore, OPN induced inflammatory signaling in human adipocytes. In conclusion, OPN activates ATM and interferes with adipocyte function. Thus these data underline the potential of OPN as a therapeutic target for obesity-induced complications.     

Fenofibrate enhances CD36 mediated endocytic uptake and degradation of oxidized low density lipoprotein in adipocytes from hypercholesterolemia rabbit

BACKGROUND: CD36 as a fatty acid transporter is predominantly expressed in adipocytes. We studied whether adipocytes could uptake and degrade OxLDL through CD36 and explored the effect of fenofibrate on OxLDL uptake in adipocytes from hypercholesterolemia rabbits. METHODS: Subcutaneous adipose tissues were collected from normal, high-cholesterol and high-cholesterol plus fenofibrate treatment rabbits for adipocytes culture. CD36 and peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA expression were evaluated by RT-PCR. RESULTS: Cellular expression of CD36 was confirmed during differentiation of adipose cell by RT-PCR. Upon incubation at 37 degrees C, (125)I-OxLDL was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by adipocytes. In binding experiments at 4 degrees C, (125)I-OxLDL exhibited specific and saturable binding to adipocytes (K(D) = 4.2 microg/mL). The endocytic uptake and degradation of (125)I-OxLDL by adipocytes were inhibited by 56 and 54% with anti-CD36 antibody. Fenofibrate treatment enhanced the (125)I-OxLDL uptake and degradation and up-regulated CD36 mRNA expression in adipocytes and suppressed PPARgamma mRNA expression in adipose tissue from hypercholesterolemia rabbits. CONCLUSION: CD36 plays a novel role in adipose tissues and adipocytes possibly involve in clearance of OxLDL in blood. Fenofibrate treatment improved the OxLDL uptake and degradation in adipocytes from hypercholesterolemia rabbits.     

Intrinsic site-specific differences in the expression of leptin in human adipocytes and its autocrine effects on glucose uptake.

Leptin, the ob gene product of adipocytes, regulates body weight by actions on the satiety center in the hypothalamus, but it may also have peripheral effects on the metabolic actions of insulin. In human mature adipocytes isolated from omental (OM) and s.c. tissue, we found that leptin (10 and 100 ng/mL) significantly reduced insulin-mediated glucose uptake by 40% (P<0.05). The effects were rapid and sustained. A U-shaped dose-response curve was obtained, and high leptin concentrations (>100 ng/mL) were without effect. Leptin did not affect basal glucose uptake in adipocytes and had no effect on insulin-stimulated glucose uptake in human preadipocytes. Because leptin may thus have autocrine effects, we examined leptin production from OM and s.c. adipocytes. Western blotting of leptin from 96-h conditioned medium showed greater leptin secretion from s.c. than OM adipocytes, with a ratio of 3.2 (SE +/-0.3, P<0.01). Long-term ceiling cultures were used to examine intrinsic differences in leptin expression under closely controlled conditions. Confocal immunofluorescence microscopy of 12- to 16-day-old ceiling-cultured adipocytes showed that sc adipocytes contained 3.4-fold more leptin (SE +/-0.5, P<0.01) than OM adipocytes, indicating an intrinsic site-specific difference in leptin production. The autocrine effects of leptin to inhibit insulin-stimulated glucose uptake and subsequent lipogenesis in adipose tissue may, therefore, be less in OM adipocytes and may play a role in determining visceral obesity.     

Short-chain fatty acids stimulate leptin production in adipocytes through the G protein-coupled receptor GPR41

Leptin is an adipose-derived hormone that regulates a wide variety of physiological processes, including feeding behavior, metabolic rate, sympathetic nerve activity, reproduction, and immune response. Circulating leptin levels are tightly regulated according to energy homeostasis in vivo. Although mechanisms for the regulation of leptin production in adipocytes are not well understood, G protein-coupled receptors may play an important role in this adipocyte function. Here we report that C2-C6 short-chain fatty acids, ligands of an orphan G protein-coupled receptor GPR41, stimulate leptin expression in both a mouse adipocyte cell line and mouse adipose tissue in primary culture. Acute oral administration of propionate increases circulating leptin levels in mice. The concentrations of short-chain fatty acids required to stimulate leptin production are within physiological ranges, suggesting the relevance of this pathway in vivo.