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At least 19 recordsLinked to original sources
PubMed1998

Autocrine secretion of TGF-beta 1 and TGF-beta 2 by pre-adipocytes and adipocytes: a potent negative regulator of adipocyte differentiation and proliferation of mammary carcinoma cells.

We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma, SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells, but not DR27 mutant epithelial cells which lack TGF-beta type II receptor. Inhibitory activity of CMs was partially abrogated by neutralizing anti-TGF-beta1 and anti-TGF-beta2 antibodies, and was removed following ultrafiltration through membranes of 10,000 Mr but not 30,000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-beta1-like and TGF-beta2-like molecules. In addition, acid-treated CM as well as purified TGF-beta inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that pre-adipocytes spontaneously activate their own secreted TGF-beta, whereas mature adipocytes do not, and suggest that activation of TGF-beta has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-beta may be an important modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment.

3T3 Cells↗
PubMed1986

A thermodynamic approach to hormone-receptor interaction; application to insulin binding to adipocytes, adipocyte plasma membranes and liposomes incorporating adipocyte insulin receptors.

The binding of insulin to its receptor in rat adipocyte and isolated plasma membranes has been measured. The adipocyte insulin receptor has been reconstituted in lecithin liposomes and the binding of insulin investigated. A method of interpreting binding data presented as binding vs. the logarithm of free insulin concentration (binding isotherms) in terms of the binding potential concept of Wyman (1965) is described, and the results are compared with the commonly used Scatchard analysis of binding. The binding potential approach enables binding constants and Gibbs energies of formation of the insulin-receptor complex to be determined as a function of insulin bound. The limiting Gibbs energies of binding at 15 degrees C to intact cells, membranes and liposomes were found to be -55, -52 and -49 kJ mol-1 respectively. The affinity of the receptor for insulin decreases smoothly with increase in binding in all three systems. For intact adipocytes the number of insulin receptors per cell is found to be approximately 43,000.

Adipose Tissue↗
PubMed1988

Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.

1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.

Adipose Tissue↗
PubMed2006

Lipid metabolism and secretory function of porcine intramuscular adipocytes compared with subcutaneous and perirenal adipocytes.

The function of adipocytes interspersed between myofiber fasciculi in skeletal muscle physiology and physiopathology is poorly documented. Because regional differences in adipocyte features have been reported in various species, we hypothesized that lipid metabolism and secretory function of intramuscular (IM) adipocytes differ from that of nonmuscular adipocytes. In the present study, adipocytes isolated from trapezius muscle were compared with subcutaneous and perirenal adipocytes in growing pigs. Between 80 and 210 days of age, gene expressions and/or activities of enzymes involved in lipogenesis or lipolysis were much lower (P < 0.05) in adipocytes isolated from muscle than in those from other locations. Insulin-induced lipogenesis and lipolytic efficiency after catecholamine addition were also the lowest (P < 0.05) in IM adipocytes. In these cells, the age-related increase (+300%) in the ratio of mRNA levels of fatty acid synthase to hormone-sensitive lipase paralleled the enlargement of adipocyte diameters (+70%, P < 0.05) and the increase in lipid content in muscle (+135%, P < 0.05) during growth. Expressions of genes coding for leptin, adiponectin, and IGF-I, as well as for various hormonal receptors, were lower (P < 0.05) in IM adipocytes than in other adipocytes, whereas levels of TNF-alpha mRNA did not differ between sites. Interestingly, IGF-II mRNA levels were higher (P < 0.05) in IM adipocytes than in other adipocytes. These data support the view that IM fat is not just an ectopic extension of other fat locations but displays specific biological features during growth.

Adipocytes↗
PubMed2005

Progressive adipocyte hypertrophy in aquaporin-7-deficient mice: adipocyte glycerol permeability as a novel regulator of fat accumulation.

Aquaporin-7 (AQP7) is a water/glycerol transporting protein expressed in adipocyte plasma membranes. We report here remarkable age-dependent hypertrophy in adipocytes in AQP7-deficient mice. Wild type and AQP7 null mice had similar growth at 0-16 weeks as assessed by body weight; however, by 16 weeks AQP7 null mice had 3.7-fold increased body fat mass. Adipocytes from AQP7 null mice of age 16 weeks were greatly enlarged (diameter 118 mum) compared with wild type mice (39 mum). Adipocytes from AQP7 null mice also accumulated excess glycerol (251 versus 86 nmol/mg of protein) and triglycerides (3.4 versus 1.7 mumol/mg of protein). In contrast, at age 4 weeks, adipocyte volume and body fat mass were comparable in wild type and AQP7 null mice. To investigate the mechanism(s) responsible for the progressive adipocyte hypertrophy, glycerol permeability and fat metabolism were studied in adipocytes isolated from the younger mice. Plasma membrane glycerol permeability measured by [(14)C]glycerol uptake was 3-fold reduced in AQP7-deficient adipocytes. However, adipocyte lipolysis, measured by free fatty acid release and hormone-sensitive lipase activity, and lipogenesis, measured by [(14)C]glucose incorporation into triglycerides, were not affected by AQP7 deletion. These data suggest that adipocyte hypertrophy in AQP7 deficiency results from defective glycerol exit and consequent accumulation of glycerol and triglycerides. Increasing AQP7 expression/function in adipocytes may reduce adipocyte volume and fat mass in obesity.

Adipocytes↗
PubMed1999

Effect of thiazolidinediones on expression of UCP2 and adipocyte markers in human PAZ6 adipocytes.

AIMS/HYPOTHESIS: Thiazolidinediones, a new class of insulin sensitizers, up-regulate the expression of uncoupling protein 2 in rodent adipocytes. It is not known, however, whether thiazolidinediones influence uncoupling protein 2 expression in human adipocytes. We therefore investigated the effect of these drugs on uncoupling protein 2 expression in the recently immortalized human PAZ6 adipocyte cell line. METHODS: Immortalized human PAZ6 preadipocytes were differentiated into adipocytes in the presence or absence of thiazolidinediones. The effect of the drugs on uncoupling protein 2 expression and adipocyte differentiation was measured by reverse transcription-polymerase chain reaction of mRNA of uncoupling protein 2 and of five adipocyte differentiation markers. RESULTS: When cells were differentiated 15 days in the presence of thiazolidinediones, uncoupling protein 2 expression was 2.1-fold higher than in the absence of the drugs. The expression of five adipocyte differentiation markers was, however, also increased by thiazolidinediones. Short-term incubation for 4 and 24 h with thiazolidinediones increased uncoupling protein 2 expression 1.35-fold and 2.3-fold, respectively. The expression of adipocyte markers studied in parallel was also augmented. CONCLUSION/INTERPRETATION: Thiazolidinediones rapidly increase the expression of uncoupling protein 2 in human PAZ6 adipocytes but the increase of uncoupling protein 2 expression is always associated with an augmentation of the expression of all adipocyte markers studied in parallel. This indicates that the effect of thiazolidinediones on uncoupling protein 2 mRNA reflects a general increase in adipocyte differentiation rather than a specific augmentation of uncoupling protein 2 gene expression.

Adipocytes↗
PubMed2001

Human breast adipocytes express interleukin-6 (IL-6) and its receptor system: increased IL-6 production by beta-adrenergic activation and effects of IL-6 on adipocyte function.

Adipocytes produce the inflammatory cytokine interleukin-6 (IL-6); however, it is not known whether these cells express the IL-6 receptor system, how the secretion of this cytokine is regulated, and whether it has a function within adipose tissue. Using cultured human breast adipocytes, we investigated the expression of IL-6 and its receptor system, the effects of IL-6 on main adipocyte functions, and the regulation of IL-6 secretion by catecholamines and glucocorticoids. In the culture system, immunohistochemistry demonstrated expression of IL-6 and its receptor system, consisting of the ligand-binding IL-6 receptor and the signal-transducing protein gp130, in mature adipocytes, but not in undifferentiated adipocyte precursor cells. In freshly isolated adipocytes, RT-PCR detected messenger ribonucleic acids encoding the above proteins. Chronic incubation of adipocytes with 1 nmol/L IL-6 during adipose differentiation reduced glycero-3-phosphate dehydrogenase (GPDH) activity, a marker of adipocyte differentiation, and triglyceride synthesis to 67 +/- 9% of the basal level (mean +/- SEM; P < 0.05) only on day 21. Incubation of differentiated adipocytes with 10 nmol/L IL-6 for 24 h also resulted in a reduction of GPDH activity to 81 +/- 5% (P < 0.05). On the other hand, 24-h exposure to 10 nmol/L IL-6 increased basal glycerol release by 42 +/- 12% (P < 0.01) and isoproterenol-induced glycerol release by 21 +/- 6% (P < 0.05). The same concentration of IL-6, however, did not alter basal or insulin-stimulated glucose transport. IL-6 secretion was acutely and chronically stimulated by 1 micromol/L isoproterenol (peak of 6.2-fold after 3 h; P < 0.001) and only moderately suppressed by 100 nmol/L cortisol (-36 +/- 10%; P < 0.001). In conclusion, human breast adipocytes release substantial amounts of IL-6 and express IL-6 receptor and gp130. The secretion of IL-6 by adipocytes is strongly stimulated by beta-adrenergic activation and is modestly suppressed by glucocorticoids. IL-6 reduces GPDH activity and stimulates lipolysis, suggesting an autocrine/paracrine role of this cytokine in human adipose breast tissue.

Adipocytes↗
PubMed1985

Alpha 2-adrenergic binding and action in human adipocytes. Comparison between binding to plasma membrane preparations and to intact adipocytes.

Binding of the alpha 2-adrenoceptor antagonist [3H]yohimbine was demonstrated on intact human adipocytes and on human adipocyte membranes. Specific binding was rapid, reversible, saturable and of high affinity in both preparations. [3H]Yohimbine binding was inhibited by various adrenergic agents in a manner which suggests that the labeled sites probably represent the alpha 2-receptor both in intact adipocytes and in the membrane fraction. In adipocyte membranes the maximal binding capacity (Bmax) was 463 +/- 38 fmol/mg protein and the binding was of high affinity with a Kd of 2.1 +/- 0.4 nM. In intact human adipocytes, Bmax was 903 +/- 139 fmol/10(6) cells (or 342 +/- 21 fmol/100 cm2) and the binding affinity was 6.6 +/- 0.8 nM. Adrenergic antagonists bound to a homogeneous class of receptors (linear Scatchard plots) both in isolated membranes and in intact adipocytes. However, agonist binding was heterogeneous in both preparations. The affinity of agonist binding was 5-10 times higher in membranes than in intact adipocytes. The physiological relevance of the binding data was evaluated by correlating the binding of yohimbine with the antilipolytic effect of clonidine. A positive and significant correlation was found between Bmax and maximal antilipolytic effect of clonidine (r = 0.70) in membranes. Furthermore, the binding affinity (Kd) was positively correlated to the sensitivity (IC50) of the clonidine-induced antilipolysis (r = 0.65). A positive and significant correlation was also found in intact adipocytes between Bmax and the maximal antilipolytic effect of clonidine (r = 0.79). However, there was no significant correlation between Kd in intact adipocytes and the IC50 of clonidine. It is concluded that both intact human adipocytes and membrane fractions are useful models to investigate the properties and regulation of alpha 2-adrenoceptors. However, it appears from our correlation studies that the binding data obtained with the membranes are the best related to the physiological effects mediated by alpha 2-receptors in human adipocytes.

Adipose Tissue↗
PubMed2003

Chronological changes in metabolism and functions of cultured adipocytes: a hypothesis for cell aging in mature adipocytes.

The growth and aging of 3T3-L1 adipocytes were investigated in a synchronized tissue-culture system. We systematically characterized several major aspects of adipocyte metabolism and functions as variables of cell age. We found that terminal differentiation of 3T3-L1 cells is followed by a near-linear hypertrophic growth (increase in triglyceride content) of the cultured adipocytes throughout a 20-day study period. However, three metabolically and functionally distinct stages are recognized. The first stage overlaps with differentiation and is represented by small immature adipocytes. The second stage is characterized by fully mature adipocytes that show peaked overall metabolic activities. The third stage is marked by cell aging, with deterioration in every major aspect of the cell's functionality except for the function of net energy storage, which is preserved even in aged adipocytes. Compared with young mature adipocytes, older cells are increasingly insulin resistant, have decreased glucose uptake and fuel consumption, and show impaired glycerokinase-mediated fatty acid reesterification. Moreover, aged adipocytes show reduced gene expression for adiponectin and leptin, each of which is important in systemic regulation of energy metabolism. The characterization of these cell age-dependent changes in adipocyte functionality provides a model for understanding dynamic changes at the tissue level and suggests that adipose tissue is modifiable via adipocyte aging.

3T3-L1 Cells↗
PubMed2001

The frequency of common progenitors for adipocytes and osteoblasts and of committed and restricted adipocyte and osteoblast progenitors in fetal rat calvaria cell populations.

In fetal rat calvaria (RC) cell populations, adipocyte differentiation is stimulated by both dexamethasone (Dex) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], whereas osteoblast differentiation is stimulated by Dex but inhibited by 1,25(OH)2D3. We examined whether the osteoblastic and adipocytic colonies were derived from a common progenitor, from committed and restricted adipocyte and osteoblast progenitors, or from both and whether the adipocyte progenitors stimulated by 1,25(OH)2D3 constitute a population of progenitors that is different from that stimulated by Dex. RC cells were isolated by sequential enzyme digestion yielding five populations designated I-V. In population I the effect of Dex on adipocyte formation was greater than that of 1,25(OH)2D3, whereas the effect of 1,25(OH)2D3 was greater than that of Dex in populations III-V. We next applied replica plating techniques to further investigate the response characteristics of individual osteoprogenitors and adipocyte progenitors by looking at the fate of duplicate colonies derived from the same progenitor under different culture conditions. RC cells were plated at 1,000-1,500 cells/100 mm culture dish and a 17-microm mesh polyester membrane overlaid onto master dishes on day 4 or day 5 and removed on day 11 or day 12. Then, replicas and master dishes were cultured separately in medium containing either Dex, 1,25(OH)2D3, or Dex plus 1,25(OH)2D3 for a further 17-21 days and then fixed and stained with both Sudan IV and the von Kossa technique. Nine hundred twenty-seven matched colonies present on both master dishes and replica membranes were screened and colonies were classified as either adipocytic, osteoblastic (bone or osteoid), or fibroblastic. Results show convincingly that most of the osteoprogenitors present in fetal RC cells are committed and restricted to the osteoblastic cell lineage (95.29%); that the 1,25(OH)2D3-responsive adipocyte progenitors are different from the Dex-responsive adipocyte progenitors, but both are restricted to form adipocytes and finally; and that a common osteoblastladipocyte progenitor is present in a low frequency (4.71% of osteoprogenitors).

Adipocytes↗
PubMed2000

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.

Adipocytes↗
PubMed1990

Expression of a 64 kD adipocyte-specific plasma membrane protein in genetically lean but not obese porcine adipocytes.

A monoclonal antibody (LA-1) to an adipocyte-specific plasma membrane protein (64 kD) was used to examine the differential expression of this protein in genetically lean and genetically obese pigs. Enzyme-linked immunosorbent assay (ELISA) implied the differential expression of the 64 kD protein in adipocyte plasma membranes having different genetic background. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of genetically lean, genetically obese, and contemporary subcutaneous adipocyte plasma membranes did not indicate any obvious qualitative differences in protein composition. Corresponding immunoblots utilizing LA-1 confirmed the presence of the 64 kD protein in contemporary and genetically lean adipocyte plasma membranes but absence in genetically obese adipocyte plasma membranes. LA-1 labelled intact adipocytes isolated from contemporary and genetically lean adipose tissue but did not react with isolated genetically obese adipocytes. The ability to bind to intact adipocytes indicates that the protein is exposed to the extracellular environment. The migration pattern of the protein was not affected by enzymatic deglycosylation by endoglycosidase-F suggesting that the protein is not highly, if at all, glycosylated. Presence of the 64 kD protein in genetically lean but not genetically obese adipocyte plasma membranes indicates the identification of a novel adipocyte-specific surface protein associated, either directly or secondary to the onset of obesity, with genetic predispositions for either genetically lean or obese body types in swine.

Adipose Tissue↗
PubMed1998

Stimulation of adipose differentiation related protein (ADRP) expression by ibuprofen and indomethacin in adipocyte precursors and in adipocytes.

Adipose differentiation related protein (ADRP) is a 50 kDa protein expressed at high level in differentiated adipocytes. ADRP expression is very low in undifferentiated adipocytes and increases rapidly and dramatically as the cells undergo adipose differentiation. In the present study, we demonstrate that ADRP expression at the mRNA and protein level is stimulated in adipocyte precursor cells in a time- and dose-dependent fashion by treatment with cyclooxygenase inhibitors, particularly indomethacin and ibuprofen. Lipoxygenase inhibitors such as AA861 and nordihydroguaiaretic acid were ineffective. Stimulation of ADRP expression was observed with 10(-5) M ibuprofen but maximal stimulation required a concentration of 3 x 10(-4) M. Nuclear run-on experiments indicated that indomethacin or ibuprofen stimulated the transcription of the ADRP gene in undifferentiated adipocytes. In addition to stimulating the induction of ADRP in undifferentiated cells, ibuprofen and indomethacin also stimulated the level of ADRP mRNA and protein in differentiated adipocytes. These experiments provide new information on the regulation of ADRP, an early inducible gene in the adipocyte differentiation programme in adipocyte precursors and in adipocytes and identify a new target for cyclooxygenase inhibitor action during adipocyte differentiation.

Adipocytes↗
PubMed1984

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes.

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.

Adipose Tissue↗
PubMed2003

Autotaxin is released from adipocytes, catalyzes lysophosphatidic acid synthesis, and activates preadipocyte proliferation. Up-regulated expression with adipocyte differentiation and obesity.

Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904-910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase d-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obese-diabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.

Adipocytes↗
PubMed2001

Bezafibrate reduces mRNA levels of adipocyte markers and increases fatty acid oxidation in primary culture of adipocytes.

The molecular mechanisms by which peroxisome proliferator-activated receptor (PPAR) activation by fibrates reduces fat deposition and improves insulin sensitivity are not completely understood. We report that exposure of a rat primary culture of adipocytes for 24 h to the PPAR activator bezafibrate increased the mRNA levels of crucial genes involved in peroxisomal and mitochondrial beta-oxidation. The mRNA levels of the peroxisomal beta-oxidation rate-limiting enzyme acyl-CoA oxidase and of the muscle-type carnitine palmitoyl transferase I (M-CPT-I), which determines the flux of mitochondrial beta-oxidation, increased by 1.6-fold (P < 0.02) and 4.5-fold (P = 0.001), respectively. These changes were accompanied by an increase in the transcript levels of the uncoupling protein-2 (UCP-2; 1.5-fold induction; P < 0.05) and UCP-3 (3.7-fold induction; P < 0.001), mitochondrial proteins that reduce ATP yield and may facilitate the oxidation of fatty acids. Furthermore, bezafibrate increased the mRNA levels of the fatty acid translocase (2-fold induction; P < 0.01), suggesting a higher fatty acid uptake into adipocytes. In agreement with these changes, bezafibrate caused a 1.9-fold induction (P < 0.02) in 9,10-[(3)H]palmitate oxidation. Moreover, bezafibrate reduced the mRNA expression of several adipocyte markers, including PPARgamma (30% reduction; P = 0.05), tumor necrosis factor-alpha (33% reduction; P < 0.05), and the ob gene (26% reduction). In contrast, adipocyte fatty acid binding protein mRNA levels increased (1.5-fold induction; P < 0.01), pointing to a mobilization of fatty acids to mitochondria and peroxisomes. The reduction of the adipocyte markers caused by bezafibrate was accompanied by an increase in the mRNA levels of the preadipocyte marker Pref-1 (1.6-fold induction; P < 0.01). Some of the changes observed in the primary culture of rat adipocytes also were studied in the epididymal white adipose tissue of bezafibrate-treated rats for 7 days. In vivo, M-CPT-I mRNA levels increased (4.5-fold induction; P = 0.001) in epididymal white adipose tissue of bezafibrate-treated rats. Similarly, fatty acid translocase (2.6-fold induction; P = 0.002) and Pref-1 (5.6-fold induction) mRNA levels increased, although differences in the latter were not significant because of huge individual variations. These results indicate that exposure of adipocytes to bezafibrate, independent of its hepatic effects, increases the degradation of fatty acids, reducing their availability to synthesize triglycerides. As a result, some degree of dedifferentiation of adipocytes to preadipocyte-like cells is achieved. These changes may be involved in the reduction in fat depots and in the improvement of insulin sensitivity observed after bezafibrate treatment.

Acyl-CoA Oxidase↗
PubMed1981

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH. 2. Stability and restoration of activity of hormone-sensitive adenylate cyclase of adipocyte plasma membrane.

The isolated intact white adipocyte of the Swiss mouse responds to both ACTH and catecholamines by an elevation of cAMP levels and an increase in lipolysis. However, in the isolated plasma membrane of the mouse adipocyte, adenylate cyclase loses its responsiveness to ACTH but retains its ability to respond to catecholamines. This lack of responsiveness to ACTH by adenylate cyclase of mouse adipocyte plasma membrane can be overcome, at least partially, by addition of GPP (NH)p, an analog of GTP, to the assay medium. The data on mouse adipocyte membrane suggests that the coupling of ACTH receptor to adenylate cyclase is dependent on GTP and that catecholamine-activation of adenylate cyclase is less dependent on this nucleotide. The isolated intact white adipocyte of adult New Zealand rabbit responds to ACTH, but does not (or only weakly) respond to catecholamines. In contrast to the mouse plasma membrane preparation, adenylate cyclase of adipocyte membrane of the rabbit responds to ACTH. And the addition of GPP(NH)P is not required to demonstrate the CTH: sensitive adenylate cyclase activity. The difference between mouse and rabbit adipocyte membrane in the requirement for GPP(NH)P in ACTH action is not readily explained. The lack of catecholamine sensitivity of rabbit membrane enzyme cannot be reversed by addition of GPP(NH)P or adenosine deaminase. These two adenylate cyclase model systems using mouse and rabbit adipocyte plasma membrane may be useful tools for the study of the specificity and mechanism of action of lipolytic hormones such as ACTH and catecholamines.

Adenylyl Cyclases↗
PubMed1988

Characterization of high-density lipoprotein binding to rat adipocytes and adipocyte plasma membranes.

The interaction of high-density lipoproteins (HDL) with adipocytes is important in the regulation of cellular cholesterol flux. To study the mechanisms of HDL binding and cellular processing, we incubated adipocytes isolated from epididymal and perirenal adipose tissue of male Wistar rats (300 g) with HDL1 (1.07-1.10 g/mL) and HDL2 (1.10-1.14 g/mL) fractions separated from rat plasma by gradient ultracentrifugation. Freshly isolated adipocytes were incubated with 125I-labeled HDL for 2 h at 37 degrees C to determine cell-associated uptake and degradation. Adipocytes from both fat regions showed significant cell-associated HDL1 and HDL2 uptake and very high medium degradation (2- to 6-fold higher than uptake). To assess 125I-labeled HDL binding independent of cellular metabolism, we purified adipocyte plasma membranes from isolated adipocytes and used them in binding assays. Binding of HDL1 and HDL2 in the membrane system was 85-95% specific, sensitive to high NaCl concentrations, and abolished by pronase treatment. In contrast to HDL2 binding, the maximum HDL1 binding to perirenal plasma membranes was significantly higher than its binding to epididymal membranes (7.2 +/- 1.3 vs. 4.4 +/- 0.2 micrograms/mg, n = 6, p less than 0.05). This increment in HDL1 binding to perirenal membranes represented an EDTA- sensitive, calcium-dependent component. These results indicate that HDL binding to adipocyte plasma membranes depends on both adipose tissue region and HDL subtype. The membrane binding characteristics, taken together with the cellular uptake results, suggest that adipocytes bind and metabolize HDL and that this interaction may involve a protein receptor.

Adipose Tissue↗