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At least 73 records · Page 4Linked to original sources
PubMed1983

Oxytocin resistance in Brattleboro rat adipocytes and comparative studies on insulin or oxytocin responsiveness in normal rat adipocytes.

We have evaluated factors, other than genetic, which might be related to the lack of an oxytocin-mediated insulinlike response (glucose oxidation; lipogenesis) in adipocytes from Brattleboro rats, homozygous for the diabetes insipidus trait (HoDI rats). The manoeuvres used in an attempt to restore the glucoregulatory responses to oxytocin in HoDI cells (increased glucose in the fat pad digestion medium; increased calcium concentration in the oxidation assay; estrogen treatment; use of [1-14C]glucose as substrate; inclusion of adenosine in the assay medium; vasopressin replacement therapy) uniformly failed to result in oxytocin activation of HoDI adipocytes. In contrast, the contractile responses of estrogenized HoDI rat uteri were indistinguishable from those of estrogenized normal rats. We conclude that the nonresponsiveness of the Brattleboro adipocytes to the glucoregulatory actions of oxytocin is not due to factors related to the conditions of the bioassay. On the other hand, in normal fat cells (from Sprague-Dawley and Long Evans rats), oxytocin responsiveness was augmented by a number of the manoeuvres mentioned above, most notably by the inclusion of either calcium (10 mM) or adenosine (10 microM) in the assay medium. Nonetheless, the maximum oxytocin responsiveness of adipocytes from Long Evans or Sprague-Dawley rats, under all conditions of assay, was still only a fraction (less than 20%) of the maximal response to insulin. The effect of adenosine on oxytocin action (increased sensitivity, without an effect on the maximum response) is in keeping with the previously observed effects of this nucleoside on the action of insulin; our results thus pointed to a new parallel in the action of insulin and oxytocin.

Adenosine↗
PubMed1991

Expression and regulation of growth hormone (GH) receptor messenger ribonucleic acid (mRNA) in rat adipose tissue, adipocytes, and adipocyte precursor cells: GH regulation of GH receptor mRNA.

The effects of hypophysectomy and hormonal replacement therapy on GH receptor (GH-R) gene expression was studied in rat adipose tissue with a cRNA probe corresponding to the amino-terminal of the hepatic GH-R. Male Sprague-Dawley rats, 50-65 days of age, were used. In all fat depots tested (epididymal, retroperitoneal, and sc), two transcripts with an estimated size of 4.0 and 1.2 kilobases (kb), respectively, were detected. An intermediate-size transcript (2.6 kb) was sometimes observed. Also, isolated adipocytes and adipocyte precursor cells from the epididymal fat pad expressed these GH-R transcripts. The pituitary dependance of GH-R gene expression was analyzed in epididymal fat. Hypophysectomies were performed at 50 days of age, and the rats were then given replacement therapy with L-T4 (10 micrograms/kg.day) and hydrocortisone (400 micrograms/kg.day). Hypophysectomy decreased the abundance of both the 4.0 and the 1.2-kb transcripts, an effect that in part was restored by GH treatment. A solution hybridization RNase protection assay was then used to further characterize the effect of GH treatment of hypophysectomized rats on GH-R gene expression. A single injection of human GH (100 micrograms/rat) increased GH-R mRNA levels within 1 h, and maximal levels were reached between 3-12 h after the injection. The increase in GH-R mRNA levels was dose dependent and was observed also after prolonged treatment (1 or 5 mg/kg.day for 6 days) with bovine GH. These results confirm that GH-R mRNAs are present in rat adipose tissue from different fat depots. GH-R transcripts of the same estimated size were detected in isolated adipocytes and adipocyte precursor cells. Furthermore, the results show that there is a rapid and GH-dependent regulation of GH-R mRNA levels in adipose tissue.

Adipose Tissue↗
PubMed1985

Assessment of initial hexose uptake in adipocytes using the physiological substrate D-glucose. Particular relevance to human adipocytes.

The characteristics of U-[14C]D-glucose uptake into human and rat adipocytes have been examined. The initial rates of U-[14C]D-glucose uptake were found to be linear during the first 30 s, both basally and with maximum insulin stimulation. Phloretin was shown to be effective in stopping glucose flux for human adipocytes, although rapid separation of cells from medium was found to be necessary for accurate measurement of initial rate of glucose uptake into rat adipocytes. The pH and temperature dependency of U-[14C]D-glucose uptake were found to be similar to those reported for 3-0-methylglucose. The use of U-[14C]D-glucose for assessment of initial rate of glucose uptake into human adipocytes provides results directly relevant to D-glucose metabolism, in addition to being technically less demanding and much less expensive than methods using the non-physiological sugar analogue 3-0-[14C]methylglucose.

Adipose Tissue↗
PubMed2000

Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity.

Severe quantitative and qualitative brown adipocyte defects are common in obesity. To investigate whether aberrant expression of tumor necrosis factor alpha (TNF-alpha) in obesity is involved in functional brown fat atrophy, we have studied genetically obese (ob/ob) mice with targeted null mutations in the genes encoding the two TNF receptors. The absence of both TNF receptors or p55 receptor alone resulted in a significant reduction in brown adipocyte apoptosis and an increase in beta(3)-adrenoreceptor and uncoupling protein-1 expression in obese mice. Increased numbers of multilocular functionally active brown adipocytes, and improved thermoregulation was also observed in obese animals lacking TNF-alpha function. These results indicate that TNF-alpha plays an important role in multiple aspects of brown adipose tissue biology and mediates the abnormalities that occur at this site in obesity.

Adaptation, Physiological↗
PubMed2003

Effect of polyunsaturated fatty acids on the expression of transcription factor adipocyte determination and differentiation-dependent factor 1 and of lipogenic and fatty acid oxidation enzymes in porcine differentiating adipocytes.

Polyunsaturated fatty acids (FA) regulate genes involved in lipid metabolism. The effects of polyunsaturated FA on the transcription factor adipocyte determination and differentiation-dependent factor (ADD) 1 and fatty acid synthase (FAS) mRNA in differentiating porcine adipocytes were measured using a stromal vascular cell culture system. Porcine stromal vascular cells were isolated from subcutaneous adipose tissues and plated in Dulbecco's modified Eagle's medium (DMEM)-nutrient mixture F-12 Ham (F-12) plus fetal bovine serum (100 ml/l) for 24 h. Then cells were differentiated in DMEM-F12 plus insulin, hydrocortisone and transferrin without or with polyunsaturated FA at 6.25, 25.00 or 100.00 microM. The ADD1 mRNA was decreased by 100.00 microM-arachidonic acid, 6.25 to 100.00 microM-docosahexaenoic acid or cis-9,trans-11-conjugated linoleic acid. The polyunsaturated FA reduced the transcription rate of FAS, but not of ADD1. All three polyunsaturated FA accelerated degradation of ADD1 and FAS mRNA to reduce the abundance of ADD1 and FAS mRNA. Results also showed that polyunsaturated FA inhibit the ADD1 expression, not only of mRNA concentration, but also of mature ADD1 protein concentration, suggesting an overall reduction of ADD1 function by polyunsaturated FA. Our present experiments demonstrate that polyunsaturated FA regulate the gene expression of ADD1 and enzymes involved in lipid metabolism in porcine adipocytes.

Adipocytes↗
PubMed1999

Regulation of peroxisome proliferator-activated receptor gamma expression by adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1: implications for adipocyte differentiation and metabolism.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor implicated in adipocyte differentiation and insulin sensitivity. We investigated whether PPARgamma expression is dependent on the activity of adipocyte differentiation and determination factor 1/sterol regulatory element binding protein 1 (ADD-1/SREBP-1), another transcription factor associated with both adipocyte differentiation and cholesterol homeostasis. Ectopic expression of ADD-1/SREBP-1 in 3T3-L1 and HepG2 cells induced endogenous PPARgamma mRNA levels. The related transcription factor SREBP-2 likewise induced PPARgamma expression. In addition, cholesterol depletion, a condition known to result in proteolytic activation of transcription factors of the SREBP family, induced PPARgamma expression and improved PPRE-driven transcription. The effect of the SREBPs on PPARgamma expression was mediated through the PPARgamma1 and -3 promoters. Both promoters contain a consensus E-box motif that mediates the regulation of the PPARgamma gene by ADD-1/SREBP-1 and SREBP-2. These results suggest that PPARgamma expression can be controlled by the SREBP family of transcription factors and demonstrate new interactions between transcription factors that can regulate different pathways of lipid metabolism.

Adipocytes↗
PubMed1992

Size distribution of adipocytes and variation in adipocyte number in lines of mice selected for high or low body fat.

1. Adipocyte size and number were measured in epididymal fat pads from lines of mice selected for high or low body fat. 2. Epididymal fat pad weight at 10 weeks of age was 4-fold greater in the high fat than the low fat mice, accompanied by a 7-fold greater adipocyte volume but there was no difference in adipocyte number. Smaller differences in fatness and cell volume were observed at younger ages. 3. The high fat mice showed one or two populations of cells up to 5 weeks of age and at 7 weeks 3 days and 10 weeks showed two or more populations. In contrast, the low fat animals at all ages showed either one or two populations. 4. These findings imply that greater lipogenesis and cell filling, not cell proliferation, is the primary mechanism of increased fatness in these selected lines of mice.

Adipose Tissue↗
PubMed1982

Hormone-sensitive lipase in human adipose tissue, isolated adipocytes, and cultured adipocytes.

Employing a perifusion technique, the activity of hormone-sensitive lipase (HSL) in human adipose tissue and isolated adipocytes was found to be significantly stimulated by 5 microM epinephrine (5-fold. P less than 0.001) or 0.5 mM theophylline (2-fold, P less than 0.03). The stimulatory effect of both, however, was not cumulative; instead, theophylline appeared to blunt the epinephrine effect. The two effectors together produced a 3-fold increase in activity over basal (P less than 0.01). Basal lipolysis was highly and significantly correlated with epinephrine and/or theophylline stimulated lipolysis in the tissue as well as in the isolated cells. The activity of the enzyme was examined in cultured human adipocytes using a cell-free system. The basal activity of HSL in the 20,000 X g supernatant (S20) fraction of cultured cells grown in fat-enriched medium was significantly higher than the value in the same fraction of cells grown in regular medium (6.06 +/- 1.49 versus 2.78 +/- 0.89 nmole glycerol/min/mg protein, mean +/- S.D., P less than 0.01), an was similar in the S20 fractions of cells grown in the enriched medium and the original tissue (6.06 +/- 1.49 versus 5.44 +/- 2.73 nmole glycerol/Min/mg protein, mean +/- S.D., P greater than 0.83). When the tissue and cells were stimulated in vitro before fractionation, the HSL activity in the S20 fraction of the original tissue increased 4-fold over basal (P less than 0.001), whereas that in the cultured cells increased 2-fold (P less than 0.01) regardless of the culture medium employed. In the S20 fractions derived from basally incubated samples, but not in those derived from the epinephrine-stimulated samples, the HSL enzyme was slightly but significantly (P less than 0.002) activated by exogenous addition of ATP, cyclic AMP, and protein kinase. The data suggest that the activity of HSL is retained in cultured human adipocytes and could be enhanced under conditions of culture which favor lipid accumulation and adipose conversion.

Adipose Tissue↗
PubMed1996

Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture.

The effects of human growth hormone (hGH) on proliferation and differentiation of primary adipocyte precursor cells isolated from rat epididymal fat pads were studied under serum-free culture conditions. hGH markedly reduced the formation of new fat cells and the expression of glycerophosphate dehydrogenase activity, a marker enzyme of adipose differentiation, in a dose-dependent manner. To find an explanation for this inhibitory effect, we investigated the action of GH on (1) cell proliferation and on (2) lipid accumulation, the latter in the absence and presence of corticosterone. In undifferentiated cells, 5 nmol/L hGH increased both cell number and [3H]-thymidine incorporation (1.3- and 2.6-fold over basal, respectively). This effect was mediated by insulin-like growth factor-I (IGF-I), since hGH stimulated IGF-I production in undifferentiated cells by 12-fold and addition of an anti-IGF-I monoclonal antibody (IGF-I MAb) abolished the mitogenic effect of hGH but did not prevent hGH-induced suppression of adipose differentiation. In developing fat cells, hGH significantly reduced cellular 2-deoxyglucose uptake and glucose incorporation into lipids. In addition, hGH exhibited a lipolytic action in the presence of insulin and triiodothyronine. These effects were not prevented by IGF-I MAb. Specific binding of [125I]-hGH to precursor cells increased significantly during adipose conversion. In differentiated cells Scatchard analysis yielded linear plots with an apparent Kd of 0.16 nmol/L and 8,400 sites per cell. Taken together, these data show that hGH reduces adipose conversion in primary cultures of rat adipocyte precursor cells while promoting cell proliferation through an increase in IGF-I production.

Adipocytes↗
PubMed2004

Regulated production of a peroxisome proliferator-activated receptor-gamma ligand during an early phase of adipocyte differentiation in 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear hormone receptor that is critical for adipogenesis and insulin sensitivity. Ligands for PPARgamma include some polyunsaturated fatty acids and prostanoids and the synthetic high affinity antidiabetic agents thiazolidinediones. However, the identity of a biologically relevant endogenous PPARgamma ligand is unknown, and limited insight exists into the factors that may regulate production of endogenous PPARgamma ligands during adipocyte development. To address this question, we created a line of 3T3-L1 preadipocytes that carry a beta-galactosidase-based PPARgamma ligand-sensing vector system. In this system, induction of adipogenesis resulted in elevated beta-galactosidase activity that signifies activation of PPARgamma via its ligand-binding domain (LBD) and suggests generation and/or accumulation of a ligand moiety. The putative endogenous ligand appeared early in adipogenesis in response to increases in cAMP, accumulated in the medium, and dissipated later in adipogenesis. Organically extracted and high pressure liquid chromatography-fractionated conditioned media from differentiating cells, but not from mature adipocytes, were enriched in this activity. One or more components within the organic extract activated PPARgamma through interaction with its LBD, induced lipid accumulation in 3T3-L1 cells as efficiently as the differentiation mixture, and competed for binding of rosiglitazone to the LBD of PPARgamma. The active species appears to be different from other PPARgamma ligands identified previously. Our findings suggest that a novel biologically relevant PPARgamma ligand is transiently produced in 3T3-L1 cells during adipogenesis.

3T3-L1 Cells↗
PubMed2004

Amino acids and leucine allow insulin activation of the PKB/mTOR pathway in normal adipocytes treated with wortmannin and in adipocytes from db/db mice.

Amino acids are nutrients responsible for mammalian target of rapamycin (mTOR) regulation in mammalian cells. The mTOR protein is mainly known for its role in regulating cell growth, notably via protein synthesis. In addition to amino acids, mTOR is regulated by insulin via a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. mTOR mediates crosstalk between amino acids and insulin signaling. We show that in freshly isolated rat adipocytes, insulin stimulates the phosphorylation of mTOR on serine 2448, a protein kinase B (PKB) consensus phosphorylation site. This site is also phosphorylated by amino acids, which in contrast to insulin do not activate PKB. Moreover, insulin and amino acids have an additive effect on mTOR phosphorylation, indicating that they act via two independent pathways. Importantly, amino acids, notably leucine, permit insulin to stimulate PKB when PI 3-kinase is inhibited. They also rescue glucose transport and the mTOR pathway. Further, leucine alone can improve insulin activation of PKB in db/db mice. Our results define the importance of amino acids in insulin signaling and reveal leucine as a key amino acid in disease situations associated with insulin-resistance in adipocytes.

Adipocytes↗
PubMed1987

Expression of the mitochondrial uncoupling protein in brown adipocytes. Absence in brown preadipocytes and BFC-1 cells. Modulation by isoproterenol in adipocytes.

The expression of the uncoupling protein has been compared in cells of BFC-1 clonal line established from mouse brown adipose tissue (BAT) and in preadipocytes, as well as in adipocytes from mouse BAT, both in primary culture. The results of immunoblots show that, after one week in culture, adipocytes have a reduced level of the 32 kD protein. This level can be raised 2-3.5-fold by a 24-h exposure to isoproterenol. Thus a direct modulation by a beta-agonist drug in the expression of the uncoupling protein is observed. Under the same conditions as well as under various other conditions, preadipocytes in primary culture and BFC-1 cells do not express the uncoupling protein. At the same time these cells are able both to differentiate into adipose cells, as demonstrated by the emergence of enzyme markers and triglyceride accumulation, and to respond to isoproterenol. Thus isoproterenol is not sufficient to trigger the expression of the uncoupling protein and behaves as a mere modulator once the cells have acquired the capacity to express it. Injection of undifferentiated BFC-1 cells into athymic mice bearing catecholamine-containing mini-osmotic pumps, or co-cultures of BFC-1 cells and pheochromocytoma PC-12 cells do not allow BFC-1 cells to express the uncoupling protein. Taken together, the results suggest that the formation of brown preadipocytes is critically linked during development to the release by sympathetic nerves of specific trophic factors acting locally.

Adipose Tissue↗
PubMed1982

Characteristics of beta-adrenergic-agonist binding to rat adipocyte membranes. Evidence that (+/-)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte beta-adrenergic receptors.

The binding characteristics of the beta-adrenergic agonist (+/)-[3H]hydroxybenzylisoproterenol to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37 degrees C (second order rate constant K1 = 1.37 . 10(7) . M-1 . min-1). Dissociation of specific binding by 0.5 mM (-)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k2 of 0.106 . min-1 and 0.011 . min-1 . [3H]Hydroxybenzylisoproterenol binding was saturable (Bmax = 690 +/- 107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [3H]hydroxybenzylisoproterenol binding sites, one having high affinity (KD = 3.5 +/- 0.7 nM) but low binding capacity (10% of the total sites) and one having low affinity (KD = 101 +/- 20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [3H]hydroxybenzylisoproterenol binding with the following order of potency = (-)-propranolol greater than (-)-isoproterenol greater than (-)-norepinephrine approximately (-)-epinephrine greater than greater than (+)-isoproterenol = (+)-propranolol, which is consistent with binding to beta 1-adrenergic receptors. Competition curves of [3H]hydroxybenzylisoproterenol binding by the beta-agonist (-)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by beta-antagonist (-)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [3H]hydroxybenzylisoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (-)-isoproterenol, the number of [3H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (-)-isoproterenol to stimulate adenylate cyclase correlated closely with the ability of (-)-isoproterenol to displace [3H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the beta-antagonist [3H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [3H]hydroxybenzylisoproterenol can be successfully used to label the beta-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [3H]dihydroalprenolol in these cells.

Adipose Tissue↗
PubMed1983

Insulin-like stimulatory effect of Fc fragments of human immunoglobulin G on rat adipocyte lipogenesis: indirect evidence for Fc receptor on adipocytes.

We have recently shown that human immunoglobulin G (IgG) exerts an insulin-like stimulatory effect on lipogenesis by rat and human adipocytes in vitro. Highly purified polyclonal IgGs prepared from normal subjects have stimulatory potencies similar to those of monoclonal IgGs obtained from patients with hIgG myeloma. These observations suggest that this effect is probably exerted by a nonspecific nonvariable portion of the IgG molecule. We now present data to show that the Fc moiety of the IgG molecule is responsible for the stimulatory effect of this IgG molecule. These data also provide evidence for the first time that adipocytes may have Fc receptors.

Adipose Tissue↗
PubMed1983

Molecular cloning of mRNA from 3T3 adipocytes. Regulation of mRNA content for glycerophosphate dehydrogenase and other differentiation-dependent proteins during adipocyte development.

We have constructed a recombinant bacterial library containing cDNA prepared from mRNA of adipose 3T3 cells. We have screened this library by several methods and isolated colonies containing sequences complementary to mRNAs for glycerophosphate dehydrogenase, two other major differentiation-dependent proteins of Mr = 28,000 and 13,000, and actin. These recombinants were identified by hybrid selection of total adipocyte mRNA, translation in vitro and subsequent immunoprecipitation, and two-dimensional electrophoresis of translated proteins. Three of the four cloned cDNAs hybridized to single adipocyte mRNA species of size close to that expected from the size of the polypeptide it specifies, but the mRNA for glycerophosphate dehydrogenase contained 3,550 bases, far larger than necessary to code for its polypeptide (Mr = 34,000). The increase in amount of mRNA during differentiation was 150-fold for the protein of Mr = 13,000 and considerably greater for glycerophosphate dehydrogenase and the protein of Mr = 28,000. The time course of mRNA accumulation was different for each of these mRNAs, indicating they do not respond synchronously during differentiation.

Adipose Tissue↗
PubMed1994

Insulin and lipogenesis in rat adipocytes. II. Effect of high levels of insulin and glucose on lipid synthesis in isolated rat adipocytes.

Insulin resistant states are characterized by receptor and post-receptor defects in insulin action. When the insulin resistant state progresses, elevated levels of insulin are accompanied by increasing levels of glucose. In a previous paper we demonstrated that treatment of isolated adipocytes with high levels of insulin led to a decrease in insulin binding as well as a decrease in basal and insulin-stimulated lipid synthesis. The results of the present study establish that the addition of high concentrations of glucose in combination with a high level of insulin, does not modify the decrease in binding of insulin to its receptor. However, the decrease in lipid synthesis previously observed in the presence of high concentrations of insulin was completely overcome by the presence of high glucose.

Adipocytes↗
PubMed1998

Leptin does not affect adipocyte glucose metabolism: studies in fresh and cultured adipocytes.

Leptin, the 16-kd hormone produced by white fat cells, regulates energy homeostasis, satiety, and multiple sites in the neuroendocrine system. Leptin receptors have been identified in the central nervous system (CNS) and are widespread in peripheral tissues, including fat. Given the association between insulin resistance and obesity, it is important to establish whether leptin has additional effects on peripheral insulin action and glucose metabolism. This study examined whether leptin has a direct autocrine/paracrine action on glucose metabolism in both freshly isolated and 24-hour cultured rat fat cells. Freshly isolated rat adipocytes were incubated for 30 minutes with 200 ng/mL recombinant murine leptin. Thereafter, basal and insulin-stimulated (10(-8) mol/L) glucose transport, glycolysis-Krebs oxidation and lipogenesis ([6-14C]glucose conversion to [14C]O2 and to [14C]triglyceride), and lipolysis were measured. Upon leptin exposure, no statistical differences were detected in glucose transport or metabolism. Increasing the leptin concentration to 1 to 2 microg/mL or prolonging the duration of preincubation with the fat cells to 60 minutes before the metabolic assays did not alter the results. Finally, using two disparate fat cell culture methods with differing substrate additions (pyruvate and high or low glucose concentrations), there was no effect of 24-hour exposure to leptin (200 ng/mL) on basal and insulin-stimulated glucose transport or lipogenesis. We conclude that leptin does not modulate basal or insulin-stimulated glucose metabolism in isolated and cultured fat cells in vitro. However, in vivo, higher pericellular leptin concentrations, as well as other cellular or soluble serum factors, may exist that might lead to a physiologically relevant autocrine action of leptin.

Adipocytes↗