RESUMEN
Physical activity improves insulin sensitivity and metabolic control in patients with type 2 diabetes. Moreover, regular exercise can reduce systemic levels of immune markers associated with diabetes development. As patients with physical impairments are not able to exercise sufficiently, the aim of this study was to investigate whether high-frequency external muscle stimulation (hfEMS) improves metabolic and immunologic parameters in patients with type 2 diabetes and might therefore serve as complementary lifestyle therapy. Sixteen patients (12 men/4 women, age 57+/-11 years (mean+/-SD); BMI 34.5+/-5.2 kg/m (2); HbA1c 7.4+/-1.1%) on oral antihyperglycaemic therapy were enrolled in this study. After a run-in phase of 2 weeks, every patient received an hfEMS device (HITOP 191, gbo-Medizintechnik AG, Rimbach/Germany) for daily treatment of femoral musculature for 6 weeks. Thereafter, patients were followed up for additional 4 weeks without hfEMS treatment. At each visit, clinical parameters were assessed and blood samples were drawn for metabolic and immunologic parameters. Immune markers (cytokines, chemokines, adipokines and acute-phase proteins) representative for the different arms of the immune system were analysed. hfEMS treatment resulted in significant reductions of body weight (-1.2 kg [-2.7 kg; -0.5 kg]; p<0.05; median [25th percentile; 75th percentile]), BMI (-0.4 kg/m (2) [-0.8 kg/m (2); -0.1 kg/m (2)]; p<0.05) and HbA1c (-0.4% [-0.9%; -0.1%]; p<0.05) which were sustained during the follow-up period. Systemic levels of IL-18 tended to be increased after hfEMS treatment (171 vs. 149 pg/ml; p=0.06), while all other immune markers remained virtually unchanged. Treatment with hfEMS in this first proof-of-principle study has beneficial effects on body weight and improves glycaemic control in patients with type 2 diabetes, which may be associated with changes in subclinical inflammation. Taken together, hfEMS might represent an additional treatment option for patients with type 2 diabetes not being able to exercise.
Asunto(s)
Glucemia/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Músculo Esquelético/fisiopatología , Desempeño Psicomotor/fisiología , Administración Oral , Adulto , Índice de Masa Corporal , Péptido C/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ejercicio Físico , Femenino , Hemoglobina Glucada/análisis , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/uso terapéutico , Interleucina-18/sangre , Masculino , Persona de Mediana Edad , Actividad Motora , Estimulación Física , Pérdida de PesoRESUMEN
Human adipose tissue is a main contributor to plasma levels of pro-inflammatory cytokine IL-6. How IL-6 expression is regulated in adipocytes remains unclear. In the current study, we investigated the effect of the HMG-CoA reductase inhibitor, cerivastatin, on the production of IL-6 from cultured human adipocytes. Cerivastatin reduced both IL-6 mRNA and secretion in a dose- and time-dependent manner. The inhibitory effect on IL-6 mRNA was prevented by the intermediates of the cholesterol synthesis pathway, mevalonate and geranyl-geranyl-phyrophosphate (GGPP) but not by farnesyl-pyrophosphate. This suggests the involvement of geranylgeranyl-modified intermediates in the effect of cerivastatin on IL-6. Moreover, cerivastatin induced an inactivation of the phosphorylation of the p65 subunit of NFkappaB which was prevented by GGPP. Our data suggest that cerivastatin exerts an anti-inflammatory effect by down-regulating IL-6 levels in adipocytes, which seems to be mediated by reduced production of GGPP and interference with the NFkappaB pathway.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Interleucina-6/antagonistas & inhibidores , Piridinas/farmacología , Adulto , Células Cultivadas , Femenino , Humanos , Interleucina-6/genética , Ácido Mevalónico/farmacología , Persona de Mediana Edad , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/metabolismo , Factor de Transcripción ReIARESUMEN
OBJECTIVE: To study the relation between body mass index (BMI) and age on the one hand and total number of human (pre-) adipocytes and preadipocyte differentiation capacity on the other hand. SUBJECTS: In total, 189 women undergoing surgical mammary reduction, age range 16-73 y, BMI range 19.7-39.7 kg/m(2). MEASUREMENTS: Differentiation of preadipocytes in primary culture was assessed by morphological criteria, and determination of glycerol-3-phosphate dehydrogenase after stimulation of the cells by standardized adipogenic conditions containing isobutyl-methylxanthine, troglitazone or both compounds. The total number of stromal cells (ie preadipocytes) and fat cells per gram of adipose tissue and per body as well as mature fat cell volume were calculated from isolated stromal cells and adipocytes, respectively, and anthropometric measures. RESULTS: BMI correlated positively to age, mature fat cell size and total number of adipocytes and stromal cells per body (r varying from 0.22 to 0.54, each P<0.05). In contrast, BMI correlated negatively to the number of adipocytes and stromal cells per gram of adipose tissue and the capacity of preadipocytes to differentiate (r varying from -0.20 to -0.37, each P<0.05). No significant correlation was observed between BMI and the ratio of stromal cells to adipocytes. The sample was also divided into three groups: BMI <25 kg/m(2) (lean), BMI 25-29.9 kg/m(2) (overweight) and BMI >/=30 kg/m(2) (obese). The overweight group showed a larger fat cell size but no increase in total fat cell or stromal cell number when compared to the lean subjects. The obese subjects showed larger stromal and fat cell numbers when compared to the lean subjects. Age did not independently correlate to the number of stromal cells or adipocytes per gram of adipose tissue or total body, nor with the capacity of preadipocytes to undergo differentiation and the ratio of stromal cells to adipocytes. CONCLUSION: There seems to be a constant ratio between the number of adipose tissue stromal cells and adipocytes independently of BMI and age in humans. During adipose tissue expansion, there seems to be both a continuous increase in fat cell size, and in stromal cell and adipocyte number, but the increase in fat cell size apparently precedes the increase in fat cell number. The differentiation capacity of the stromal cells appears to decrease with increasing BMI.
Asunto(s)
Adipocitos/citología , Tejido Adiposo/citología , Tiazolidinedionas , 1-Metil-3-Isobutilxantina/farmacología , Adipocitos/enzimología , Tejido Adiposo/enzimología , Adolescente , Adulto , Anciano , Índice de Masa Corporal , Diferenciación Celular , Células Cultivadas , Cromanos/farmacología , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Humanos , Persona de Mediana Edad , Células del Estroma/citología , Tiazoles/farmacología , TroglitazonaRESUMEN
The recently discovered C825T polymorphism of the G-protein beta 3 subunit has been reported to be associated with the development of hypertension and obesity. The aim of our study was to investigate the relationship between the C825T polymorphism and functional aspects of human adipose cells, particularly with regard to adipose differentiation and lipolysis. Adipose tissue samples were collected from 65 women with a BMI ranging from 19.7 to 39.7 kg/m 2 undergoing surgical mammary reduction. The stromal cells were allowed to undergo differentiation in primary culture using adipogenic media of defined composition. No significant difference was observed between the CC carriers and the carriers of the T allele under all adipogenic conditions with differentiation capacity related to the genotype. In a subgroup of patients (n = 20), lipolysis in isolated fat cells was determined by measurement of glycerol in the culture medium upon catecholamine exposure. Glycerol release after 10(-7) mmol/l isoproterenol was significantly higher in fat cells from the 10 CC carriers than in adipocytes from the T allele carriers when expressed as percentage of basal glycerol release (increase above baseline: CC: 809 +/- 174 %, T allele carriers: 247 +/- 88 %, p = 0.01), while basal glycerol concentrations were no different according to genotype after controlling for either age or BMI. In conclusion, this study provides the first evidence that the GNB3 825T allele is associated with an impairment of the beta-adrenergic control of lipolysis.
Asunto(s)
Adipocitos/metabolismo , Proteínas de Unión al GTP Heterotriméricas/genética , Lipólisis/genética , Mutación Puntual , Adipocitos/citología , Adolescente , Agonistas Adrenérgicos beta/farmacología , Adulto , Anciano , Alelos , Índice de Masa Corporal , Catecolaminas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Genotipo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Isoproterenol/farmacología , Lipólisis/efectos de los fármacos , Persona de Mediana Edad , Obesidad/genética , Células Madre/citología , Células Madre/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
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.
Asunto(s)
Adipocitos/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Gliceraldehído 3-Fosfato/metabolismo , Leptina/farmacología , Obesidad/metabolismo , Adipocitos/metabolismo , Adipocitos/fisiología , Índice de Masa Corporal , División Celular , Células Cultivadas , Colagenasas/metabolismo , Femenino , Glucosa/metabolismo , Humanos , Lipólisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Angiotensin (Ang) II is the active component of the renin-angiotensin-system (RAS), but its degradation products have also been shown to exhibit biological activity. This system, which mainly controls blood pressure and electrolyte homeostasis, was recently found to be completely expressed in human adipose tissue. The major determinant in the fibrinolytic system is the plasminogen activator inhibitor-1 (PAI-1). Both PAI-1 and components of the RAS are over-expressed in the obese state. We have recently shown that Ang II is able to induce PAI-1 expression and release via the AT1-receptor in human fat cells in primary culture, and have provided the first evidence that two metabolites, Ang III and Ang IV, may have a similar stimulatory effect on PAI-1 release. We have now performed additional experiments to further characterize the role of the angiotensin peptides in the production of PAI-1. Ang III and Ang IV showed a time- and dose-dependent stimulation of PAI-1 protein release. Concomitantly, mRNA-levels were markedly elevated. Using specific receptor blockers, all angiotensin peptides seem to induce PAI-1 expression via the angiotensin receptor subtype 1. However, components of the renin-angiotensin-system seem to play an important role in the control of fibrinolysis in adipose tissue. We conclude that PAI-1 production by adipose tissue may contribute to the elevated thromboembolic risk in obesity.
Asunto(s)
Adipocitos/fisiología , Angiotensina III/farmacología , Angiotensina II/análogos & derivados , Angiotensina II/farmacología , Inhibidor 1 de Activador Plasminogénico/genética , Adipocitos/citología , Adulto , Células Cultivadas , Femenino , Fibrinólisis/fisiología , Expresión Génica/efectos de los fármacos , Humanos , Hipertensión/fisiopatología , Persona de Mediana Edad , Obesidad/fisiopatología , Sistema Renina-Angiotensina/fisiologíaRESUMEN
Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic of the obese state and may contribute to the association between obesity and cardiovascular disease. In this study, we measured the rate of secretion of PAI-1 antigen in isolated subcutaneous and omental abdominal adipocytes from severely obese and non-obese individuals and studied the effect of selected cytokines on PAI-1 release using a suspension culture technique. PAI-1 secretion was approximately 2-fold greater in isolated fat cells from the obese versus non-obese subjects. In addition, PAI-1 mRNA levels were higher in adipose tissue samples from obese versus non-obese individuals (P < .05). PAI-1 release was also approximately 2-fold greater in omental versus subcutaneous adipocytes from both obese and non-obese subjects (each P < .05). A 24-hour exposure to 1 nmol/L tumor necrosis factor alpha (TNF-alpha) slightly increased PAI-1 release from both subcutaneous and omental adipocytes (30% +/- 21% and 17% +/- 18%, respectively, nonsignificant [NS]). Transforming growth factor beta 1 (TGF-beta1) induced a significant dose-dependent increase of PAI-1 release into the medium. Exposure to 400 pmol/L TGF-beta1 of subcutaneous and omental fat cells from both obese and non-obese individuals elevated PAI-1 secretion by 2-fold. These data provide evidence that human fat cells release a substantial amount of PAI-1 in a depot-specific manner and that TGF-beta1 particularly contributes to the regulation of PAI-1 secretion.
Asunto(s)
Adipocitos/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adipocitos/metabolismo , Adulto , Anciano , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana Edad , Epiplón/citología , Epiplón/efectos de los fármacos , Epiplón/metabolismo , Piel/citología , Piel/efectos de los fármacos , Piel/metabolismo , SuspensionesRESUMEN
Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are characteristic for obesity and are associated with increased risk of thromboembolic complications. PAI-1 recently was reported to be expressed and secreted by human adipocytes, but little is known about regulation of PAI-1 in human adipose tissue. Therefore, we examined the effects of selected cytokines present in adipose tissue on expression and secretion of PAI-1 in in vitro, differentiated subcutaneous human adipocytes in primary culture. Transforming growth factor-beta1 (TGF-beta1) increased PAI-1 secretion in a dose- and time-dependent manner. PAI-1 protein increased by 3.2-fold and PAI-1 mRNA by 1.9-fold after a 6-hour exposure to 400 pmol/L TGF-beta1. This effect is probably mediated by TGF-beta1 type 2 and 3 receptors, which were found to be expressed in cultured human adipocytes. Moreover, TNF-alpha and interkeukin-1beta (IL-1beta) also exerted a stimulatory effect on PAI-1 release and increased PAI-1 mRNA levels. As assessed by a semiquantitative reverse transcription-polymerase chain reaction technique, TGF-beta1 mRNA is expressed by differentiation of human preadipocytes and is moderately upregulated by TNF-alpha and IL-1beta. In conclusion, our results clearly indicate that TGF-beta1 is a potent inducer of PAI-1 production in subcutaneous human adipocytes. In addition, data suggest that TNF-alpha and IL-1beta also have stimulatory effects on PAI-1 protein secretion and may contribute to the elevated PAI-1 levels observed in obesity.
Asunto(s)
Adipocitos/metabolismo , Citocinas/fisiología , Regulación de la Expresión Génica , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Diferenciación Celular , Medios de Cultivo Condicionados , Citocinas/farmacología , Humanos , Interleucina-1/administración & dosificación , Interleucina-1/farmacología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
AIMS/HYPOTHESIS: Increased plasma plasminogen activator inhibitor-1 (PAI-1) concentrations are characteristic for subjects with insulin resistance and could contribute to the increased cardiovascular risk in this state. In this study, we investigated the effect of troglitazone, a ligand of the nuclear receptor peroxisome proliferator activated receptor-gamma, on PAI-1 expression and secretion in human adipocytes. METHODS: We used two models: in vitro differentiated subcutaneous and omental adipocytes cultured under serum-free conditions and isolated subcutaneous and omental fat cells kept in suspension culture. Plasminogen activator inhibitor-1 protein was measured by ELISA, PAI-1 mRNA by a semiquantitative RT-PCR technique. RESULTS: Exposure of in vitro differentiated subcutaneous adipocytes from young normal-weight females to 1 microgram/ml troglitazone for 72 h caused a reduction of both PAI-1 secretion (by 29 +/- 5%; p < 0.01) and PAI-1 mRNA expression (by 26 +/- 3%; p < 0.05). In cultures from severely obese subjects, troglitazone induced a decrease of PAI-1 antigen secretion from newly differentiated omental adipocytes by 49 +/- 8% (p < 0.01) and from subcutaneous adipocytes by 30 +/- 7% (p < 0.05). Exposure of freshly isolated subcutaneous and omental adipocytes in suspension culture to troglitazone induced a similar reduction of PAI-1 concentration in the culture medium (by 35 +/- 11%, p < 0.05, and 33 +/- 8%, p < 0.05 compared with control, respectively). CONCLUSION/INTERPRETATION: This study provides evidence that troglitazone reduces PAI-1 production in human adipocytes, probably at the transcriptional level. This observation could point to a new beneficial effect of troglitazone, particularly in obese subjects, which could be associated with a reduced cardiovascular risk.
Asunto(s)
Adipocitos/metabolismo , Cromanos/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Tiazoles/farmacología , Tiazolidinedionas , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adulto , Diferenciación Celular , Células Cultivadas , Medios de Cultivo/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Obesidad/metabolismo , Obesidad/patología , Epiplón , Concentración Osmolar , Inhibidor 1 de Activador Plasminogénico/genética , ARN Mensajero/metabolismo , Valores de Referencia , Piel , TroglitazonaRESUMEN
We wished to determine whether the capacity of the sugar uptake mechanisms of guard cells of the Argenteum mutant of pea (Pisum sativum L.) sufficed to support a concurrent stomatal opening movement. Sugar uptake by guard cell protoplasts was determined by silicone-oil-filtering centrifugation. The protoplasts took up [(14)C]glucose, [(14)C]fructose, and [(14)C]sucrose (Suc), apparently in symport with protons. Mannose, galactose, and fructose competed with Glc for transport by a presumed hexose carrier. The uptake of Glc saturated with a K(m) of 0.12 mM and a V(max) of 19 fmol cell(-1) h(-1). At external concentrations <1 mM, the uptake of Suc was slower than that of Glc. It exhibited a saturating component with a K(m) varying between 0.25 and 0.8 mM and a V(max) between 1 and 10 fmol cell(-1) h(-1), and at external concentrations >1 mM, a non-saturating component. At apoplastic sugar concentrations below 4 mM, sugar import was estimated to be mainly in the form of hexoses and too slow to support a simultaneous stomatal opening movement. If, however, during times of high photosynthesis and transpiration, the apoplastic Suc concentration rose and entered the range of non-saturating import, absorbed Suc could replace potassium malate as the osmoticum for the maintenance of stomatal opening.
RESUMEN
Tumor necrosis factor-alpha (TNF) is known to inhibit fat cell development in vitro and to be expressed in adipose tissue suggesting that it may act as an auto-/paracrine regulator of adipose tissue mass in vivo. We demonstrate here that endogenous TNF-mRNA expression of cultured human preadipocytes and adipocytes is suppressed by the unspecific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which is frequently used to trigger the differentiation process. As assessed by the measurement of glycerophosphate dehydrogenase, IBMX stimulated the differentiation of human preadipocytes in a dose dependent manner up to threefold but remained ineffective when cells were simultaneously treated with 1 nM TNF. These results suggest that the adipogenic effect of IBMX is mediated by suppression of endogenous TNF production.
Asunto(s)
1-Metil-3-Isobutilxantina/farmacología , Adipocitos/metabolismo , Inhibidores de Fosfodiesterasa/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adipocitos/efectos de los fármacos , Adulto , Diferenciación Celular , Células Cultivadas , Femenino , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Persona de Mediana Edad , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: In differentiating human preadipocytes glucose uptake in the presence of insulin is a prerequisite for lipid accumulation. The aim of this study was to characterize the insulin-regulated glucose transport system during and after differentiation. DESIGN AND METHODS: Human adipocyte precursor cells kept in primary culture were allowed to differentiate into fat cells under serum-free hormone-supplemented conditions. 2-Deoxy-glucose uptake was measured as a functional parameter of the glucose transport system, the amount of GLUT1 and GLUT4 protein was determined by Western blotting. RESULTS: In the undifferentiated state, cells did not increase 2-deoxy-glucose uptake in response to insulin. On day 16, when cells have acquired the adipocyte phenotype, there was a 3-4-fold stimulation of glucose transport by insulin compared to basal rates, whereas basal glucose uptake was dramatically diminished. Measurement of GLUT4 protein in cell extracts, showed a marked increase in the amount of this insulin-regulated transporter isoform during the differentiation period. On average, the amount of GLUT4 was 16.7-fold greater after than before differentiation. In contrast, the amount of GLUT1 protein decreased during differentiation to almost undetectable levels on day 16. When newly developed adipocytes were maintained in culture for another 14d, the stimulation of glucose uptake and the amount of GLUT4 remained stable. CONCLUSION: Differentiating human fat cells in primary culture develops an insulin-responsive glucose transport system which exhibits a high stability, thereby providing a valuable model for long-term studies of glucose transport and GLUT4 expression in human adipocytes.
Asunto(s)
Adipocitos/metabolismo , Diferenciación Celular , Glucosa/metabolismo , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Células Madre/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adulto , Transporte Biológico , Células Cultivadas , Femenino , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Humanos , Células Madre/citología , Factores de TiempoRESUMEN
Tumor necrosis factor (TNF)-alpha is postulated to play a major role in the pathogenesis of obesity-linked insulin resistance, probably resulting from an interaction with insulin signaling pathways. This cross talk has now been investigated in human adipocytes at the level of phosphatidylinositol (PI) 3-kinase, and the TNF receptors (TNFRs) mediating these processes have been identified. Equilibrium binding studies using human adipocytes from mammary tissue indicated the presence of two populations of TNFR with apparent affinity constants of 13 pmol/l and 1.6 nmol/l, respectively. Interaction of TNF-alpha with insulin signaling was determined by quantification of insulin receptor substrate (IRS)-1-associated PI 3-kinase activity. Under control conditions, PI 3-kinase was activated about 10-fold in response to insulin (10[-7] mol/l, 5 min). Preincubation of adipocytes with 5 nmol/l TNF-alpha for 15 min resulted in a 60-70% reduction of insulin action, reaching a stable inhibition (40%) after longer incubation with the cytokine. The inhibitory action of TNF-alpha was dose-dependent, already detectable at 10 pmol/l, and was correlated to inhibition of tyrosine phosphorylation of IRS-1 with an unaltered autophosphorylation of the insulin receptor beta-subunit. The modulation of insulin signaling by TNF-alpha was found to be paralleled by a comparable inhibition of insulin-stimulated glucose transport. An agonistic TNFR1 antibody completely mimicked the inhibitory action of TNF-alpha on insulin signaling, whereas at 100 pmol/l TNF-alpha, a nonagonistic p80 TNFR antibody, was shown to ameliorate the inhibitory action of the cytokine. These findings indicate that in human adipocytes, low concentrations of TNF-alpha induce a rapid inhibition of insulin signaling at the level of PI 3-kinase. We suggest that under these conditions, the p80 TNFR is essential for initiating the intracellular cross talk that involves signaling by the p60 TNFR.
Asunto(s)
Adipocitos/metabolismo , Antígenos CD/metabolismo , Insulina/fisiología , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Adolescente , Adulto , Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Femenino , Glucosa/metabolismo , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Tumour necrosis factor alpha (TNF alpha) has been found to cause a delipidation of fat cells and a decrease of the adipose tissue mass. In the present study, we tried to elucidate some of the mechanisms responsible for this phenomenon by investigating the action of TNF alpha on specific pathways which are involved in lipid storage. Cultured stromal cells from human adipose tissue were induced to differentiate into adipose cells by exposure to adipogenic factors and subsequently used for studying the effects of TNF alpha on fat cell metabolism. Presence of 5 nmol/l TNF alpha for 24 h resulted in a complete loss of the stimulatory effect of insulin on 2-deoxy-glucose transport. This inhibitory action was paralleled by a decrease of GLUT4 protein and mRNA levels. The amount of cellular GLUT4 protein was reduced by 49 +/- 3% after a 24-h exposure and by 82 +/- 18% after a 72-h exposure to 5 nmol/l TNF alpha. GLUT4 mRNA was almost undetectable after a 24-h incubation with 5 nmol/l TNF alpha. In a similar time-dependent manner, TNF alpha dramatically reduced the lipoprotein lipase mRNA content of the cells. Furthermore, incubation of cultured human fat cells with TNF alpha resulted in a marked dose-dependent stimulation of lipolysis, assessed by glycerol release, by up to 400% above controls, which became apparent after a 6-h exposure at the earliest.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/metabolismo , Desoxiglucosa/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Factor de Necrosis Tumoral alfa/farmacología , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Adolescente , Adulto , Transporte Biológico/efectos de los fármacos , Diferenciación Celular , Fraccionamiento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Humanos , Lipoproteína Lipasa/biosíntesis , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas Recombinantes/farmacologíaRESUMEN
We investigated the effects of epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) on the differentiation of human adipocyte precursor cells and some metabolic aspects of newly formed fat cells kept in primary culture. Exposure of stromal cells from human adipose tissue to EGF (0.01-100 ng mL-1) resulted in a dose- and time-dependent decrease in the number of developing fat cells and the activity of glycerol-3-phosphate dehydrogenase (GPDH), a marker of adipose differentiation. Continuous presence of EGF completely blocked lipid accumulation with a ED50 in the range of 0.2 ng mL-1. This inhibitory action of EGF was associated with a potent stimulation of cell proliferation, up to 8-fold compared with cultures in the absence of EGF. PDGF (0.1-50 ng mL-1) and FGF (0.1-100 ng mL-1) provoked a less marked suppression of GPDH activities which was significant at concentrations of 10 ng mL-1 and higher. A 12 day exposure to EGF of differentiated cells was followed by a suppression of GPDH and, again, a significant increase in cell number. Concomitantly, a distinct loss of cellular lipids was observed in the newly formed adipocytes. This effect could be partly explained by a stimulation of lipolysis, since EGF caused an increase of glycerol in the culture medium. Addition of PDGF or FGF to newly developed fat cells had no effect on lipolysis but, at higher concentrations, also decreased GPDH activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Adipocitos/citología , Factor de Crecimiento Epidérmico/farmacología , Factores de Crecimiento de Fibroblastos/farmacología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Adipocitos/enzimología , Adulto , Biomarcadores/análisis , División Celular/efectos de los fármacos , Células Cultivadas , Femenino , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Lipólisis/fisiología , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
In order to improve our understanding of the control of adipose tissue development in man by adipogenic and antiadipogenic factors we studied the effect of transforming growth factor beta (TGF beta) on the differentiation of human adipocyte precursor cells cultured in a serum-free medium. Addition of TGF beta at concentrations ranging from 5 to 100 pmol/l for 24 h at the beginning of the differentiation process resulted in a dose-dependent reduced expression of glycero-3-phosphate dehydrogenase (GPDH) activity. Continuous exposure of cells to TGF beta completely blocked differentiation even at a concentration of 5 pmol/l. Under these conditions cells were not able to accumulate lipid droplets. This inhibitory effect of TGF beta was not mediated by stimulation of cell proliferation. Exposure of newly developed fat cells to TGF beta was also associated with a significant suppression of GPDH activity. A 10-day incubation of cells with TGF beta reduced GPDH activity by more than 80% compared to controls. Despite this suppressive effect cells retained the typical fat cell-like morphology. In conclusion, these data clearly suggest that TGF beta exerts an inhibitory action on human adipose tissue development and reduces the activity of a lipogenic key enzyme in newly formed fat cells.
Asunto(s)
Adipocitos/citología , Diferenciación Celular , Células Madre/citología , Factor de Crecimiento Transformador beta/farmacología , Adolescente , Adulto , Mama , División Celular , Células Cultivadas , Medios de Cultivo , Femenino , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Insulina/farmacología , Triyodotironina/farmacologíaRESUMEN
Cytochemical stains and morphologic characteristics were essential in making the correct diagnosis in a dog with acute myelomonocytic leukemia, which initially was diagnosed as lymphoblastic leukemia. Response to a treatment regimen of cytosine arabinoside, thioguanine, vincristine, and doxorubicin, besides accomplishing complete remission, helped revise the initial diagnosis to acute myelomonocytic leukemia. After remission was achieved, the dog died of a fulminating viral infection.