RESUMEN
Few human cell strains are suitable and readily available as in vitro adipocyte models. We used resected lipoma tissue from a patient with germline phosphatase and tensin homolog (PTEN) haploinsufficiency to establish a preadipocyte cell strain termed LipPD1 and aimed to characterize cellular functions and signalling pathway alterations in comparison to the established adipocyte model Simpson-Golabi-Behmel-Syndrome (SGBS) and to primary stromal-vascular fraction cells. We found that both cellular life span and the capacity for adipocyte differentiation as well as adipocyte-specific functions were preserved in LipPD1 and comparable to SGBS adipocytes. Basal and growth factor-stimulated activation of the PI3 K/AKT signalling pathway was increased in LipPD1 preadipocytes, corresponding to reduced PTEN levels in comparison to SGBS cells. Altogether, LipPD1 cells are a novel primary cell model with a defined genetic lesion suitable for the study of adipocyte biology.
Asunto(s)
Adipocitos/metabolismo , Haploinsuficiencia , Fosfohidrolasa PTEN/genética , Adipocitos/citología , Adipogénesis/genética , Tejido Adiposo Blanco/metabolismo , Biomarcadores , Diferenciación Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Metabolismo de los Lípidos , Lipoma/etiología , Lipoma/metabolismo , Lipoma/patología , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Adiponectin is an adipocytokine with profound anti-diabetic and anti-atherogenic effects. Even though adiponectin expression is restricted to adipocytes, serum levels are paradoxically decreased in obesity. We characterized how adiponectin expression and regulation relates to adipocyte differentiation in a human adipocyte cell culture model. Adiponectin was not expressed by human preadipocytes. Differentiation into adipocytes was necessary to induce an increasing expression of adiponectin (359 +/- 64-fold, P < 0.001) in parallel to an increasing expression of adipocyte differentiation markers. Adiponectin protein synthesis and secretion occurred specifically in mature adipocytes and may thus serve as a distinctive marker of adipocyte differentiation. Addition of serum during the course of differentiation as well as acutely to mature adipocytes significantly and concentration-dependently suppressed adiponectin to almost non-detectable levels (to 9.8 +/- 0.03%, P = 0.0043), suggesting a strong humoral serum component of adiponectin down-regulation. This serum component is present in both obese and lean individuals with a tendency to a stronger effect in obese men and women. Separation by molecular size suggests that higher molecular weight (>30 kDa) fractions exert inhibition of adiponectin. Withdrawal of adipogenic ingredients from the culture medium also resulted in a decrease of adiponectin expression and secretion to 62.01 +/- 0.09% and 70.86 +/- 0.05%, respectively. We identified insulin as a critical component to maintain adiponectin expression with a down-regulation to 61.6 +/- 0.1% (P = 0.0011) in the absence of insulin. These dynamic changes of adiponectin expression and regulation with adipocyte differentiation are of physiological interest in the light of the paradoxical decrease of adiponectin levels and the continuous recruitment of preadipocytes for differentiation in obesity.
Asunto(s)
Adipocitos/metabolismo , Adiponectina/metabolismo , Diferenciación Celular/fisiología , Suero/química , Adipocitos/ultraestructura , Secuencia de Bases , Células Cultivadas , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Femenino , Expresión Génica , Humanos , Insulina/metabolismo , Masculino , Peso Molecular , Obesidad/metabolismo , Obesidad/patología , Suero/metabolismoRESUMEN
The ob gene product, leptin, is synthesized by adipocytes. In rodents, its main role is to regulate energy expenditure and food intake. The growth hormone (GH)-insulin-like growth factor (IGF) system is also ubiquitously expressed, is also involved in energy homeostasis and shares some of the signaling molecules of leptin and its receptors. Therefore, we have asked whether or not leptin interacts with the GH-IGF system in an in vitro model. SK-N-MC cells were chosen for further investigation since they express IGF-I, IGF-I receptor and IGFBP-2 mRNA and secrete IGF-I and IGFBP-2 protein. Specific leptin receptor mRNA, both short and long isoform transcripts, were identified in SK-N-MC cells by RT-PCR. Secondly and most importantly, when SK-N-MC cells were cultured in the presence of leptin, neither IGF-I, nor IGF-I receptor or IGFBP-2 mRNA expression was different than in the absence of leptin. In addition, an increase in leptin did not alter secretion of immunoreactive IGF-I or IGFBP-2 protein into cell culture medium. In conclusion, (1) human SK-N-MC neuroepithelioma cells express ob and leptin receptor mRNA and secrete leptin. (2) Added exogenous leptin does not affect IGF-I, IGFBP-2 or IGF-I receptor mRNA expression and IGF-I and IGFBP-2 secretion by SK-N-MC cells in vitro under the conditions studied. We hypothesize that leptin and the IGF system do not interact directly in a cell culture model of neuroepithelioma cells.
Asunto(s)
Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Leptina/metabolismo , Células Neuroepiteliales/citología , Receptor IGF Tipo 1/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Hormona del Crecimiento/metabolismo , Humanos , Ligandos , Hibridación de Ácido Nucleico , Isoformas de Proteínas , ARN/metabolismo , ARN Mensajero/metabolismo , Radioinmunoensayo , Ratas , Receptores de Superficie Celular/metabolismo , Receptores de Leptina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: We have developed a rapid, sensitive and quantitative in vitro assay for leptin based upon its ability to bind to the soluble extracellular domain of the leptin receptor (sOB-R). Such an assay is theoretically capable of differentiating between physiologically active leptin molecules from those with modified, either enhanced or reduced, binding activity. METHODS: A preparation of sOB-R was immobilized to capture leptin from serum samples or standards. Anti-leptin antibodies that had been raised in rabbits were added in a second incubation step to identify leptin molecules bound to sOB-R. Signal detection was performed in a third incubation step by anti-rabbit IgG labeled with peroxidase. The immunofunctional assay (IFA) was clinically validated by the comparison of leptin levels in adolescents (n = 41, age range 9-18 years, BMI range 13.4-33.8 kg/m(2) and adults (n = 80, age range 18-77 years, BMI range 16.4-54.7 kg/m(2) measured using the IFA with data of an in-house RIA performed with the same standards and leptin antibodies. RESULTS: The functional sensitivity of the IFA was 0.4 ng/ml and comparable to the data of the RIA. Intra- and interassay coefficients of variation were below 12.5 % in both methods. Leptin levels correlated well with the BMI of the subjects studied (r = 0.70 for RIA, r = 0.72 for IFA; p < 0.0001) as well as between IFA (y) and RIA (x) (y = x -1.31 ng/ml; r = 0.97, p < 0.0001). The median of the quotient between IFA and RIA levels was 0.86 (quartile range 0.60-1.10) for all samples. CONCLUSIONS: So far, only at the most minor differences between leptin measurements using the newly developed IFA and those using a conventional RIA have been detected. Additional studies using the IFA method are required to investigate whether or not discrepant results with the IFA will be seen in various states of relative leptin resistance and whether or not such differences are of biological relevance.