Tarik Issad

The use of resonance energy transfer in high-throughput screening: BRET versus FRET

Bioluminescence resonance energy transfer has developed in recent years as a new technique to study protein-protein interactions. Protein partners of interest are tagged with either luciferase or green fluorescent protein (GFP). Non-radiative energy transfer between the excited luciferase and the GFP permits the study of spatial relationships between the two partners. This technique constitutes an important tool for the study of the functional activity of different types of receptors, and can be used in sensitive, homogenous high-throughput screening assays.

Effect of dexamethasone on adipocyte differentiation markers and tumour necrosis factor-α expression in human PAZ6 cells

Aims/hypothesis. Adipose tissue-derived tumour necrosis factor-α (TNF-α) has been implicated in the insulin resistance observed in animal models of obesity. Moreover, TNF-α has inhibitory effects on adipocyte differentiation. Glucocorticoids play important roles in the regulation of insulin sensitivity and adipose tissue distribution. We therefore studied the effect of dexamethasone on TNF-α expression and adipocyte differentiation in human PAZ6 cells. Methods. The expression of TNF-α and adipocyte differentiation markers was assessed by reverse-transcription polymerase chain reaction in PAZ6 cells. Results. In cells cultured for 15 days in the presence of dexamethasone, adipocyte differentiation marker expression was higher and TNF-α expression was lower than in cells cultured in the absence of dexamethasone. The presence of dexamethasone was necessary during the whole period of differentiation because removal of dexamethasone during the second week resulted in poorly differentiated adipocytes that express higher levels of TNF-α.

The involvement of leptin in humans revealed by mutations in leptin and leptin receptor genes

Support for our work comes mostly from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the University of Paris VII, the Ministry for Research and Technology. We are also grateful for help from the Ligue Nationale contre le Cancer, the Fondation pour la Recherche Medicale Francaise and last but not least, the Association pour la Recherche contre le Cancer.

Desensitization of the β-Adrenergic Response in Human Brown Adipocytes1

Activation of adenylyl cyclase by β-adrenergic receptors (βARs) plays a major role in adipose tissue homeostasis. The increase in cAMP promotes lipolysis in white adipose tissue, activates both thermogenesis and lipolysis in brown adipose tissue (BAT), and induces BAT hypertrophy. Previous studies indicated that among the three βAR subtypes present in adipose tissue, β3AR could be a potential target for antiobesity treatments in humans. We studied immortalized human brown adipocytes (PAZ6 adipocytes) as a model ofβ -adrenergic response in human BAT. PAZ6 adipocytes and freshly isolated mature human brown adipocytes display the same proportions ofβ AR subtypes, with β3AR being the most abundant (∼80% of the total). However, β3AR was poorly coupled to the adenylyl cyclase pathway in PAZ6 cells, contributing to only 10% of the isoproterenol-induced accumulation of cAMP, whereas 20% and 70% of the signal depended on β1- andβ 2-subtypes, respectively. Upon isoproterenol stimulation, β1- and β2AR down-regulated...

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. [Diabetologia (1999) 42: 527–533]

Bioluminescence resonance energy transfer to monitor protein-protein interactions.

The bioluminescence resonance energy transfer (BRET) methodology allows for the study of protein-protein interactions as well as conformational changes within proteins or molecular complexes. BRET is a highly versatile technique that can be applied to in vitro studies using purified proteins, crude cell membranes, cell fractions obtained by centrifugation on a density gradient, as well as permeabilized cells. Importantly, BRET also allows for monitoring of protein-protein interactions, in real time, in intact living cells that can be submitted to various stimuli. Moreover, quantitative BRET analysis also permits a pharmacological approach of protein-protein interactions, allowing one to determine whether a given stimulus induces a conformational change within preassociated partners or increases the association (recruitment) between two separated partners. Determination of the proportion of the dimeric vs monomeric form of a protein in the cell also is possible. Therefore, the BRET technology can be considered as a new and powerful tool in the field of protein-protein interactions.

Looking for an insulin pill? Use the BRET methodology!

Insulin exerts its biological effects through a plasma membrane receptor that possesses a tyrosine-kinase activity. This tyrosine-kinase activity depends on the autophosphorylation of the receptor on tyrosine residues and on its dephosphorylation by protein tyrosine-phosphatases. The discovery of pharmacological agents that specifically stimulate the autophosphorylation of the insulin receptor or inhibit its dephosphorylation will be of great importance for the treatment of insulin resistant or insulin deficient patients. Bioluminescence Resonance Energy Transfer (BRET) has developed in recent years as a new technique to study protein-protein interactions. In the BRET technique, one partner is fused to Renilla luciferase, whereas the other partner is fused to a fluorescent protein (e.g. YFP, Yellow Fluorescent Protein). The luciferase is excited by addition of its substrate, coelenterazine. If the two partners interact, resonance energy transfer occurs between the luciferase and the YFP, and a fluorescent signal, emitted by the YFP, can be detected. Our work indicates that this methodology could be an important tool for the search of molecules that activate insulin receptor autophosphorylation or that inhibit its dephosphorylation. Indeed, we first showed that the activation of the insulin receptor by different ligands can be monitored using a chimeric receptor with one B-subunit fused to Renilla luciferase and the other B-subunit fused to YFP. The conformational changes induced by different ligands could be detected as an energy transfer (BRET signal) between the luciferase and the YFP, that reflects the activation state of the receptor. This methodology allows for rapid analysis of the effects of agonists on insulin receptor activity and may therefore be used in high-throughput screening for the discovery of molecules with insulin-like properties. More recently, we demonstrated that the BRET methodology could also be used to monitor the interaction of the insulin receptor with protein tyrosine-phosphatase 1B, one of the main tyrosine-phosphatase that controls its activity. HEK cells were co-transfected with the insulin receptor fused to Renilla luciferase and a substrate-trapping mutant of PTP1B (PTP1B-D181A) fused to YFP. Insulin-induced BRET signal could be followed in real time for more than 30 min. Therefore, this methodology can also be used in high-throughput screening for the search of molecules that will specifically disrupt the interaction between the insulin receptor and PTP1B.

Rat uncoupling protein 2 (UCP2): Expression in obese ventromedial hypothalamus (VMH)-lesioned animals

BACKGROUND: The family of uncoupling proteins is thought to play an important role in the regulation of energy metabolism by uncoupling the respiratory chain reactions from ATP synthesis. The recently discovered uncoupling protein 2 (UCP2) is upregulated in genetically obese rodent models and during long term high fat feeding.AIM: We have examined the UCP2 mRNA levels in liver, heart and white adipose tissue (WAT) of obese ventromedial hypothalamus (VMH)-lesioned rats, during the dynamic and the early stage of the static phase of obesity, before the appearance of most of the metabolic perturbations associated with long term established obesity.RESULTS: The amount of UCP2 mRNA was not increased in any tissue of VMH-lesioned rats relative to control animals during the dynamic phase nor during the early static phase of obesity.CONCLUSION: These results indicate that in the rat, obesity does not necessarily lead to an increase in UCP2 expression and suggest that the up-regulation of UCP2 described in other models may be secondary to metabolic perturbations, rather than to a direct adaptative response to the increased adipose tissue content of the organism.