Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

Abstract

OBJECTIVE: Human adipocytes can be obtained in vitro by differentiation of human preadipocytes or mesenchymal stem cells (hMSC). Although functionally similar to freshly isolated cells, no detailed comparison of the different cell types has been performed. The antilipolytic a2A-adrenoceptor (AR) and the cAMP-degrading enzyme Phosphodiesterase-3B (PDE3B) have been implicated in the fine-tuning of lipolysis but little is known regarding their role in human adipocytes nor whether their expression and/or function differs in fat cells from different precursors. METHODS:The effects of a2A-AR and PDE3B inhibition in mature adipocytes was determined and compared to that in differentiated preadipocytes and hMSC-derived fat cells. Gene expression was determined by real-time PCR and protein expression by Western blot. RESULTS: Noradrenaline (NA) stimulated lipolysis in preadipocytes and mature adipocytes but markedly reduced lipolysis in differentiated hMSC derived-adipocytes. This was due to a potent stimulation of a2A-AR since co-incubation with NA and the a2-AR-inhibitor yohimbine restored NA-induced lipolysis. The order of Yohimbine response was hMSC4preadipocytes4mature adipocytes. Although a2-AR mRNA expression was highest in mature adipocytes there was no difference in a2A-AR protein levels between the cell types. In contrast, Ga i 2 mRNA and protein expression was significantly higher in MSC-derived adipocytes, suggesting that differences in the response to a2A-AR inhibition reside at the postreceptor level. Incubation with the cAMPanalog 8-bromo(8b) cAMP increased lipolysis in hMSC-derived fat cells while co-incubation with the PDE3-specific inhibitor OPC3911 did not alter the lipolytic effect. In contrast, OPC3911 increased 8bcAMP-induced lipolysis significantly in preadipocytes and mature adipocytes. The response to PDE3B inhibition was; mature adipocytes4preadipocytes4hMSC a finding that correlated significantly with both PDE3B mRNA expression and enzymatic activity. CONCLUSION: Although differentiated adipocytes of different origins display similar functional characteristics there are important differences in the regulation of lipolysis with a marked a2A-AR and less pronounced PDE3B effect in fat cells from MSCs.

Liver X Receptor (LXR) Regulates Human Adipocyte Lipolysis

“…This cell system has been shown to display a similar lipolysis regulation to human adipocytes in vivo (25). 3T3-L1 cells were cultured and differentiated to adipocytes as previously described (26).…”

“…qRT-PCR was performed using an iCycler IQ TM (Bio-Rad) with Taqman probes (Assays-on-Demand, Applied Biosystems, Foster City, CA) for all genes except HSL and PLIN1, for which Sybrgreen detection method was used. PCR conditions and primers have been described previously (25,31). The mRNA levels of the different genes were determined by the 2 Ϫ⌬⌬C T comparative method (32).…”

MicroRNA profiling links miR-378 to enhanced adipocyte lipolysis in human cancer cachexia

“…RT-qPCR was performed using commercial Taqman probes or SYBR Green-based technology. PCR conditions and primers for detection of LIPE transcript have been described previously (13,28). The 18S and low-density lipoprotein receptorrelated protein 10 were used as reference genes with similar results.…”

Mesenchymal Stromal Cells; Role in Tissue Repair, Drug Discovery and Immune Modulation

“…Nevertheless such approaches have found application in osteoarthritis research, where modulators of early chondrogenic differentiation may be measured by the expression of SOX-9 in MSC pellet cultures [92]. Although not strictly drug discovery, MSC differentiation along the adipogenic pathway [93][94] might also be used to probe environmental features of pollutants, environmental screening or bio distribution/accumulation in fatty tissue in ADME studies [95]. MSC derived adipocytes can also be used to screen lipolytic agents, or to assess modifiers of lipid accumulation, supporting discovery of novel therapeutic interventions in obesity and diabetes [95].…”

“…Although not strictly drug discovery, MSC differentiation along the adipogenic pathway [93][94] might also be used to probe environmental features of pollutants, environmental screening or bio distribution/accumulation in fatty tissue in ADME studies [95]. MSC derived adipocytes can also be used to screen lipolytic agents, or to assess modifiers of lipid accumulation, supporting discovery of novel therapeutic interventions in obesity and diabetes [95]. The selection of readouts for immunological indicators will also be very dependent on the disease indication of interest.…”