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Digital archive of theses discussed at the University of Pisa


Thesis etd-04052010-122803

Thesis type
Tesi di dottorato di ricerca
Thesis title
“ Obesity serum factors affect human platelets SERT: we need a cellular model to investigate “
Academic discipline
Course of study
tutor Prof. Giannaccini, Gino
  • Meg-01
  • obesity
  • SERT
Graduation session start date
Amongst molecules active in modulating 5-HT transmission, SERT exerts a main function by promoting 5-HT clearance and re-uptake into the pre-synaptic terminal, therefore controlling either the duration and extent of the transmitter action at specific targets after release or its “reserve” inside pre-synaptic vesicles. For this reason, SERT expression and function is under the control of several intracellular phosphorylation systems (Ramamoorthy et al. 2007). More recently, a relationship between obesity and inflammatory-immune response has been suggested (Matarese and La Cava, 2004), indicating a cross-talk between CNS and periphery in the control of body weight. In a preliminary attempt to elucidate these networks, the present study was thus designed to investigate the influence of 5-HT on body weight by measuring the equilibrium binding parameters (maximal binding capacity, Bmax and dissociation constant, Kd) of the high affinity SERT ligand [3H]-paroxetine in platelets from 174 subjects recruited on the basis of their body mass index (BMI), starting from 18 to > 40 kg/m2. The high affinity ligand [3H]-paroxetine was used as a selective tracer of SERT and subjects recruited to obtain five groups as regards to BMI: 1. normal weight (18-25 kg/ m2), 2. overweight (26-30 kg/ m2), 3. obese, I grade, (31-35 kg/ m2), 4. obese, II grade, (36-40 kg/ m2); 5. obese, III grade, (>40 kg/ m2). All subjects were also accurately monitored for clinical-biochemical parameters, including insulinemia and leptinemia blood glucose, insulin, leptin cytokines and other clinical-chemical parameters. We have chosen to study platelets as these non nucleate cells display an identical SERT to the brain one as well as several 5-HT receptor subtypes. For such reasons, platelets are considered “indicators” of 5-HT-regulated CNS-periphery connections and have been extensively studied in psychiatric-affective disorders since many years (Mellerup et al, 1983; Martini C. et al., 2004). Finally, platelets respond to agonist-induced activation by rapid phosphorilation and represent, therefore, a good model to study neuronal signal transduction events in periphery (Aharanovitz and Granot, 1996 ).
Our study is the first that showed a significant reduced SERT density in platelets of obese subjects (p<0.05), providing new insights in the role of 5-HT system and obesity.
Moreover, no BMI-dependent changes in [3H]-paroxetine Kd (nM) values, but a significant inverse correlation (p<0,0001) between [3H]-paroxetine Bmax values (fmol/mg protein) and BMI.
Consequently, a negative significant correlation was observed between [3H]-paroxetine Bmax values and: blood leptin, TNF-α, insulin, glicemia, PAS, aptoblobin and triglycerides.
In conclusion, these findings show that platelet SERT number is reduced in obese subjects and such a decrease could be linked, in part, to the efficiency of the insulin/adipokine-related downstream signaling cascade in periphery. These results could explain that adipose tissue entity are able to modulate SERT expression, but not his functionality, in platelets membrane.
Identification of isolated cellular model, capable to express SERT and having biochemical characteristics of platelet, could be useful to study the modulation of serotonin transporter by different factors related with obesity. We identified this model in MEG-01, a human megakaryoblastic leukaemia cell line, which specifically retains the morphological and functional properties of bone marrow megakaryocytes. This cell line is considered to be the most suitable one for evaluating human megakaryocytic maturation and differentiation into platelet-like cells (Isakari Y. et al 2007).
The evidence that SERT is present in megakaryoblasts suggests the physiological relevance of a balanced control of 5-HT levels during the differentiation processes leading to platelet formation (Liu YS et al 2006; Yang M et al 2007). After 8 days of MEG-01 cell culture with -TPA, significant cell morphological variations were observed, accompanied by changes in SERT immunostaining, from a perinuclear (undifferentiated cells) to a cytoplasm-diffuse (differentiated cells) fluorescence pattern. In -TPA activated MEG-01 cells a maximal increase of SERT mRNA was observed after 3 day of stimulation, reaching the steady state plateau; a moderate increase in protein expression was noticeable after 8 days of culture in-TPA, as shown by densitometric analysis of SERT immunoblot band and, even if not significantly, by 5-HT uptake results. 5-HT re-uptake experiments have shown that SERT is present in the plasma membrane of MEG-01 cells. Western blot revealed a single SERT band in control and treated cells, with an apparent size of 71 KDa, a M.W. comparable to that commonly reported for SERT in human brain and platelets (67-75 KDa). These findings demonstrate that megakaryoblastic cells increase SERT expression during megakaryocytopoiesis and that differentiation into megakaryocyte and proplatelet-like formation ensures an adequate SERT reserve and 5-HT storage in circulating platelets. The enhancement of SERT expression in -TPA treated cells seems in apparent contradiction with -TPA early events observed in platelets (Marazziti D et al 1999c; Jayanthi LD et al.2005; Carneiro AM et al. 2006). Nevertheless, some authors have reported that 1 M -TPA is able either to reduce (after 2h) or to stimulate (after 16 h) SERT uptake velocity in JAR placental cells (Ramamoorthy JD et al 1995). Jiang and coauthors (2002) have observed that both fibronectin and protein kinase C-dependent ERK1/2 MAPK activities are essential to promote megakaryoblastic differentiation. These authors have demonstrated that activation of protein kinase C alone (serum free -TPA stimulation) is not able to promote a full megakaryocytopoiesis process. Thus, the observed SERT increase might depend from -TPA-protein kinase C pathways and other signaling cascades. The protein kinase network and downstream signal convergent MAPK pathways could play a key role in SERT modulation. Proteomic analysis and gene expression studies together the use of selective protein kinase (also protein kinase C) or MAPK inhibitors will clarify the signaling pathways involved. On the basis of results we hypothesize that our findings derived from an equilibrium between different SERT regulatory effects: differentiation events, inducing morphological changes and leading to the formation of cytoskeleton-organized cells with 5-HT storage vesicles (Ogura M et al 1998), increase total SERT protein density (necessity to accumulate 5-HT in dense granules); phosphorylation, down-regulation and trafficking mechanisms (linked to cytoskeleton formation) can start having influence on SERT protein expression and function, especially in late events of megakaryocytopoiesis. This could explain the observed discrepancy between SERT mRNA and protein expression after 8 days of treatment with -TPA. We cannot, in fact, exclude that our Western blot protein extracts were enriched fractions of cytoplasm and plasma membrane components. These data seem to be in accordance with binding results obtained on cell membranes. Confocal microscopy of MEG-01 cells activated by TPA will verify SERT localization during early and late differentiation events. Since diverse protein regulatory patterns are present in immature megakaryoblasts cells as regards to pro-platelets and platelets (Tytgat GAM et al. 2002), the MEG-01 model should be primarily applied as a developmental model, for investigations regarding molecular differentiation events. For instance, it should be mentioned that a SERT regulatory endoproteolytic cleavage has been observed in human platelets, producing fragments of different size after immunoblot analysis, whereas a single SERT band was resolved in MEG-01 cell extracts, using the same primary antibodies, as here reported.

Megakaryoblastic MEG-01 differentiation is a complex phenomenon leading to up or down-regulation of a variety of genes (Isakari Y et al 2009). Nevertheless, taking into account all limits, the use of MEG-01 cells can be the starting point for many investigations as the evaluation of hormone and transmitter effects on megakaryoblastic differentiation.
Moreover, this cell line could be a cellular model to screen those of obesity serum factors are mostly important for long-term SERT down or up-regulation.