Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids.

Fluorescent lipid probes were used to track lipid trafficking between parent RBL cells and exosomes. We have checked the intracellular labeling of exosomes ("in vivo labeling") from parent cell incubated with either Bodipy-Cer, Bodipy-PC, or NBD-PC. Bodipy-PC labeled equally cells and exosomes, whereas Bodipy-Cer, a Golgi marker, was enriched in exosomes. Golgi membranes participated effectively in exosome biogenesis since cell incubation with brefeldin A leads to a modified phospholipid/protein ratio in exosomes. At the opposite, NBD-PC, a plasma membrane marker weakly labeled exosome membranes. Sorting of subpopulations indicated that the MHC-II containing exosomes were enriched in Bodipy-PC, whereas tetraspanin(CD 63 or CD81)-containing exosomes are essentially labeled with Bodipy-Cer and Bodipy-PC. These results indicated that RBL released two main subpopulations of exosomes that can be discriminated by their protein and lipid contents. When the bulk of exosomes was labeled after their purification ("in vitro labeling") with either of the above-mentioned lipid probes, the Bodipy-Cer was the only one to incorporate noticeably in all the subpopulations, indicating that the previous results obtained during "in vivo labeling" monitored real intracellular lipid trafficking between organelles and exosomes. Bodipy-Cer was further used as a tool to measure the respective amounts of each subpopulations. CD63, MHC II, and CD81-containing exosomes accounted for 47%, 32%, and 21%, respectively, of total exosomes.

Tyramine and benzylamine partially but selectively mimic insulin action on adipose differentiation in 3T3-L1 cells.

Biogenic amines like tyramine, methylamine and the non-naturally occuring amine, benzylamine, have been described to promote adipose conversion of murine 3T3 preadipocytes. To further investigate these novel effects of amines, we studied whether they selectively mimic the long-term adipogenic action of insulin. To this aim, we decided to use the 3T3-L1 cell line since this model needs a complex combination of inducers to trigger the differentiation programme: insulin, isobutylmethylxanthine (IBMX, an activator of cAMP-signal transduction pathway) and the synthetic glucocorticoid, dexamethasone. A cell culture protocol was designed, by which each component of the differentiation cocktail was replaced with either benzylamine or tyramine, in order to determine whether these amine oxidase substrates could substitute any of the differentiation inducers in 3T3-L1 cells. The incomplete lipid accumulation found in cells grown under IBMX- or dexamethasone-free conditions was not improved by the daily addition of amines to the culture medium. Insulin was the only component of adipose differentiation cocktail of 3T3-L1 that could be replaced, although partially, by tyramine or benzylamine. When used at 0.5 mM, these amines resulted in a significant increase of triacylglycerol accumulated eight days after confluence, when compared to cells kept without insulin. This partial insulin replacement was totally abolished by SSAO-inhibitors, while MAO-blockade did not reduce lipid accumulation. As previously reported for other insulin-sensitive processes, such as stimulation of glucose transport or lipolysis inhibition in mature adipocytes, the stimulation of adipogenesis by tyramine and benzylamine was an SSAO-dependent mechanism that apparently shared common signaling pathways with insulin.

Effect of prolonged treatment with tyramine on glucose tolerance in streptozotocin-induced diabetic rats.

The biogenic amine tyramine has been reported to stimulate in vitro glucose transport in adipocytes, cardiomyocytes and skeletal muscle, and to improve in vivo glucose utilization in rats. These effects were dependent on amine oxidation, since they were blocked by inhibitors of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO). We thus tested in this work whether a prolonged treatment with tyramine could improve glucose tolerance in streptozotocin-induced diabetic rats. First, tyramine content of standard rodent chow was determined by HPLC and daily tyramine intake of control rats was estimated to be around 26 micromol/kg body weight. Then, tyramine was administred during 3 weeks in streptozotocin-induced diabetic rats at 29 micromol/kg by daily i.p. injection alone or together with vanadate 0.02 micromol/kg. In another group of diabetic rats, tyramine was subcutaneously delivered at 116 micromol/kg/day by osmotic minipumps. All tyramine treatments resulted in a decrease of the hyperglycemic responses to an i.p. glucose load. Adipocytes isolated from either untreated or treated diabetic rats were sensitive to the stimulation of glucose uptake by tyramine. However, diabetic animals receiving tyramine for three weeks did not recover from their hyperglycemia, hypoinsulinemia and glucosuria. These results show that the improvement of glucose tolerance induced by prolonged tyramine administration occurs in an insulin-depleted model and probably results from peripheral insulin-like actions of the oxidation of MAO/SSAO substrates, such as the stimulation of glucose uptake into adipocytes.