No abstract

Using mature adipocytes and preadipocytes from genetically obese Zucker rats, we investigated the cells' ability to maintain abnormal fat storage capacity when withdrawn from their in vivo environment. Long-term adipocyte cultures from obese rats displayed an increase in both glucose consumption (GC) and enzyme activities, including fatty acid synthase (4-fold), glycerol-3-phosphate dehydrogenase (4.5-fold), lipoprotein lipase (LPL; 6-fold), and malic enzyme (2.5-fold). Fully differentiated obese predipocytes exhibited a twofold increase in these enzyme activities, together with higher glucose metabolism. In obese cells, LPL mRNA was increased in both adipocytes (6-fold) and differentiated preadipocytes (2-fold). Insulin mediated an increase in GC and lipogenic enzymes in both adipocytes and preadipocytes regardless of the genotype; this effect was more marked in obese cells. Examining cultured adipocytes from rats fed a high-fat diet, we showed that the nutritional effect upon GC and lipogenic enzymes was abolished after culture. These results demonstrated that fatty mutation may be intrinsically expressed in prolonged cultured mature adipocytes and in newly differentiated adipocytes.

Platelets achieve bleeding arrest at sites of vascular injury via secretion of secretory proteins from their storage granules, termed ␣-granules. We have recently analyzed granule targeting of platelet factor 4 (PF4), a secretory ␣-granule chemokine, and demonstrated that PF4 ␣-granule storage relied upon determinants within PF4 mature sequence. To define these determinants, PF4 mutants fused to the fluorescent reporter protein green fluorescent protein were generated by progressive deletions and site-directed mutagenesis. They were then transfected in AtT20 cells and assessed for granule targeting by colocalization with ACTH-containing granules, using laser scanning confocal microscopy. This strategy identified the amino acid 41-50 (LIATLKNGRK) sequence as most critical for PF4 granule targeting and/or storage; its deletion from PF4 induced a marked decrease in granule storage (from 81 ؎ 2% to 17 ؎ 3%, p < 0.0001). Ala-scanning mutagenesis of LIATLKNGRK narrowed down the targeting motif to LKNG. A direct role for LKNG in ␣-granule targeting was confirmed in the thrombopoietin-induced human megakaryocytic Dami cells, in which the LKNGgreen fluorescent protein chimera exhibited an 82.5 ؎ 1.8% colocalization with the ␣-granule proteins von Willebrand factor and P-selectin. LKNG is poorly conserved within the chemokine family. However three-dimensional alignments of the human ␣-granule chemokines Nap-2 (neutrophil-activating peptide) and RANTES (Regulated upon Activation Normal T Cell Expressed and Secreted) with PF4 revealed that LKNG, a surface-exposed hydrophilic turn/loop, matched Nap-2 (LKDG) and RANTES (TRKN) peptides with similar features. Moreover Nap-2 and RANTES peptides exhibited the same ␣-granule targeting efficiency than LKNG. We therefore postulate that the three-dimensional and physicochemical characteristics of PF4 LKNG are of general relevance to ␣-granule targeting of chemokines and possibly of other ␣-granule proteins.

Summary.  The storage mechanism of endogenous secretory proteins in megakaryocyte α‐granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well‐known platelet α‐granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4‐GFP), were transfected in the well‐characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno‐electronmicroscopy. In preliminary experiments, GFP, a non‐secretory protein (no signal peptide), localized in the cytoplasm, while PF4‐GFP colocalized with adrenocorticotropin hormone (ACTH)‐containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4‐GFP localized in α‐granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify α‐granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4‐GFP), GFP was not stored into granules in spite of its efficient translocation to the ER‐Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in α‐granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4‐GFP appears as an appropriate probe with which to analyze α‐granule biogenesis and its alterations in the congenital defect gray platelet syndrome.