N-Acetylated alpha-linked acidic dipeptidase expressed in rat adipocytes is localized in the insulin-responsive glucose transporter (GLUT4) intracellular compartments and involved in the insulin-stimulated GLUT4 recruitment.
The GLUT4-containing vesicles purified from rat adipocyte contain many protein species of unknown identity, some of which are likely to play a critical role in the trafficking of GLUT4. Presently, we describe an 85-kDa protein in GLUT4-vesicles of rat adipocytes as a potential GLUT4 traffic regulatory protein. MALDI-TOF MS, RT-PCR, gene cloning, protein sequence analysis, and immunoreactivity assay have identified this protein as N-acetylated alpha-linked acidic dipeptidase (NAALADase) expressed in rat adipocytes. NAALADase in rat adipocytes was mostly membrane-associated and colocalized in discrete GLUT4-compartments with enrichment in putative GLUT4-sorting endosomes (G4G(L)). Total cell lysates of adipocytes exhibited NAALADase activity. Next, we treated rat adipocytes with 2-[phosphonomethy]pentanedionic acid (2-PMPA), a potent NAALADase inhibitor, and studied its effect on the distribution of GLUT4 and 3-O-methyl glucose (3OMG) flux. In 2-PMPA-treated adipocytes, there was a significant reduction (by 40%) in the insulin-stimulated GLUT4 translocation to the plasma membrane. The 3OMG flux in insulin-stimulated adipocytes was also delayed (51% of control) by 2-PMPA treatment, indicating that 2-PMPA impairs insulin-stimulated GLUT4 recruitment and the uptake of glucose. It is suggested that NAALADase may function as a regulator required for the insulin-stimulated GLUT4 vesicle movement and/or its exocytosis, thus may regulate insulin-induced GLUT4 recruitment in rat adipocytes.
Park
Seung Y
SY
The Department of Biochemistry, College of Medicine, Dongguk University, Kyungju, Kyungpook 780-714, Republic of Korea.
Ha
Byoung G
BG
Choi
Geum H
GH
Lee
Wan
W
D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D000273:Adipose Tissue / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051275:Glucose Transporter Type 4; D043425:Glutamate Carboxypeptidase II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010446:Peptide Fragments / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013347:Subcellular Fractions / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism
C402107:2-(phosphonomethyl)pentanedioic acid; D004791:Enzyme Inhibitors; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D011994:Recombinant Proteins; C495694:Slc2a4 protein, rat; D043425:Glutamate Carboxypeptidase II