-Cell Calcium-Independent Group VIA Phospholipase
A
2
(iPLA
2
)
Tracking iPLA
2
Movements in Response to Stimulation
With Insulin Secretagogues in INS-1 Cells
Shunzhong Bao,
1
Chun Jin,
1
Sheng Zhang,
1
John Turk,
1
Zhongmin Ma,
2
and Sasanka Ramanadham
1
Evidence that group VIA cytosolic calcium-independent
phospholipase A
2
(iPLA
2
) participates in -cell signal
transduction includes the observations that inhibition
of iPLA
2
with the bromoenol lactone suicide substrate
suppresses glucose-stimulated insulin secretion and
that overexpression of iPLA
2
amplifies insulin secre-
tory responses in INS-1 insulinoma cells. Immunofluo-
rescence analyses also reveal that iPLA
2
accumulates
in the perinuclear region of INS-1 cells stimulated with
glucose and forskolin. To characterize this phenomenon
further, iPLA
2
was expressed as a fusion protein with
enhanced green fluorescent protein (EGFP) in INS-1
cells so that movements of iPLA
2
are reflected by
changes in the subcellular distribution of green fluores-
cence. Stimulation of INS-1 cells overexpressing
iPLA
2
-EGFP induced greater insulin secretion and
punctate accumulation of iPLA
2
-EGFP fluorescence in
the perinuclear region. To determine the identity of
organelles with which iPLA
2
might associate, colocal-
ization of green fluorescence with fluorophores associ-
ated with specific trackers targeted to different
subcellular organelles was examined. Such analyses
reveal association of iPLA
2
-EGFP fluorescence with
the ER and Golgi compartments. Arachidonate-contain-
ing plasmenylethanolamine phospholipid species are
abundant in -cell endoplasmic reticulum (ER) and are
excellent substrates for iPLA
2
. Arachidonic acid pro-
duced by iPLA
2
-catalyzed hydrolysis of their sub-
strates induces release of Ca
2
from ER stores—an
event thought to participate in glucose-stimulated insu-
lin secretion. Diabetes 53 (Suppl. 1):S186 –S189, 2004
P
hospholipase A
2
(PLA
2
) is a diverse group of
enzymes that catalyze hydrolysis of the sn-2
substituent from glycerophospholipid substrates
to yield a free fatty acid and a 2-lysophospholipid
(1,2). Among the PLA
2
s is an 84-kDa cytosolic PLA
2
that
does not require Ca
2
for catalysis and is designated group
VIA cytosolic calcium-independent phospholipase A
2
(iPLA
2
) (3–5).
A role for iPLA
2
in signal transduction in insulin-
secreting -cells is suggested by the observations that
inhibition of iPLA
2
activity with the bromoenol lactone
(BEL) suicide substrate of iPLA
2
suppresses insulin
secretion, and overexpression of iPLA
2
amplifies glucose-
and forskolin-stimulated insulin secretion from pancreatic
islets and INS-1 insulinoma cells (6,7). Stimulation of
iPLA
2
-overexpressing INS-1 cells with cAMP-elevating
agents also induces translocation of iPLA
2
to the perinu-
clear region (7). This is of interest because glucose pro-
motes both -cell insulin secretion and proliferation, and
glucose-induced INS-1 cell mitogenesis is cAMP depen-
dent (8). To characterize iPLA
2
subcellular movements
further, we developed INS-1 cell lines that overexpress
iPLA
2
as a fusion protein with enhanced green fluores-
cent protein (EGFP) so that green fluorescence associated
with EGFP reflects the location of iPLA
2
. Simultaneous
monitoring of florescence associated with various tracking
molecules allowed us to identify specific subcellular or-
ganelles with which iPLA
2
associates when cells are
stimulated.
RESEARCH DESIGN AND METHODS
Preparation of a construct for expressing iPLA
2
as a fusion protein
with EGFP and selection of stably transfected clones. Full-length iPLA
2
cDNA was amplified by PCR using the following primer set: sense, 5
AGCTTCGAATTCATGCAGTTCTTTGGACGC-3, and antisense, 5-TTC
GATATCGGGAGATAGCAGCAGCTGG-3. The amplified full-length iPLA
2
from the pMSCV-neo-iPLA
2
constructs were then subcloned into the
pEGFP-N2 (Clontech, Palo Alto, CA) after the immediate early promoter of
cytomegalovirus major and before EGFP coding sequences in the same
code-reading frame with EGFP. The EGFP-N2 control vector and the con-
struct encoding iPLA
2
-EGFP (FPN2) fusion protein were transfected into
INS-1 cells with a Gene PORTER transfection system according to the
manufacture’s instructions (Gene Therapy Systems, San Diego, CA). Stably
transfected clones were selected using G418 (0.4 mg/ml). Stably transfected
cells obtained with EGFP-N2 control vector and FPN2 construct are designed
as N2 cells and FPN2 cells, respectively.
From
1
Mass Spectrometry Resource, Division of Endocrinology, Diabetes, and
Metabolism, Department of Medicine, Washington University School of Med-
icine, St. Louis, Missouri; and the
2
Division of Experimental Diabetes and
Aging, Mount Sinai School of Medicine, New York, New York.
Address correspondence and reprint requests to Sasanka Ramanadham,
Washington University School of Medicine, Department of Medicine, Box
8127, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: sramanad@im.wustl.edu.
Received for publication 14 March 2003 and accepted in revised form 7 May
2003.
This article is based on a presentation at a symposium. The symposium and
the publication of this article were made possible by an unrestricted educa-
tional grant from Les Laboratoires Servier.
BEL, bromoenol lactone; EGFP, enhanced green fluorescent protein; FITC,
fluorescein isothiocyanate; iPLA
2
, group VIA cytosolic calcium-independent
phospholipase A
2
; PLA
2
, phospholipase A
2
.
© 2004 by the American Diabetes Association.
S186 DIABETES, VOL. 53, SUPPLEMENT 1, FEBRUARY 2004