Am J Physiol Endocrinol Metab
281: E147–E154, 2001.
Increased expression of GPI-specific phospholipase D
in mouse models of type 1 diabetes
MARK A. DEEG,1 ROSARIO F. BOWEN,1 MONET D. WILLIAMS,1
L. KARL OLSON,2 ELIZABETH A. KIRK,3 AND RENÉE C. LEBOEUF3
1
Departments of Medicine and of Biochemistry and Molecular Biology, Indiana University
School of Medicine and the Richard L. Roudebush Veterans Affairs Medical Center,
Indianapolis, Indiana 46202; 2Department of Physiology, Michigan State University,
East Lansing, Michigan 48824; and 3Departments of Pathobiology, Nutritional
Sciences, and Medicine, University of Washington, Seattle, Washington 98125
Received 30 October 2000; accepted in final form 5 March 2001
Deeg, Mark A., Rosario F. Bowen, Monet D. Williams,
L. Karl Olson, Elizabeth A. Kirk, and Renée C.
LeBoeuf. Increased expression of GPI-specific phospholipase
D in mouse models of type 1 diabetes. Am J Physiol Endocrinol
Metab 281: E147–E154, 2001.—Glycosylphosphatidylinositolspecific phospholipase D (GPI-PLD) is a high-density lipoprotein-associated protein. However, the tissue source(s) for
circulating GPI-PLD and whether serum levels are regulated
are unknown. Because the diabetic state alters lipoprotein
metabolism, and liver and pancreatic islets are possible
sources of GPI-PLD, we hypothesized that GPI-PLD levels
would be altered in diabetes. GPI-PLD serum activity and
liver mRNA were examined in two mouse models of type 1
diabetes, a nonobese diabetic (NOD) mouse model and lowdose streptozotocin-induced diabetes in CD-1 mice. With the
onset of hyperglycemia (2- to 5-fold increase over nondiabetic
levels), GPI-PLD serum activity and liver mRNA increased 2to 4-fold in both models. Conversely, islet expression of GPIPLD was absent as determined by immunofluorescence. Insulin may regulate GPI-PLD expression, because insulin
treatment of diabetic NOD mice corrected the hyperglycemia
along with reducing serum GPI-PLD activity and liver
mRNA. Our data demonstrate that serum GPI-PLD levels
are altered in the diabetic state and are consistent with liver
as a contributor to circulating GPI-PLD.
nonobese diabetic mouse; streptozotocin; glycosylphosphatidylinositol; apolipoproteins; pancreatic islets
SERUM GLYCOSYLPHOSPHATIDYLINOSITOL-specific
phospholipase D (GPI-PLD) is a 110- to 120-kDa N-glycosylated
protein abundant in mammalian serum (5, 18), where
it associates with high-density lipoproteins (HDL). A
paucity of knowledge exists regarding the regulation
and function of this enzyme in serum. The association
with HDL raises the possibility that GPI-PLD may be
involved in lipid/lipoprotein metabolism, and we are
currently examining this potential role for GPI-PLD.
GPI-PLD is capable of cleaving GPI-anchored proteins
in vitro. However, adding crude or purified GPI-PLD to
intact cells will not lead to cleavage of GPI-anchored
Address for reprint requests and other correspondence: M. A.
Deeg, Division of Endocrinology, 111E, Roudebush VAMC, 1481 W.
10th St., Indianapolis, IN 46202-2883 (E-mail: mdeeg@iupui.edu).
http://www.ajpendo.org
proteins unless detergent or cholesterol binding agents
are added to perturb the cell membrane (1, 17). In
addition, others have suggested that GPI-PLD is catalytically inactive in serum because bicarbonate inhibits
GPI-PLD enzymatic activity in vitro (27). Although
GPI-PLD mRNA has been identified in a number of
tissues and cell types, the source(s) of circulating GPIPLD and factors regulating its expression are essentially unknown.
GPI-PLD cDNA has been isolated from both liver
and pancreatic cDNA libraries from multiple species
(14, 23, 28). Comparing these sequences reveals 90%
homology between cDNAs. Analysis of the primary
amino acid sequence reveals multiple potential posttranslational modifications, including phosphorylation, N-glycosylation, and myristoylation sites (8, 14).
Liver has the highest expression of GPI-PLD mRNA
(14, 26). Some studies have suggested that liver contributes to circulating GPI-PLD on the basis of changes
in serum GPI-PLD compared with liver function in
patients (19, 22). However, the amino acid sequence of
human serum GPI-PLD is consistent with the pancreatic and not liver GPI-PLD cDNA (10, 28). This suggests that pancreatic islets may contribute to circulating GPI-PLD. We and others have demonstrated that
GPI-PLD is expressed and secreted from pancreatic
islets and ␤-cell lines (9, 21). In addition, GPI-PLD
appears to colocalize with insulin in the secretory granule (9), raising the possibility that pathological conditions such as diabetes may be associated with changes
in islet production of GPI-PLD.
Considerable changes in lipoprotein metabolism occur in diabetic patients and animals. Alterations include changes in the expression of many apolipoproteins and in the levels of lipoprotein fractions (11). The
increased risk of atherosclerosis observed for diabetic
patients derives in part from these alterations in lipoprotein metabolism (2). Recently, the role of minor
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society
E147
E148
INCREASED GPI-PLD IN DIABETES
HDL-associated proteins, including apoJ and paraoxonase, in the development of atherosclerosis has been
appreciated (4, 12, 24, 25). Because serum GPI-PLD
behaves as a minor HDL-associated protein (7, 10), its
potential role in diabetes and atherosclerosis needs
further investigation.
One goal in this report was to determine whether
GPI-PLD expression is perturbed due to diabetes. We
found that GPI-PLD expression was markedly increased in two mouse models for type 1 diabetes; these
are nonobese diabetic (NOD) mice, known to develop
spontaneous insulitis, and low-dose streptozotocin
(STZ)-induced diabetes in CD-1 mice. A role for regulation of GPI-PLD expression by insulin is proposed on
the basis of these studies, combined with results of
GPI-PLD expression in mice under different nutritional status. A second goal was to determine whether
islets contribute significantly to serum GPI-PLD levels. In the mouse models studied in the present investigation, GPI-PLD mRNA levels in the liver mirrored
the changes in serum GPI-PLD. Overall, this is the
first report showing that GPI-PLD levels can be regulated. Furthermore, our data are consistent with liver
as a contributor to circulating GPI-PLD.
MATERIALS AND METHODS
Animals. CD-1 male mice (8 wk old) were purchased from
Harlan Sprague Dawley (Indianapolis, IN). NOD mice (female) were from an inbred colony maintained at the University of Washington (NOD/RL). These mice were established
from mating pairs originally provided by Dr. Pia Reich (Department of Medicine, Yale University) and are in their 23rd
generation intercross. The frequency of diabetes is greater
than 80% among female mice by 3 mo of age (13). Agematched nondiabetic NOD mice were used as controls.
CD-1 mice were maintained in a temperature-controlled
(25°C) facility with a strict 12:12-h light-dark cycle and were
given free access to food and water. Mice were fed rodent
chow pellets (Wayne Rodent BLOX 8604, Teklad, Madison,
WI). NOD mice were maintained under specific pathogenfree conditions at 25°C with a strict 12:12-h light-dark cycle
and had free access to food (autoclaved) and water (acid
treated). Food was removed from the mice 4 h before the
collection of blood from the retroorbital sinus (15). Serum was
used immediately or stored at ⫺70°C before analysis. Mice
were killed by cervical dislocation. This project was approved
by the Animal Care and Use Committees of Indiana University and the University of Washington.
Drug treatment. STZ was dissolved in sterile citrate buffer
(50 mM sodium citrate, pH ⫽ 4.5) and injected intraperitoneally into CD-1 mice (40 mg/kg, ⬃20 ␮l) within 5 min of
preparation. STZ or citrate buffer alone was administered for
5 consecutive days during the 1st wk of this study. These
mice were not treated with insulin.
NOD mice were monitored for diabetes by weekly weighing
and urinary glucose testing (Diastix, Eli Lilly, Indianapolis,
IN). Minimal insulin replacement therapy (Regular Iletin II
porcine insulin, Eli Lilly) was initiated after a positive urine
glucose test. Insulin dosage was determined on the basis of
urinary glucose output and body weight and was designed to
maintain body weight comparable to that of nondiabetic
littermates. Insulin doses ranged from 20 to 600 U/kg every
other day. Nondiabetic littermates were not treated with
insulin at any time during this study.
GPI-PLD enzymatic activity. GPI-PLD activity was determined using the [3H]myristate-labeled membrane form of
variant glycoprotein as the substrate (9). Phosphatidic acid
was confirmed as the only reaction product by thin-layer
chromatography (data not shown, but see examples in Refs.
5, 9, and 18). One unit of activity was arbitrarily designated
as the amount of enzyme converting 1% of substrate per
minute.
Analytical procedures. Cholesterol, total triglyceride
(which includes glycerol), and glucose concentrations were
determined using commercially available kits (catalog numbers: 352–20, 339–10, and 315–100, respectively, Sigma, St.
Louis, MO). Insulin and glucagon assays were performed by
the Indiana University Diabetes Research and Training Center Immunoassay Core with commercially available kits
(Linco Research, St. Charles, MO).
Fast performance liquid chromatography. Plasma lipoproteins were separated by fast performance liquid chromatography (FPLC) size-exclusion chromatography using a Superose 6 column (Pharmacia, Piscataway, NJ). One hundred
microliters of pooled serum (from 2 or 3 mice) were loaded
onto the column and eluted with buffer consisting of 150 mM
NaCl, 20 mM HEPES (pH ⫽ 7.4), and 0.02% NaN3 at a flow
rate of 0.25 ml/min at 4°C. Sixty fractions, 500 ␮l each, were
collected and assayed for cholesterol, triglyceride, and GPIPLD activity.
Western blot analysis. GPI-PLD and apolipoprotein (apo)
A-I were detected by immunoblotting after SDS-PAGE using
7 or 12% acrylamide gels, respectively. Nitrocellulose membranes (Schleicher and Schuell, Keene, NH) were used for the
protein transfer, and specific proteins were visualized by
chemiluminescence using commercial reagents (SuperSignal,
Pierce, Rockford, IL). Band intensities were compared by
densitometry using a Bio-Rad GS-700 Imaging Densitometer
and Molecular Analyst software (Bio-Rad, Hercules, CA).
GPI-PLD immunoreactivity was identified using a GPI-PLD
antibody by Western blotting, and changes relative to nondiabetic samples were determined.
GPI-PLD was identified using anti-GPI-PLD771, a peptidespecific rabbit polyclonal antibody generated against a peptide sequence corresponding to amino acids 771–790 in human serum GPI-PLD (M. Deeg, unpublished observations).
ApoA-I was identified using a polyclonal rabbit anti-mouse
antibody (BioDesign International, Kennebunk, ME).
Insulitis. Pancreatic tissues were isolated from NOD or
CD-1 mice and fixed in 4% paraformaldehyde for 4 h. Tissues
were embedded in paraffin blocks, and 5-␮m sections were
mounted on polylysine-coated slides. The degree of insulitis
was determined from multiple nonsequential slides from
individual mice. Approximately 5–10 islets/section were
scored. Each islet was assigned a score, as previously described (6): 0 ⫽ no lymphocytic infiltration; 1 ⫽ peri-insulitis;
2 ⫽ ⬍50% islet infiltration; and 3 ⫽ ⬎50% islet infiltration.
The sum of all scores for each mouse was divided by the
number of islets analyzed to derive a final insulitis score.
Immunofluorescence. To determine GPI-PLD expression in
islets, paraffin sections (see above) were deparaffinized and
rehydrated with xylene and graded ethanol. GPI-PLD, insulin, and glucagon were identified as previously described (9)
using anti-GPI-PLD771 sera (1/50 dilution), guinea pig antibovine insulin (1/50 final dilution; Sigma), and a murine
monoclonal anti-glucagon (1/100 final dilution; Sigma). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA): fluorescein-conjugated goat anti-rabbit antiserum at a final concentration of
150 ␮g protein/ml; Cy5-conjugated goat anti-guinea pig antiserum at a final concentration of 150 ␮g protein/ml; and
E149
INCREASED GPI-PLD IN DIABETES
Table 1. Summary of metabolic, hormone, and lipid values in nondiabetic, diabetic,
and insulin-treated diabetic NOD mice
nonDM (5)
DM (5)
DM ⫹ insulin (7)
Glucose,
mg/dl
Insulin,
ng/ml
Glucagon,
pg/ml
Total Cholesterol,
mg/dl
Total Triglycerides,
mg/dl
apoA-I,
mg/ml
173 ⫾ 26
860 ⫾ 167*
154 ⫾ 149
1.03 ⫾ 0.02
0.56†
24.1, 48.3
52.3 ⫾ 9.8
ND
51.4 ⫾ 1.7
90 ⫾ 9
144 ⫾ 43*
107 ⫾ 26
115 ⫾ 32
160 ⫾ 58
189 ⫾ 86*
3.6 ⫾ 0.4
4.7 ⫾ 0.8
4.5 ⫾ 0.5
Blood was collected after a 4-h fast from nondiabetic (nonDM), diabetic (DM), and insulin-treated diabetic (DM ⫹ insulin) nonobese diabetic
(NOD) mice. apoA-I, apo-lipoprotein A-I. Metabolic, hormone, and lipid assays were determined as described in MATERIALS AND METHODS.
Values are individual or means ⫾ SD for no. of animals in parentheses. ND, not determined. * P ⬍ 0.05 vs. nonDM. † Pool from 3 animals.
Texas Red-conjugated sheep anti-mouse antiserum at a final
concentration of 140 ␮g protein/ml.
The immunostained samples were examined with a BioRad MRC 1024 laser-scanning confocal microscope (Hercules, CA) mounted on a Nikon Diaphot 300 platform (Renal
Imaging Facility, Indiana University, Indianapolis, IN). The
fluorescent tags were individually excited at 488, 522, or 588
nm for fluorescein, Texas Red, and Cy5, respectively, and
monitored at emission wavelengths of 522, 588, or 680, respectively. Islets were graded as positive or negative for
insulin and GPI-PLD immunoreactivity. All emission wavelengths were monitored at each excitation wavelength to
ensure that the observed fluorescence did not result from the
wrong fluorescent tag.
mRNA quantification. Total RNA was extracted from liver
using a commercial kit (PolyAtract mRNA isolation system
IV, Promega, Madison, WI). mRNA specific for GPI-PLD was
identified by Northern blotting by use of a full-length GPIPLD cDNA (3.4 kb) obtained from a library generated in
pBluescript II SK⫹ (Stratagene) from a mouse glucagonoma
cell line ␣TC6 (14). Three major transcripts are seen for
GPI-PLD. The major band is 3.9 kb in length with variable
minor bands at 5.4 and 8.0 kb. Only the 3.9-kb transcript was
quantitated; ␤-actin mRNA was used to normalize loading.
Statistics. Values are reported as means ⫾ SD, with the
number of animals (n) in parentheses. Statistical differences
between groups were determined by one-way ANOVA. P ⬍
0.05 was accepted as statistically significant.
to diabetes offers another model in which to study
GPI-PLD expression. For CD-1 mice, changes in diabetic parameters at 0, 7, 14, and 28 days after STZ
treatment were monitored, where day 0 is the first STZ
treatment day (Fig. 1). Plasma insulin levels decreased
⬃30% by day 7 and were ninefold lower than on day 0
by day 28. Concomitant hyperglycemia was observed at
day 10 (not shown) and by day 14 was increased over
twofold from day 0. Glucagon levels were significantly
elevated at days 14 and 28. By 7 days, STZ-treated
CD-1 mice showed extensive insulitis, consistent with
the deterioration of diabetic parameters. Insulitis was
evident as early as day 1 (not shown).
Diabetes in NOD and CD-1 mice results in increased
GPI-PLD expression. Diabetes onset in NOD mice resulted in significant increases in GPI-PLD expression
(Fig. 2). Serum enzyme activity and immunoreactivity
were elevated 2- and 3.5-fold, respectively, in diabetic
RESULTS
Diabetic phenotypes in NOD and CD-1 mice. NOD
mice develop a spontaneous autoimmune destruction
of ␤-cells within islets that is similar in character to
type 1 diabetes in humans (29). This results in hyperglycemia and hypoinsulinemia, as shown in Table 1.
Diabetic NOD mice had a fivefold increase in glucose
and an ⬃50% reduction in insulin levels compared with
nondiabetic mice. Plasma cholesterol and triglyceride
concentrations were increased by 1.4-fold and 1.6-fold,
respectively. Diabetic mice treated with insulin maintained reduced plasma glucose, cholesterol, and glucagon levels, although triglyceride levels remained elevated compared with nondiabetic mice. This may
reflect the every-other-day dosing of insulin and the
variation in glycemic control (Table 1). The triglyceride
levels may also be influenced by the individual variation in nutritional status. Levels of plasma apoA-I, the
primary protein found on HDL, remained unchanged.
STZ treatment also results in hyperglycemia and
hypoinsulinemia and inflammatory processes that are
targeted primarily to the pancreas (16). Thus this route
Fig. 1. Effect of streptozotocin treatment on glucose, hormones, and
insulitis in CD-1 mice. CD-1 mice were treated daily for 5 days with
streptozotocin. Blood and tissue were harvested on days 0, 7, 14, and
28 (first day of injection is considered day 0). Insulitis, glucose, and
hormones were determined as described in MATERIALS AND METHODS.
Results are means ⫾ SD; n ⫽ 8–10 at each time point. *P ⬍ 0.05 vs.
day 0.
E150
INCREASED GPI-PLD IN DIABETES
For CD-1 mice, serum GPI-PLD immunoreactivity
and liver mRNA (Fig. 3) mirrored increases as seen for
glucose and glucagon. There were significant increases
for serum GPI-PLD immunoreactivity and liver mRNA
levels of ⬃1.5-fold at day 14 and ⱖ2-fold at day 28.
Overall, GPI-PLD expression was elevated by hypoinsulinemic phenotypes.
Tissue sources of GPI-PLD. The origin of circulating
GPI-PLD is controversial and has not been previously
examined in diabetic mice. Two likely sources for serum GPI-PLD are liver and pancreatic islets. Destruction of pancreatic ␤-cells by autoimmune disease or
STZ treatment would be expected to result in decreased amounts of GPI-PLD from this tissue source.
Pancreatic sections from nondiabetic and diabetic
NOD mice were triple-stained for GPI-PLD, glucagon,
and insulin. For nondiabetic mice, glucagon and insulin were readily identified in pancreatic islets (Fig. 4).
GPI-PLD immunoreactivity was also identified and
colocalized with insulin, suggesting that GPI-PLD is
expressed primarily in ␤-cells. In diabetic NOD mice,
Fig. 2. Serum glycosylphosphatidylinositol-phospholipase D (GPIPLD) and liver GPI-PLD mRNA steady-state levels in nondiabetic,
diabetic, and insulin-treated nonobese diabetic (NOD) mice. Serum
and liver samples were harvested from nondiabetic (nonDM), diabetic (DM), and insulin-treated diabetic NOD mice (DM ⫹ insulin)
after a 4-h fast, and serum GPI-PLD activity (open bars), immunoreactivity (immuno, hatched bars), and liver GPI-PLD mRNA levels
(solid bars) were determined as described in MATERIALS AND METHODS.
Representative Northern blots for GPI-PLD and ␤-actin are shown at
top. The serum GPI-PLD activity in nonDM animals was 17 ⫾ 1
U/␮l. Results are from the animals characterized in Table 1 and are
expressed as means ⫾ SD. * P ⬍ 0.05 vs. nonDM group.
mice compared with nondiabetic mice. Hence, there is
a decrease in the specific activity of GPI-PLD in the
diabetic mice. This decrease in specific activity is likely
secondary to the increase in serum lipids, which interfere with the GPI-PLD assay (data not shown). Therefore, measuring GPI-PLD immunoreactivity more
accurately reflects changes in serum GPI-PLD concentrations, although we cannot eliminate the possibility
that changes in GPI-PLD immunoreactivity may also
occur in the diabetic state. Hepatic mRNA levels for
GPI-PLD were increased fourfold as a result of diabetes. Treatment of diabetic mice with insulin reversed
these changes, reducing GPI-PLD levels to those of
nondiabetic mice. For the three groups of NOD mice,
Western blot analysis of serum for GPI-PLD showed
the presence of the expected 115-kDa protein only
(data not shown).
Fig. 3. Serum GPI-PLD immunoreactivity and liver mRNA in streptozotocin-treated CD-1 mice. Serum GPI-PLD immunoreactivity
(hatched bars) and liver GPI-PLD mRNA levels (solid bars) were
determined in steptozotocin-treated CD-1 mice, as described in Fig.
2 legend. Representative Northern blots for GPI-PLD and ␤-actin are
shown at top. Results are from animals in Fig. 1. *P ⬍ 0.05 vs. day 0.
INCREASED GPI-PLD IN DIABETES
E151
Fig. 4. Comparison of glucagon, GPI-PLD, and insulin
expression in islets from nondiabetic and diabetic
NOD mice. Pancreata from nondiabetic (insulitis
score ⫽ 0) and diabetic (insulitis score ⫽ 3) mice were
isolated, and glucagon, insulin, and GPI-PLD were
identified by immunofluorescence, as described in MATERIALS AND METHODS. Slides were excited at each
individual wavelength for each secondary antibody
and monitored at all 3 emission wavelengths. Excitation at the wavelength for the insulin antibody did not
reveal any signal at the emission wavelength used for
GPI-PLD (not shown). Results are representative of 5
nondiabetic and 5 diabetic NOD mice.
glucagon was readily identified, but GPI-PLD and insulin expression were below the level of detection in
nearly all islets (Fig. 4 and Table 2). Faint staining for
insulin in the islet from the diabetic animal shown
(Fig. 4) has the same distribution as glucagon, suggesting that this image derives from the glucagon probe.
Quantification of islet numbers showing immunoreactivity for insulin and GPI-PLD in islets from diabetic
and nondiabetic NOD mice compared with normal islets from CD-1 mice substantiate a reduction in islet
production of both insulin and GPI-PLD due to diabetes (Table 2). For CD-1 mice, the majority of islets
examined contained insulin and/or GPI-PLD compared
with 50–65% for nondiabetic NOD and 5% for diabetic
NOD mice. The reduction in staining seen for nondiabetic NOD vs. CD-1 mice may reflect ongoing insulitis,
which occurs before onset of frank diabetes (3). The
main point is that, because islet GPI-PLD expression is
lost upon frank diabetes onset but serum GPI-PLD is
increased in diabetic NOD and STZ-treated CD-1 mice
(Figs. 2 and 3), the liver may likely be the primary
source for GPI-PLD at least in the diabetic state.
GPL-PLD expression may be controlled by insulin.
Diabetes onset in NOD and CD-1 mice is associated
with a myriad of metabolic changes, including hyperTable 2. Insulin and GPI-PLD staining of islets from
CD-1, nondiabetic, and diabetic NOD mice
CD-1
nonDM
DM
Islets, n
Insulin ⫹
GPI-PLD ⫹
36
24
21
35 (97%)
15 (65%)
1 (5%)
29 (81%)
12 (52%)
1 (5%)
Pancreata were isolated from CD-1 (not treated with streptozotocin and used as a positive control), nondiabetic (nonDM), and diabetic (DM) NOD mice and stained for insulin and glycosylphosphatidylinositol-phospholipase D (GPI-PLD) as described in MATERIALS
AND METHODS. The no. of islets examined is from 5 mice/group.
Staining was graded as ⫹ or ⫺, and no. of positively stained islets is
shown with % positive in parentheses. Three of five mice in the
nonDM group had histological evidence of insulitis.
glycemia, hypoinsulinemia, alterations in circulating
lipids, and potentially other parameters that may regulate GPI-PLD expression. It is tempting to speculate
that hypoinsulinemia is the driving factor in the increased expression of GPI-PLD seen for diabetic NOD
and CD-1 mice. Because severe starvation can induce
many of the same metabolic and hormonal changes as
diabetes except for hyperglycemia, we used nutritional
status as a tool to test for a potential role of insulin in
GPI-PLD expression.
Serum GPI-PLD levels, liver GPI-PLD mRNA levels,
and various hormonal markers were determined for
CD-1 mice that were fasted for 4 h (fed) and 72 h
(severe starvation). As expected, there was no difference in serum glucose, but insulin levels decreased
markedly (Table 3). Severe starvation resulted in a
small increase in serum GPI-PLD activity, a 50% increase in serum GPI-PLD immunoreactivity, and a
greater than twofold increase in liver GPI-PLD mRNA
levels.
GPI-PLD is primarily associated with HDL in nondiabetic and diabetic states. In normal serum, GPIPLD behaves as an apolipoprotein, forming a complex
with apoA-I (7, 10). Because the concentration and
composition of many lipoproteins are altered in the
diabetic state, it is possible that GPI-PLD may redistribute among circulating lipoprotein fractions in our
diabetic mice.
GPI-PLD associations with lipoproteins were examined using serum taken from nondiabetic, diabetic, and
insulin-treated diabetic NOD mice. Lipoprotein profiles obtained by FPLC showed marked differences
among the groups (Fig. 5). For all groups of mice, the
majority of cholesterol eluted as HDL (fractions 28–35)
and overall levels were increased in diabetic mice,
consistent with the 40% elevation in total cholesterol
level (Table 1). Conversely, triglycerides, levels of
which were elevated in diabetic and insulin-treated
mice (Table 1), were nearly exclusively associated with
very low-density lipoprotein (VLDL) particles eluting
E152
INCREASED GPI-PLD IN DIABETES
Table 3. Effect of fasting on GPI-PLD serum levels and liver mRNA levels
GPI-PLD
Fed (2)
Fast (3)
Activity,
U/ml
Immunoreactivity,
degree of increase
Liver mRNA,
degree of increase
Glucose,
mg/dl
Insulin,
ng/ml
Glucagon,
pg/ml
37, 39
56 ⫾ 1.3
1.0
1.50 ⫾ 0.10
1.0
2.65 ⫾ 0.88
168, 183
152 ⫾ 39
1.62, 0.58
0.20 ⫾ 0.13
43.14*
58, 57
CD-1 mice were fasted 4 (fed) or 72 h (fast), and serum hormone levels and GPI-PLD activity and immunoreactivity, as well as liver
GPI-PLD mRNA levels, were determined as described in MATERIALS AND METHODS. Nos. of animals used are shown in parentheses, and results
are individual values or means ⫾ SD. * Serum pooled from 2 animals.
near the column void volume (fractions 10–20). The
majority (97%) of GPI-PLD activity eluted just before
HDL (fractions 21–27). This relationship of GPI-PLD
complexes migrating before HDL is a consistent finding and is also seen in human serum (7). Overall, the
lipoprotein patterns are consistent with total lipid
measurements and suggest that GPI-PLD is associated
with a specific subfraction of HDL in both diabetic and
nondiabetic states.
NOD mice with insulin led to a rapid and complete
return of GPI-PLD liver mRNA and serum levels to
those of nondiabetic mice. Because of the complex metabolic alterations induced by diabetes and fasting, we
DISCUSSION
There were two main findings in this report. First,
hypoinsulinemic states led to increases in liver expression of GPI-PLD. Both hepatic mRNA levels and serum
immunoreactivity and enzymatic activity were increased. The finding that levels of GPI-PLD can be
regulated by altered physiological states of diabetes
and fasting constitutes the first evidence that GPI-PLD
expression is not merely constitutive. It is clear from
our previous studies and this report that the regulation
of the GPI-PLD gene is complex and involves genetic
factors (14), nutritional status, and extent and type of
diabetes. Second, our data are consistent with the
possibility that the liver is the main source of circulating GPI-PLD. Overall, changes in liver mRNA levels
paralleled changes in serum immunoreactivity in all
cases studied here. Although a variety of cell types can
produce GPI-PLD, secretion into plasma appears proportionately greater from liver.
In the mouse, the liver is the most abundant source
of mRNA for GPI-PLD (14). However, in other species,
the source of circulating GPI-PLD is less clear. Immunohistochemical detection of this protein has been
found in a variety of cell types (20, 30). Furthermore, a
confusing observation in studies of human GPI-PLD is
that the amino acid sequence reported for serum GPIPLD matches that predicted from human pancreatic
and not liver GPI-PLD cDNA (10, 28). This latter
difference could be explained by genetic polymorphisms that may exist between the individuals from
whom liver and pancreas samples were taken for cloning or mistakes in sequencing (14). Future studies are
needed in which serum, liver, and pancreas samples
can be derived from individual subjects for final determinations of GPI-PLD sources in humans.
It is tempting to speculate that insulin regulates
GPI-PLD expression. Circumstantial evidence for this
includes the increased mRNA levels seen in models of
hypoinsulinemia studied here. Treatment of diabetic
Fig. 5. Gel filtration of lipoproteins and GPI-PLD complexes from
nondiabetic, diabetic, and insulin-treated NOD mice. Lipoproteins in
serum obtained from nondiabetic, diabetic, and insulin-treated diabetic NOD mice were separated by gel filtration, and cholesterol,
triglyceride, and GPI-PLD activity was determined in each fraction
as described in MATERIALS AND METHODS. Serum was pooled from 2 or
3 mice. Results are representative of 2 separate fast performance
liquid chromatography runs.
INCREASED GPI-PLD IN DIABETES
cannot rule out other modulators of GPI-PLD expression. It is unclear from this work whether GPI-PLD is
regulated at the level of transcription and/or whether
posttranscriptional processes are involved in controlling mRNA levels for GPI-PLD. Further studies of the
direct effects of insulin on GPI-PLD in hepatic cell
systems, changes due to nutritional status in other
mouse models, and the mode of regulation are needed.
In addition, sequencing of the promoter regions of
GPI-PLD is needed to establish whether insulin response elements exist.
Altered turnover of GPI-PLD in serum could also
affect its circulating concentrations. Our studies of
NOD mice showed that both serum HDL and GPI-PLD
levels were elevated by diabetes onset and reduced
after insulin administration. Because GPI-PLD associates with HDL, one interpretation of this concentration
data is that serum GPI-PLD was elevated by diabetes
because of the increase in its binding pool. Thus GPIPLD concentration merely reflects HDL concentration.
There are two arguments against this idea. First, in a
previous study (14), HDL and GPI-PLD activity levels
did not correlate. For C57BL/6 mice fed an atherogenic
diet, HDL levels were decreased twofold, yet GPI-PLD
activities remained the same. Second, apoA-I concentrations were essentially unchanged among the groups
of NOD mice (Table 1). Diabetic NOD mice showed
increased HDL cholesterol levels, suggesting that although particle composition was altered, the number of
HDL particles did not change. In humans, GPI-PLD is
associated with a small, discrete HDL-containing
apoA-I and apoA-IV that accounts for no more than
0.2% of the total apoA-I (7). (Note: Preliminary data
suggest that the mouse GPI-PLD complexes have similar properties to those of humans.) Hence, it is conceivable that this GPI-PLD-containing subclass of
HDL may change without affecting total HDL. Further
work is needed to confirm whether HDL turnover, or
turnover of specific HDL subfractions, contributes to
determining GPI-PLD levels.
Although we still do not know the primary function
of serum GPI-PLD, the observations reported here
provide some important fundamental information regarding liver as a source of serum GPI-PLD. In addition, our results suggest that serum GPI-PLD levels
may be regulated and altered in pathological conditions. Finally, serum GPI-PLD behaves very much like
an apolipoprotein and joins the growing list of minor,
but often important, metabolically active proteins associated with HDL particles.
We thank Mark Caldwell for excellent technical assistance.
This work was supported by a National Institutes of Health (NIH)
Diabetes New Investigator Award from the Diabetes Research Center, University of Washington (NIH DK-17047, D. Porte Jr., Principal Investigator); the Diabetes Research Council, Seattle, WA; an
American Diabetes Association Career Development Award (M. A.
Deeg); an NIH grant (HL-52848 to R. C. LeBoeuf); and research
awards from the American Diabetes Association and the Juvenile
Diabetes Foundation (L. K. Olson).
E153
REFERENCES
1. Bergman AS and Carlsson SR. Saponin-induced release of
cell-surface-anchored Thy-1 by serum glycosylphosphatidylinositol-specific phospholipase D. Biochem J 298: 661–668, 1994.
2. Bierman EL. George Lyman Duff Memorial Lecture. Atherogenesis in diabetes. Arterioscler Thromb 12: 647–656, 1992.
3. Calafiore R, Pietropaolo M, Basta G, Falorni A, Picchio
ML, and Brunetti P. Pancreatic beta-cell destruction in nonobese diabetic mice. Metabolism 42: 854–859, 1993.
4. Calero M, Tokuda T, Rostagno A, Kumar A, Zlokovic B,
Frangione B, and Ghiso J. Functional and structural properties of lipid-associated apolipoprotein J (clusterin). Biochem J
344: 375–383, 1999.
5. Davitz MA, Hereld D, Shak S, Krakow J, Englund PT, and
Nussenzweig V. A glycan-phosphatidylinositol-specific phospholipase D in human serum. Science 238: 81–84, 1987.
6. Debussche X, Lormeau B, Boitard C, Toublanc M, and
Assan R. Course of pancreatic beta cell destruction in prediabetic NOD mice: a histomorphometric evaluation. Diabete Metab
20: 282–290, 1994.
7. Deeg MA, Bierman EL, and Cheung MC. Glycosylphosphatidylinositol-specific phospholipase D associates with an apolipoprotein AI- and AIV-containing complex in human plasma. J
Lipid Res 42: 442–451, 2001.
8. Deeg MA and Davitz MA. Structure and function of the glycosylphosphatidylinositol-specific phospholipase D. In: SignalActivated Phospholipases, edited by M Liscovitch. Austin, TX:
RG Landes, 1994, p. 125–138.
9. Deeg MA and Verchere CB. Regulation of GPI-specific phospholipase D secretion from ␤TC3 cells. Endocrinology 138: 819–
826, 1997.
10. Hoener MC and Brodbeck U. Phosphatidylinositol-glycanspecific phospholipase D is an amphiphilic glycoprotein that in
serum is associated with high-density lipoproteins. Eur J Biochem 206: 747–757, 1992.
11. Howard BV and Howard WJ. Dyslipidemia in non-insulindependent diabetes mellitus. Endocr Rev 15: 263–274, 1994.
12. Jordan-Starck TC, Lund SD, Witte DP, Aronow BJ, Ley
CA, Stuart WD, Swertfeger DK, Clayton LR, Sells SF,
Paigen B, and Harmony JAK. Mouse apolipoprotein J: characterization of a gene implicated in atherosclerosis. J Lipid Res
35: 194–210, 1994.
13. Kirk EA and LeBoeuf RC. Diabetes accelerates atherosclerosis in non-obese diabetic (NOD) mice (Abstract). Circulation 94:
I-37, 1996.
14. LeBoeuf RC, Caldwell M, Guo Y, Metz C, Davitz MA, Olson
LK, and Deeg MA. Mouse glycosylphosphatidylinositol-specific
phospholipase D (Gpld1) characterization. Mamm Genome 9:
710–714, 1998.
15. LeBoeuf RC, Caldwell M, and Kirk E. Regulation by nutritional status of lipids and apolipoproteins A-I, A-II, and A-IV in
inbred mice. J Lipid Res 35: 121–133, 1994.
16. Like AA, Appel MC, Williams RM, and Rossini AA. Streptozotocin-induced pancreatic insulitis in mice. Morphologic and
physiologic studies. Lab Invest 38: 470–486, 1978.
17. Low MG and Huang KS. Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the
membrane anchors of cell surface proteins. Biochem J 279:
483–493, 1991.
18. Low MG and Prasad ARS. A phospholipase D specific for the
phosphatidylinositol anchor of cell-surface proteins is abundant
in plasma. Proc Natl Acad Sci USA 85: 980–984, 1988.
19. Maguire GA and Gossner A. Glycosyl phosphatidyl inositol
phospholipase D activity in human serum. Annu Clin Biochem
32: 74–78, 1995.
20. Metz CN, Thomas P, and Davitz MA. Immunolocalization of a
glycosylphosphatidylinositol-specific phospholipase D in mast
cells found in normal tissue and neurofibromatosis lesions. Am J
Pathol 140: 1275–1281, 1992.
21. Metz CN, Zhang Y, Guo Y, Tsang TC, Kochan JP, Altszuler
N, and Davitz MA. Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans. J Biol
Chem 266: 17733–17736, 1991.
E154
INCREASED GPI-PLD IN DIABETES
22. Rhode H, Lopatta E, Schulze M, Pascual C, Schulze HP,
Schubert K, Schubert H, Reinhart K, and Horn A. Glycosylphosphatidylinositol-specific phospholipase D in blood serum:
is the liver the only source of the enzyme? Clin Chim Acta 281:
127–145, 1999.
23. Scallon BJ, Fung WJC, Tsang TC, Li S, Kado-Fong H,
Huang KS, and Kochan JP. Primary structure and functional
activity of a phosphatidylinositol-glycan-specific phospholipase
D. Science 252: 446–448, 1991.
24. Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG, Fogelman AM, and Lusis
AJ. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394: 284–287,
1998.
25. Shih DM, Xia YR, Wang XP, Miller E, Castellani LW, Subbanagounder G, Cheroutre H, Faull KF, Berliner JA, Witztum JL, and Lusis AJ. Combined serum paraoxonase knockout/apolipoprotein E knockout mice exhibit increased lipoprotein
oxidation and atherosclerosis. J Biol Chem 275: 17527–17535,
2000.
26. Stadelmann B, Zurbiggen A, and Brodbeck U. Distribution
of glycosylphosphatidylinositol-specific phospholipase D mRNA
in bovine tissue sections. Cell Tissue Res 274: 547–552, 1993.
27. Stieger S, Diem S, Jakob A, and Brodbeck U. Enzymatic
properties of phosphatidylinositol-glycan-specific phospholipase C from rat liver and phosphatidylinositol-glycan-specific phospholipase D from rat serum. Eur J Biochem 197:
67–73, 1991.
28. Tsang TC, Fung WJ, Levine J, Metz CN, Davitz MA, and
Burns DK. Isolation and expression of two human glycosylphosphatidylinositol phospholipase D (GPI-PLD) cDNAs (Abstract).
FASEB J 6: A1922, 1992.
29. Wicker LS, Miller BJ, Coker LZ, McNally SE, Scott S,
Mullen Y, and Appel MC. Genetic control of diabetes and
insulitis in the nonobese diabetic (NOD) mouse. J Exp Med 165:
1639–1654, 1987.
30. Xie M, Sesko AM, and Low MG. Glycosylphosphatidylinositolspecific phospholipase D is localized in keratinocytes. Am J
Physiol Cell Physiol 265: C1156–C1168, 1993.