help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deeg, M. A.
Right arrow Articles by Verchere, C. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deeg, M. A.
Right arrow Articles by Verchere, C. B.
Endocrinology Vol. 138, No. 2 819-826
Copyright © 1997 by The Endocrine Society

Articles

Regulation of Glycosylphosphatidylinositol-Specific Phospholipase D Secretion from ßTC3 cells1

Mark A. Deeg and C. Bruce Verchere

Division of Endocrinology and Metabolism, Department of Medicine, Indiana University School of Medicine, and the Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202; and the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, University of Washington, and the Seattle Veterans Affairs Medical Center (C.B.V.), Seattle, Washington 98108

Address all correspondence and requests for reprints to: Mark Deeg, M.D., Ph.D., Division of Endocrinology and Metabolism (111E), Roudebush Veterans Affairs Medical Center, 1481 West 10th Street, Indianapolis, Indiana 46202-2884. E-mail: deeg.mark{at}indianapolis.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in mammalian serum, but the source of the circulating enzyme is unknown. Pancreatic islets have been reported to contain and secrete GPI-PLD. In this report we examined the regulation of GPI-PLD secretion from ßTC3 cells, a mouse insulinoma cell line. In the absence of glucose, phorbol myristic acid (0.1 µM) stimulated insulin secretion by 2.5-fold and GPI-PLD secretion by 2-fold. Carbachol (5 µM), glucagon-like peptide I-(7–36) amide (0.1 µM), and isobutylmethylxanthine (0.1 mM) had no significant effect on insulin or GPI-PLD secretion in the absence of glucose. Glucose (16.7 mM) stimulated both GPI-PLD and insulin secretion from ßTC3 cells by 55% and 235%, respectively. In addition, glucose potentiated the secretagogue effect of isobutylmethylxanthine, phorbol myristic acid, and glucagon-like peptide I on both insulin and GPI-PLD secretion. By immunohistochemistry and confocal microscopy, ßTC3 cells contain both insulin and GPI-PLD, which generally colocalized intracellularly. However, GPI-PLD secretion differed from insulin secretion by a higher rate of basal release (2.8% vs. 0.23%/h), a lower magnitude of response to secretagogues, and a more prolonged period of increased secretion. These results demonstrate that ßTC3 cells secrete GPI-PLD in response to insulin secretagogues and suggest that GPI-PLD may be secreted via the regulated pathway in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUMEROUS proteins have been identified that are anchored to the external leaflet of the plasma membrane by glycosylphosphatidylinositols (GPI), a lipid modification whose structure has been determined for a number of GPI anchors (1). These GPI-anchored proteins have a diverse array of functions and include catalytic enzymes, lymphocyte antigens, adhesion molecules, protozoa antigens, nutrient receptors, and complement regulatory proteins (2). Although the functional utility of membrane anchoring via a GPI is unknown, the potential for cleavage by a phospholipase mechanism exists. Soluble forms of GPI-anchored proteins have been identified in serum with a structure consistent with phospholipase cleavage (3, 4). Two GPI-specific phospholipases have been purified and cloned, a GPI-specific phospholipase C from Trypanosome brucei (5) and a GPI-specific phospholipase D from mammalian sources (6, 7).

GPI-specific phospholipase D (GPI-PLD) has been purified from serum (8, 9), the most abundant source of GPI-PLD, and cloned from both human liver and pancreatic complementary DNA (cDNA) libraries (7). Despite an extensive understanding of the protein biochemistry of this enzyme (reviewed in 10 , the physiological role and regulation of GPI-PLD remain unclear. Adding exogenous GPI-PLD, crude or purified, to cells does not cleave membrane-bound GPI-anchored proteins unless the plasma membrane is perturbed by detergents (11) or cholesterol-binding agents (12). Circulating GPI-PLD has been shown to associate with high density lipoprotein particles (13, 14), and apolipoprotein AI has been reported to stimulate GPI-PLD activity in vitro (15), but a role for GPI-PLD in lipoprotein action or metabolism has not been examined. Many cells have been found to contain GPI-PLD (10), including hepatocytes, pancreatic islets, keratinocytes, and myeloid cells lines, but only islets (16) and myeloid cell lines (17) have been demonstrated to secrete GPI-PLD. Pancreatic islets appear to contribute to the circulating pool of GPI-PLD in humans, as the amino acid sequence of serum GPI-PLD corresponds to that predicted by the human pancreatic GPI-PLD cDNA (7, 13). One approach we have taken to understand the function of GPI-PLD is to examine the regulation of GPI-PLD secretion from cells.

In addition to pancreatic islets, mouse-derived insulinoma (ßTC3) and glucagonoma ({alpha}TC6) cell lines have been shown to secrete GPI-PLD (16). ßTC3 cells were derived from transgenic mice expressing the simian virus 40 tumor antigen using the insulin promoter (18) and secrete insulin in a regulated manner very similar, but not identical, to that of intact ß-cells (19). These studies were undertaken to examine GPI-PLD secretion from islets using ßTC3 cells as a model, so that secretion could be examined in a relatively pure cell population. The data indicate that numerous insulin secretagogues also stimulate GPI-PLD secretion from ßTC3 and suggest that ß-cells may contribute to the circulating GPI-PLD activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Glucagon-like peptide I-(7–36) [GLP-I-(7–36)] amide was a gift from Dr. David D’Alessio (University of Washington). Phorbol 12-acetate 13-myristate (PMA) was purchased from LC Services (Woburn, MA). Nutridoma SP was purchased from Boehringer Mannheim (Indianapolis, IN). Myristic acid, isobutylmethylxanthine (IBMX), dimyristoylphosphatidic acid, BSA, carbachol, and 1,10-phenanthroline were obtained from Sigma Chemical Co. (St. Louis, MO). Cycloheximide was purchased from Calbiochem (La Jolla, CA).

Cell culture
ßTC3 cells (passages 45–52) were plated at 5 x 105 cells/35-mm dish in 2 ml DMEM containing 100 mg/dl glucose and 10% FBS. After 48 h, the dishes were rinsed three times with 1 ml DMEM and incubated for an additional 24 h in 2 ml DMEM containing 100 mg/dl glucose and 1% Nutridoma. Nutridoma is a serum substitute that contains no GPI-PLD activity (our unpublished observation). On the day of the experiment, the cells were rinsed twice with 1 ml Krebs-Ringer bicarbonate buffer with 0.1% BSA (KRB) containing no glucose and preincubated for an additional 1 h in the same media. The incubation was initiated by removing the media and adding 2 ml KRB containing the desired glucose and secretagogue concentrations, as indicated in the figures. After incubating for the length of time indicated in the figure legends, the media were removed, centrifuged, and aliquoted for insulin and GPI-PLD determinations. The cells were scraped and pooled with two 750-µl aliquots of KRB and centrifuged for 5 min at 2000 x g, and the supernatant was discarded. Lysis buffer (100 µl PBS containing 0.1% Nonidet P-40, 1 mM benzamidine, 5 µg/ml aprotinin, 0.2 mM phenylmethylsulfonylfluoride, and 5 µg/ml leupeptin) was added to the pellet and frozen at -70 C. Extra plates of cells were grown identically and treated as the control cells during the incubations. These extra plates were extracted with 95% ethanol containing 225 mM HCl at the end of the incubation for total cellular insulin and DNA determinations (20). Immunoreactive insulin and islet amyloid polypeptide (IAPP)-like immunoreactivity were determined by previously described RIAs (21, 22).

Immunohistochemistry and confocal microscopy
ßTC3 cells were grown in chamber slides and incubated for 4 h in KRB without glucose as described above. Cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, then washed three times with PBS. After washing, the cells were pretreated for 15 min with 10 mM Na2PO4 (pH 7.4) and 0.01% NaN3 (buffer A) with 0.1% Triton X-100, and then blocked with buffer A containing 1% BSA and 3% normal goat serum. For double labeling, cells were incubated sequentially with primary antibodies: first, rabbit polyclonal antiinsulin (Biogenex, San Ramon, CA) followed by a monoclonal anti-GPI-PLD [612c (23), a gift from Michael Davitz, New York University, New York, NY] in buffer A containing 1% BSA and 0.3% Triton X-100. After incubating with the primary antibodies, the samples were incubated sequentially with sheep antirabbit antibody coupled to fluorescein (Cappel Laboratories, Cochranville, PA) and goat antimouse antibody coupled with rhodamine (Jackson ImmunoResearch Laboratories, West Grove, PA) in buffer A containing 1% BSA and 0.3% Triton X-100. The immunostained samples were examined with a Bio-Rad MRC 1024 laser scanning confocal microscope (Hercules, CA) mounted on a Nikon Diaphot 300 platform (Renal Imaging Facility, Indiana University, Indianapolis, IN). For double labeling, images were pseudocolored green or red using MetaMorph (Universal Imaging, Westchester, PA). In the merged images, cells appeared red, green, or, in the case of colocalization, yellow. Final image processing was performed using MetaMorph.

GPI-PLD activity
GPI-PLD activity was determined using the membrane form of the variant surface glycoprotein (mfVSG) radiolabeled with [3H]myristate as the substrate, as previously described (8). Briefly, 100 µl medium or 20 µl cell lysate and 80 µl glass-distilled water were aliquoted into 1.7-ml microcentrifuge tubes, and the incubation was initiated by the addition of 100 µl reaction buffer [40 mM HEPES (pH 7.0), 2 mM CaCl2, 0.02% Nonidet P-40, and approximately 10,000 cpm [3H]mfVSG; 3–4 µg protein]. After incubating for 3 h at 37 C, the reaction was terminated by the addition of 500 µl n-butanol saturated with 1 M NH4OH, vortexed, and centrifuged at 16,000 x g for 5 min, and the amount of radioactive product in 350 µl of the organic-rich upper phase was determined by liquid scintillation. One unit of activity was arbitrarily defined as the amount of enzyme that hydrolyzed 1% of the substrate in 1 h at 37 C.

TLC
To determine the radioactive product(s) generated during the GPI-PLD assay, media or lysate samples were incubated with [3H]mfVSG as described above, and 400 µl of the organic phase were dried under N2. The samples were resuspended in 150 µl CHCl3-CH3OH (2:1, vol/vol) containing 25 µg each of myristic acid, dimyristoylglycerol, and dimyristoylphosphatidic acid as carriers. The samples were spotted on silica gel 60 TLC plates (E. M. Merck, Gibbstown, NJ), and the plates were developed with CHCl3-CH3OH-0.25% KCl (55:45:5, vol/vol/vol). The lanes were scanned using a Berthhold Imaging system (Berthold Analytical Instruments, Inc., Nashua, NH) for 120 min/lane. Carrier standards were identified by iodine vapors.

Phosphatidic acid, the expected product from a phospholipase D-mediated cleavage, accounted for over 95% of the radiolabeled products in both the medium and lysate samples (Fig. 1Go). Two other peaks of radioactivity were identified that comigrated with myristic acid and dimyristoylglycerol. A small amount of dimyristoylglycerol is present in the blank, which is a minor contaminant generated during the preparation of the substrate. When 250 µM 1,10-phenanthroline, a known inhibitor of GPI-PLD (9), was included during the incubation, no phosphatidic acid was present in either the medium or lysate, whereas the myristic acid and dimyristoylglycerol contents did not change (Fig. 1Go).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 1. Analysis of GPI-PLD assay products from ßTC3 media and cellular lysates. Media or cellular lysates from ßTC3 cells under basal conditions were incubated with [3H]mfVSG in the presence or absence of 1,10-phenanthroline as described in Materials and Methods. The reaction products were separated by TLC, and the products were detected using a Berthold imaging system. The detector response is expressed in arbitrary units (counts). The origin (O), solvent front (F), and migration of dimyristoylphosphatidic acid (DMPA), myristic acid (MA), or dimyristoylglycerol (DAG) standards are indicated in the top panel. Blank panels present the reaction mixture without the addition of samples.

 
Statistical methods
Statistical significance was determined using Student’s paired t test or one-way ANOVA; P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ßTC3 cells secrete GPI-PLD activity
As pancreatic islets are a mixture of endocrine cells and nonendocrine cells, ßTC3 cells were chosen as a ß-cell model to study GPI-PLD secretion. These cells secrete insulin and mimic many of the characteristics of normal ß-cells (19). To determine whether GPI-PLD secretion could be stimulated in these cells, ßTC3 cells were stimulated to secrete insulin with 16.7 mM glucose or strongly stimulated with a mixture of secretagogues (16.7 mM glucose, 5 µM carbachol, and 0.1 mM IBMX). Insulin concentration and GPI-PLD activity (see Fig. 1Go for analysis of reaction products from GPI-PLD assay) in the media were determined after 4 h of stimulation. Glucose stimulated both insulin (55%) and GPI-PLD (55%) secretion from these cells (Fig. 2Go), although glucose stimulation of insulin secretion was variable (see Tables 1Go and 2Go). When the cells were strongly stimulated with a mixture of secretagogues, insulin secretion increased nearly 20-fold compared to basal release in the absence of glucose, whereas GPI-PLD secretion increased over 4-fold. This amount of stimulated secretion corresponds to approximately 20% and 29% of the total insulin and GPI-PLD activity, respectively, recovered in both the medium and cell lysate (Table 1Go). The increase in GPI-PLD activity in the medium with stimulation did not result from loss of cell integrity or vesiculation, because there was no lactate dehydrogenase detected in the medium before or after stimulation, and centrifugation (100,000 x g for 60 min) did not alter medium GPI-PLD activity (data not shown). Stimulation of ßTC3 cells was associated with a 30% decrease in the GPI-PLD activity associated with the cell lysate (Fig. 2Go).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. Stimulation of GPI-PLD and insulin secretion from ßTC3 cells. ßTC3 cells were plated and grown as described in Materials and Methods. After 1 h of preincubation in KRB, the cells were incubated in KRB without glucose (control), in KRB with 16.7 mM glucose (glucose), or in KRB containing 16.7 mM glucose, 5 µM carbachol, and 0.1 mM IBMX (stimulated). The insulin (B) and GPI-PLD (A) contents of the media (open bars) and GPI-PLD activity in the cellular lysate (A, hatched bars) were determined as described in Materials and Methods. Values are the mean ± SD of quintuplet samples assayed in duplicate from three independent experiments. *, P < 0.05 compared to control (by paired Student’s t test). Control values (mean ± SD) for GPI-PLD in the medium and lysate were 176 ± 137 mU/ml·µg DNA and 2980 ± 1560 U/µg DNA, respectively. There was a small, but significant, decrease in the total recovered GPI-PLD activity (medium plus lysate) between control and strongly stimulated cells (mean ± SD, 3.16 ± 1.58 and 2.74 ± 1.52 U/µg DNA, respectively; n = 3; P < 0.01).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of sorting index for insulin and GPI-PLD in ßTC3 cells

 

View this table:
[in this window]
[in a new window]
  		Table 2. Effects of glucose and other insulin secretagogues on GPI-PLD and insulin secretion

 
Time course of GPI-PLD secretion
To further characterize stimulated secretion of GPI-PLD from ßTC3 cells, the time courses of GPI-PLD and insulin secretion under basal and stimulated conditions were compared. Under basal conditions (0 mM glucose), a small amount of insulin secretion occurred (Fig. 3Go and Table 1Go). After 4 h of static incubation, the net accumulation of insulin in the medium was 0.9 ± 0.4% (mean ± SD; n = 6) of the total insulin or, on the average, 0.2% of the total insulin/h. During this same period, 11.3 ± 8.1% (mean ± SD; n = 6) of the total GPI-PLD activity accumulated in the medium, or approximately 2.8%/h. When ßTC3 cells were strongly stimulated with a mixture of secretagogues, as described above, the rate of both insulin and GPI-PLD secretion increased. Insulin secretion increased rapidly, and over 95% of the insulin secretion during the 4-h incubation occurred during the first hour. During the second hour of stimulation, the rate of insulin secretion returned to the basal rate (Fig. 3BGo, inset). In comparison, secretagogue stimulation increased the rate of GPI-PLD secretion for 2 h, with the maximal increase in rate occurring during the first hour of stimulation (Fig. 3AGo, inset). After 2 h, the secretion rate returned to the basal rate. The cellular GPI-PLD activity did not show a significant decrease until after 2 h of stimulation (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Time course of insulin and GPI-PLD secretion from ßTC3 cells. ßTC3 cells were incubated for the times indicated in KRB with 0 mM glucose (open circles) or stimulated with 16.7 mM glucose, 5 µM carbachol, and 0.1 mM IBMX (closed circles), and the medium contents of GPI-PLD (A) and insulin (B) were determined as described in Fig. 2Go. Each point represents the mean ± SD of triplicate samples assayed in duplicate from three independent experiments. Error bars for the insulin control are within the size of the symbol. The inset in each panel depicts the rate of protein secretion per h for four different time periods in the following order: basal conditions, 0–1, 1–2, or 2–4 h of stimulation. The units for the y-axis in A and B insets are milliunits per ml/µg DNA·h and microunits x 10-3 per ml/h, respectively.

 
Like other endocrine cells, ßTC3 cells have both a regulated and a constitutive pathway of secretion. Insulin is secreted primarily through the regulated pathway, although compared to intact islets, ßTC3 cells have a significant amount of (pro)insulin secretion that occurs through a constitutive pathway (24). To compare the efficiency at which insulin and GPI-PLD were sorted between these pathways, a sorting index was calculated, which determines the fractional increase in secretion stimulated by a secretagogue compared to the basal unstimulated rate of secretion (25). Proteins secreted via a regulated pathway have a high sorting index; conversely, a low sorting index is characteristic of constitutively released proteins. When strongly stimulated, the sorting index for insulin was 21.4 ± 9.8 (mean ± SD) and was over 12-fold higher than the sorting index of 1.7 ± 1.1 for GPI-PLD (Table 1Go). These results suggest that sorting of GPI-PLD and insulin in ßTC3 cells may differ.

Effects of insulin secretagogues on GPI-PLD secretion
To determine which insulin secretagogues also stimulate GPI-PLD secretion, ßTC3 cells were stimulated with various insulin secretagogues in the absence and presence of glucose, and the insulin and GPI-PLD contents of the medium were compared. The secretagogues chosen varied in their mechanism of action, including two agents that interact with cell surface receptors (carbachol and GLP-I) and two that bypass receptor activation (IBMX and PMA). In addition, two of the agents (GLP-I and IBMX) worked primarily by activating cAMP-dependent mechanisms, whereas the others involved protein kinase C. In the absence of glucose, PMA (0.1 µM) stimulated the secretion of insulin (2.5-fold) and GPI-PLD (2-fold; Table 2Go). Carbachol also stimulated insulin secretion in the absence of glucose to a small extent (25–30%; Table 2Go). Glucose (16.7 mM) stimulated both insulin and GPI-PLD secretion by 235% and 55%, respectively.

Glucose also potentiated the effect of most insulin secretagogues on GPI-PLD secretion. In contrast to their ineffectiveness in the absence of glucose, both IBMX and GLP-I stimulated GPI-PLD secretion by 40% over glucose alone. These agents increased insulin secretion by 7.0- and 2.3-fold, respectively, in the presence of 16.7 mM glucose. PMA increased GPI-PLD and insulin secretion 2.4- and 12.3-fold, respectively, compared to glucose alone. However, in the presence of 16.7 mM glucose, carbachol increased insulin secretion 2.7-fold above glucose alone, but did not increase GPI-PLD secretion.

Effects of cycloheximide on GPI-PLD and insulin secretion
In addition to secreting stored protein, newly synthesized protein can make a significant contribution to the amount of protein secreted. To determine whether new protein synthesis contributes to the GPI-PLD secreted during stimulation, ßTC3 cells were preincubated for 60 min with cycloheximide (20 µg/ml), then stimulated. Cells were stimulated with glucose or strongly stimulated (glucose, carbachol, and IBMX, as described above) in the continued presence of cycloheximide, and the medium contents of insulin, GPI-PLD, and IAPP were determined. IAPP is secreted by ßTC3 cells (26) and was included as an additional protein to monitor the effects of cycloheximide. After 4 h of stimulation, glucose (16.7 mM) stimulated GPI-PLD (74%), insulin (210%), and IAPP (320%) secretion (Table 3Go). However, in the presence of cycloheximide, glucose-stimulated release of these proteins was abolished. When cells were strongly stimulated (glucose, carbachol, and IBMX), GPI-PLD, insulin, and IAPP secretion increased 2.6-, 25-, and 16.6-fold, respectively, during a 4-h incubation. Cycloheximide inhibited this stimulation of GPI-PLD, insulin, and IAPP secretion by 25%, 40%, and 50%, respectively. These results suggest that newly synthesized protein may make a significant contribution to the increase in the medium content of GPI-PLD, insulin, and IAPP seen during ß-cell stimulation.


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of cycloheximide on GPI-PLD, insulin, and IAPP secretion from ßTC3 cells

 
Immunohistochemical localization of GPI-PLD and insulin
ßTC3 cells were examined for the presence of insulin and GPI-PLD by immunostaining. ßTC3 cells contain both insulin and GPI-PLD immunoreactivity, but the cellular content of insulin or GPI-PLD, based on the intensity of immunostaining, varied from cell to cell (Fig. 4Go, A and B). To determine whether insulin and GPI-PLD colocalize to the same cells, ßTC3 cells were double stained, and the images were merged. Using double staining, the majority of cells contained both insulin and GPI-PLD immunoreactivity (Fig. 4CGo). Both GPI-PLD and insulin immunoreactivity were located primarily in the cytosol and had a similar punctate pattern of staining. A similar pattern of punctate staining was observed with a polyclonal antibody to the C-terminal portion of human pancreatic GPI-PLD (data not shown). Moreover, immunostaining for insulin and GPI-PLD appears to occur in the same intracellular region (Fig. 4CGo). However, there are a few areas where the immunostaining does not colocalize. These results suggest that the observed differences in insulin and GPI-PLD secretion cannot be explained by separate populations of insulin- and GPI-PLD-containing cells.



View larger version (147K):
[in this window]
[in a new window]
  		Figure 4. Immunohistochemical localization of GPI-PLD and insulin. ßTC3 cells were fixed and immunostained as described in Materials and Methods. GPI-PLD-immunoreactive material is shown in A (red), insulin-immunoreactive material is shown in B (green), and the merged image is shown in C. Secondary antibodies alone are shown in D. Bar = 10 µm. Results are representative of five microscopic fields.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mechanisms for regulating the amount and activity of GPI-PLD in serum are unknown and have not been examined in part because the source of circulating GPI-PLD has not been fully determined. Pancreatic islets may contribute to the circulating GPI-PLD activity, because pancreatic islets secrete GPI-PLD (16), and the amino acid sequence of human serum GPI-PLD matches that obtained from the human pancreatic cDNA (7, 13). As an initial approach to studying GPI-PLD secretion from islets, we examined GPI-PLD secretion from ßTC3 cells, a mouse-derived insulinoma cell line. The results presented here demonstrate that ßTC3 cells secrete GPI-PLD along with insulin in response to ß-cell secretagogues.

A number of known insulin secretagogues were found to stimulate GPI-PLD secretion, including glucose, GLP-I, PMA, and IBMX. Glucose had a small effect on both insulin and GPI-PLD secretion, and it is likely that this weak effect is secondary to the loss of glucose responsiveness that occurs with passage of these cells (27). In addition, glucose potentiated the effects of GLP-I, IBMX, and PMA on insulin and GPI-PLD secretion. Carbachol stimulated insulin release in the presence of glucose, but had no significant effect on GPI-PLD secretion in the absence or presence of glucose. This apparent dissociation of insulin and GPI-PLD secretion with carbachol stimulation is not unique to GPI-PLD; previous studies suggest that carbachol may preferentially stimulate insulin release over other granular peptides (28). Alternatively, higher concentrations of carbachol may be required to induce a detectable increase in GPI-PLD secretion, because stimulating protein kinase C, a mediator of carbachol action, with PMA also increased GPI-PLD secretion. The overlap in insulin and GPI-PLD secretagogues suggests that there are common elements in the signal transduction pathways involved in mediating the secretion of these two proteins.

The overlap in secretagogues and other observations suggest that GPI-PLD may be stored and released via the regulated secretory pathway. First, agents (glucose, GLP-I, PMA, and IBMX) that stimulate insulin secretion by exocytosis of secretory granule contents also stimulate GPI-PLD secretion. A second observation consistent with secretion of a stored protein is that the cellular content of GPI-PLD activity decreases with strong stimulation concomitant with GPI-PLD activity appearing in the medium. Third, an increased medium content of GPI-PLD can be observed after as little as 5 min of strong stimulation (preliminary data), consistent with the secretion of a stored protein via the regulated secretory pathway. One possible intracellular store for GPI-PLD is the insulin secretory granule. Immunohistochemical colocalization of GPI-PLD and insulin immunoreactivity in the same intracellular region supports this suggestion, but more definitive experiments are needed to determine the subcellular localization of GPI-PLD.

A portion of the secreted GPI-PLD, insulin, and IAPP during 4 h of stimulation may derive from new protein synthesis, as cycloheximide inhibits more than 25% of the secretagogue-stimulated secretion of these proteins and completely blocks their glucose-stimulated release. This cycloheximide effect on insulin secretion in ßTC3 cells is comparable to the findings of studies in intact islets, which demonstrated that newly synthesized insulin is preferentially secreted after prolonged stimulation with glucose (29). In addition, perfusion of isolated pancreas with cycloheximide inhibits glucose-stimulated insulin secretion by 35%, but inhibition only occurs after 1–2 h of stimulation (30). The complete inhibition by cycloheximide of glucose-stimulated GPI-PLD, insulin, and IAPP release may reflect a property of ßTC3 cells to preferentially secrete newly synthesized propeptides (24, 26), as cycloheximide impairs proinsulin synthesis (31). The significant cycloheximide effect on GPI-PLD secretion in ßTC3 cells contrasts with the minor inhibitory (10–15%) effects of cycloheximide on GPI-PLD secretion from J774 cells (17) or GPI-PLD content in keratinocytes (32), suggesting that GPI-PLD turnover is much higher in ßTC3 cells. Alternatively, cycloheximide may inhibit the synthesis of a protein with rapid turnover that is essential for ß-cell protein secretion.

Although the patterns of insulin and GPI-PLD secretion are similar, the secretion of these two proteins was found to diverge under certain conditions. This divergence was most evident in the time course of secretion during strong stimulation, where the increased rate of GPI-PLD secretion was much more prolonged than that of insulin secretion. Preliminary studies indicate that this divergence becomes evident after as little 30 min of stimulation. Another difference is the increased basal rate of GPI-PLD secretion, which was over 10-fold higher than the basal insulin release based on the percentage of total cellular GPI-PLD or insulin (2.8% vs. 0.2% of the total/h). This high basal release of GPI-PLD may occur via a classical constitutive pathway (33), a hypothesis supported by our finding of a higher sorting index for insulin than GPI-PLD. Further, during strong stimulation by secretagogues, the GPI-PLD/insulin ratio in the medium decreased 5- to 10-fold compared to that under basal conditions (data not shown). The ratio of proteins secreted from ß-cells can vary (28, 34) and may reflect different granular contents (35), pools of secretory granules, or pathways of secretion (constitutive vs. regulated) (36). It is likely that with strong stimulation, secretion reflects predominantly release from granules in the regulated pathway; the decrease in the GPI-PLD/insulin ratio with stimulation suggests that GPI-PLD is less efficiently sorted toward this pathway. Alternatively, the dissociation of GPI-PLD and insulin secretion might also be due to the secretion of GPI-PLD via a constitutive-like pathway of secretion that has been described in intact islets and derives from vesicle budding off immature secretory granules (37, 38) or may reflect an abnormal secretory characteristic of ßTC3 cells exhibited by the preferential secretion of proinsulin (24).

It is difficult to extrapolate the results in ßTC3 cells to intact islets and nontransformed ß-cells primarily because of the abnormal secretory characteristics ßTC3 cells exhibit compared to intact islets. In addition, the cell type(s) within intact islets that secretes GPI-PLD is still unknown. However, the present data suggest that ß-cells may be a contributing source of circulating GPI-PLD. In support of this, another ß-cell line (HIT) also secretes GPI-PLD in response to glucose and other insulin secretagogues (our preliminary data). However, other islet cell types may also secrete GPI-PLD, as we found that two glucagon-secreting cell lines ({alpha}TC6 and HIP) secrete GPI-PLD as well (preliminary data).

The function and site of action of GPI-PLD are still unclear. Is it possible that GPI-PLD is active within the ß-cell secretory granule? GPI-PLD has enyzmatic properties (e.g. calcium and pH dependence) that would allow GPI-PLD to be active within the mature granule (10, 39, 40). Interestingly, GPI-anchored proteins have been identified in pancreatic acinar (41) and chromaffin secretory granules (42) and in {alpha}- and ß-cells (43), but GPIs have not yet been identified in islet secretory granules; this remains one interesting possible sight of action for GPI-PLD. As these studies suggest that GPI-PLD secretion can be regulated from ß-cells, one might also speculate a role for GPI-PLD and GPI metabolism in nutrient utilization. Further characterization of GPI-PLD secretagogues and GPIs in islets may provide additional clues to the function and regulation of GPI-PLD.


   Acknowledgments
 
The authors thank Jeanette Teague and Chare Vathanaprida for expert technical assistance, Dr. Alain Baron for critical review of the manuscript, Dr. Steven Kahn for IAPP assays, and Drs. Denis Baskin and Reuben Sandoval for assistance with immunostaining and confocal imagining, respectively.


   Footnotes
 
1 This work was supported by a postdoctoral fellowship from the Medical Research Council of Canada (to C.B.V.), a Diabetes New Investigator Award from the Diabetes Research Center, University of Washington (NIH DK-17047, to D. Porte, Jr.), the Diabetes Research Council (Seattle, WA), and an American Diabetes Association Career Development Award (to M.A.D.). A portion of this work was performed while M.A.D. was a Pfizer Postdoctoral Fellow at the University of Washington under the direction of Edwin L. Bierman, M.D. Back

Received June 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Englund PT 1993 The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu Rev Biochem 62:121–138[CrossRef][Medline]
  2. Brown D, Waneck GL 1992 Glycosyl-phosphatidylinositol-anchored membrane proteins. J Am Soc Nephrol 3:895–906[Abstract]
  3. Nakano Y, Noda K, Endo T, Kobata A, Tomita M 1994 Structural study on the glycosyl-phosphatidylinositol anchor and the asparagine-linked sugar chain of a soluble form of CD59 in human urine. Arch Biochem Biophys 311:117–126[CrossRef][Medline]
  4. Solter PF, Hoffmann WE 1995 Canine corticosteroid-induced alkaline phosphatase in serum was solubilized by phospholipase activity in vivo. Am J Physiol 269:G278–G286
  5. Mensa-Wilmot K, Englund PT 1992 Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli. Mol Biochem Parasitol 56:311–322[CrossRef][Medline]
  6. Scallon BJ, Fung W-JC, Tsang TC, Li S, Kado-Fong H, Huang K-S, Kochan JP 1991 Primary structure and functional activity of a phosphatidylinositol-glycan-specific phospholipase D. Science 252:446–448[Abstract/Free Full Text]
  7. Tsang TC, Fung WJ, Levine J, Metz CN, Davitz MA, Burns DK 1992 Isolation and expression of two human glycosylphosphatidylinositol phospholipase D (GPI-PLD) cDNAs. FASEB J 6:A1922
  8. Davitz MA, Hom J, Schenkman S 1989 Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma. J Biol Chem 264:13760–13764[Abstract/Free Full Text]
  9. Huang K-S, Li S, Fung W-JC, Hulmes JD, Reik L, Pan Y-CE, Low MG 1990 Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D. J Biol Chem 265:17738–17745[Abstract/Free Full Text]
  10. Deeg MA, Davitz MA 1994 Structure and function of the glycosylphosphatidylinositol-specific phospholipase D. In: Liscovitch M (ed) Signal-Activated Phospholipases. Landes, Austin, pp 125–138
  11. Low MG, Huang K-S 1991 Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the membrane anchors of cell surface proteins. Biochem J 279:483–493
  12. Bergman A-S, Carlsson SR 1994 Saponin-induced release of cell-surface-anchored Thy-1 by serum glycosylphosphatidylinositol-specific phospholipase D. Biochem J 298:661–668
  13. Hoener MC, Brodbeck U 1992 Phosphatidylinositol-glycan-specific phospholipase D is an amphiphilic glycoprotein that in serum is associated with high-density lipoproteins. Eur J Biochem 206:747–757[Medline]
  14. Deeg MA 1994 GPI-specific phospholipase D as an apolipoprotein. Brazil J Med Biol Res 27:375–381[Medline]
  15. Hoener MC, Golli R, Brodbeck U 1993 Glycosyl-phosphatidylinositol-specific phospholipase D: interaction with and stimulation by apolipoprotein A-I. FEBS 327:203–206[CrossRef][Medline]
  16. Metz CN, Zhang Y, Guo Y, Tsang TC, Kochan JP, Altszuler N, Davitz MA 1991 Production of the glycosylphosphatidylinositol-specific phospholipase D by the islets of Langerhans. J Biol Chem 266:17733–17736[Abstract/Free Full Text]
  17. Xie M, Low MG 1994 Expression and secretion of glycosylphosphatidylinositol-specific phospholipase D by myeloid cell lines. Biochem J 297:547–554
  18. Efrat S, Linde S, Kofod H, Spector D, Delannoy M, Grant S, Hanahan D, Baekkeskov S 1988 Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci USA 85:9037–9041[Abstract/Free Full Text]
  19. D’Ambra R, Surana M, Efrat S, Starr RG, Fleischer N 1990 Regulation of insulin secretion from ß-cell lines derived from transgenic mice insulinomas resembles that of normal ß-cells. Endocrinology 126:2815–2822[Abstract/Free Full Text]
  20. Fujimoto WY, Teague J 1977 Fibroblast monolayer cultures in scintillation counting vials: metabolic and growth experiments using radioisotopes and a microfluorometric DNA assay. In Vitro 13:237–244[Medline]
  21. Zaharko DS, Beck LV 1968 Studies of a simplified insulin immunoassay using cellulose powder. Diabetes 17:444–457[Medline]
  22. Kahn SE, D’Alessio DA, Schwartz MW, Fujimoto WY, Ensinck JW, Taborsky Jr GJ, Porte Jr D 1990 Evidence of cosecretion of islet amyloid polypeptide and insulin by ß-cells. Diabetes 39:634–638[Abstract]
  23. Metz CN, Thomas P, Davitz MA 1992 Immunolocalization of a glycosylphosphatidylinositol-specific phospholipase D in mast cells found in normal tissue and neurofibromatosis lesions. Am J Pathol 140:1275–1281[Abstract]
  24. Nagamatsu S, Steiner DF 1992 Altered glucose regulation of insulin biosynthesis in insulinoma cells: mouse ßTC3 cells secrete insulin-related peptides predominantly via a constitutive pathway. Endocrinology 130:748–754[Abstract/Free Full Text]
  25. Schmidt WK, Moore H-PH 1994 Synthesis and targeting of insulin-like growth factor-I to the hormone storage granules in an endocrine cell line. J Biol Chem 269:27115–17124[Abstract/Free Full Text]
  26. Nagamatsu S, Nishi M, Steiner DF 1991 Biosynthesis of islet amyloid polypeptide: elevated expression in mouse ßTC3 cells. J Biol Chem 266:13737–13741[Abstract/Free Full Text]
  27. Knaack D, Fiore DM, Surana M, Leiser M, Laurance M, Fusco-DeMane D, Hegre OD, Fleischer N, Efrat S 1994 Clonal insulinoma cell line that stably maintains correct glucose responsiveness. Diabetes 43:1413–1417[Abstract]
  28. Stridsberg M, Sandler S, Wilander E 1993 Cosecretion of islet amyloid polypeptide (IAPP) and insulin from isolated rat pancreatic islets following stimulation or inhibition of ß-cell function. Regul Pept 45:363–370[CrossRef][Medline]
  29. Rhodes CJ, Halban PA 1987 Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway. J Cell Biol 105:145–153[Abstract/Free Full Text]
  30. Curry DL 1971 The effects of cycloheximide and 2-deoxyglucose on the diphasic pattern of insulin secretion. Acta Diabetol Lat 8:48–65[CrossRef][Medline]
  31. Sando H, Borg J, Steiner DF 1972 Studies on the secretion of newly synthesized proinsulin and insulin from isolated rat islets of Langerhans. J Clin Invest 51:1476–1485
  32. Xie M, Sesko AM, Low MG 1993 Glycosylphosphatidylinositol-specific phospholipase D is localized in keratinocytes. Am J Physiol 265:C1156–C1168
  33. Kelly RB 1985 Pathways of protein secretion in eukaryotes. Science 230:25–32[Abstract/Free Full Text]
  34. Guest PC, Pipeleers D, Rossier J, Rhodes CJ, Hutton JC 1989 Co-secretion of carboxypeptidase H and insulin from isolated rat islets of Langerhans. Biochem J 264:403–508[Medline]
  35. Johnson KH, O’Brien TD, Betsholtz C, Westermark P 1992 Islet amyloid polypeptide: mechanisms of amyloidogenesis in the pancreatic islets and potential roles in diabetes mellitus. Lab Invest 66:522–535[Medline]
  36. Kahn SE, Verchere CB, D’Alessio DA, Cook DL, Fujimoto WY 1993 Evidence for selective release of rodent islet amyloid polypeptide through the constitutive secretory pathway. Diabetologia 36:570–573[Medline]
  37. Arvan P, Kuliawat R, Prabakaran D, Zavacki A-M, Elahi D, Wang S, Pilkey D 1991 Protein discharge from immature secretory granules displays both regulated and constitutive characteristics. J Biol Chem 266:14171–14174[Abstract/Free Full Text]
  38. Kuliawat R, Arvan P 1992 Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment. J Cell Biol 118:521–529[Abstract/Free Full Text]
  39. Heller M, Bütikofer P, Brodbeck U 1994 Generation by limited proteolysis of a catalytically active 39-kDa protein from the 115-kDa form of phosphatidylinositol-glycan-specific phospholipase D from bovine serum. Eur J Biochem 224:823–833[Medline]
  40. Li JY, Hollfelder K, Huang KS, Low MG 1994 Structural features of GPI-specific phospholipase D revealed by proteolytic fragmentation and Ca2+ binding studies. J Biol Chem 269:28963–28971[Abstract/Free Full Text]
  41. Scheele GA, Fukuoka S-I, Freedman SD 1994 Role of the GP2/THP family of GPI-anchored proteins in membrane trafficking during regulated exocrine secretion. Pancreas 9:139–149[Medline]
  42. Fouchier F, Bastiani P, Baltz T, Aunis D, Rougon G 1988 Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells. Biochem J 256:103–108[Medline]
  43. Kajimoto Y, Miyagawa J-i Ishihara K, Okuyama Y, Fujitani Y, Itoh M, Yoshida H, Kaisho T, Matsuoka T-a, Watada H, Hanafusa T, Yamasaki Y, Kamada T, Matsuzawa Y, Hirano T 1996 Pancreatic islet cells express BST-1, a CD38-like surface molecule having ADP-ribosyl cyclase activity. Biochem Biophys Res Commun 219:941–946[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
N. S. Raikwar, W. K. Cho, R. F. Bowen, and M. A. Deeg
Glycosylphosphatidylinositol-specific phospholipase D influences triglyceride-rich lipoprotein metabolism
Am J Physiol Endocrinol Metab, March 1, 2006; 290(3): E463 - E470.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
M. A. Deeg, R. F. Bowen, M. D. Williams, L. K. Olson, E. A. Kirk, and R. C. LeBoeuf
Increased expression of GPI-specific phospholipase D in mouse models of type 1 diabetes
Am J Physiol Endocrinol Metab, July 1, 2001; 281(1): E147 - E154.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
X. Du and M. G. Low
Down-Regulation of Glycosylphosphatidylinositol-Specific Phospholipase D Induced by Lipopolysaccharide and Oxidative Stress in the Murine Monocyte- Macrophage Cell Line RAW 264.7
Infect. Immun., May 1, 2001; 69(5): 3214 - 3223.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Deeg, M. A.
Right arrow Articles by Verchere, C. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Deeg, M. A.
Right arrow Articles by Verchere, C. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals