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Endocrinology, doi:10.1210/en.2003-0719
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*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CYCLOHEXIMIDE
*GLUCOSE
*PALMITIC ACID
*SODIUM PALMITATE
*TRITIUM
Endocrinology Vol. 144, No. 12 5232-5241
Copyright © 2003 by The Endocrine Society

Nutrient Modulation of Palmitoylated 24-Kilodalton Protein in Rat Pancreatic Islets

Satoko Yamada, Mitsuhisa Komatsu, Yoshihiko Sato, Keishi Yamauchi, Toru Aizawa and Itaru Kojima

Institute for Molecular and Cellular Regulation (S.Y., I.K.), Gunma University, Maebashi 371-8512, Japan; Department of Aging Medicine and Geriatrics (M.K., Y.S., K.Y.), School of Medicine, and Center for Health Services (T.A.), Shinshu University, Matsumoto 390-8621, Japan

Address all correspondence and requests for reprints to: Itaru Kojima, M.D., Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan. E-mail: ikojima{at}showa.gunma-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein acylation in glucose stimulation of insulin secretion in the ß-cells has been implicated. Accordingly, we attempted to identify the target(s) of acylation in the pancreatic islets. Rat pancreatic islets were labeled with [3H]palmitic acid for 1 h at 37 C, and the whole cell lysate was analyzed by SDS-PAGE and two-dimensional gel electrophoresis. The labeling of the proteins by [3H]palmitic acid was shown to be palmitoylation by chemical analyses. Palmitoylation of four distinct bands was recognized, and the palmitoylation was significantly reduced in all of them when the labeling was performed with high glucose. Quite interestingly, the degree of attenuation was particularly dominant for a 24-kDa doublet. Palmitoylation of the 24-kDa doublet was preferentially attenuated also by the mitochondrial fuels and an acylation inhibitor, cerulenin. The half-life of the labeling of the doublet was apparently shorter (approximately 45 min) than that of other bands on pulse chasing of the islets, irrespective of the presence or absence of high glucose. High glucose attenuation of the palmitoylation of the 24-kDa doublet was partially blocked by 20 mM mannoheptulose, a glucokinase inhibitor. Two-dimensional gel electrophoresis revealed that the doublet was composed of acidic peptides, and, by immunoprecipitation, it was shown not to be synaptosome-associated protein of 25 kDa. We identified rapidly turning over palmitoylated 24-kDa acidic proteins distinct from synaptosome-associated protein of 25 kDa in the pancreatic islets, which are preferentially modulated by fuel secretagogues. The data suggested a functional role of the palmitoylated 24-kDa doublet in nutrient stimulation of insulin secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MECHANISM OF glucose-induced insulin secretion by the pancreatic ß-cell has been extensively studied. Nutrients including glucose stimulate insulin release both via ATP-sensitive potassium (KATP) channel-dependent and -independent pathways (1, 2, 3, 4). The KATP channel-dependent signal triggers insulin release, leading to first-phase insulin release. Concomitantly, glucose causes expansion of the readily releasable pool of insulin, which is conceptually called potentiation (1), augmentation (2), or amplification (3), through the KATP channel-independent pathway(s). The latter leads to generation of a gradually increasing second phase (1, 2, 3, 4), which is a salient feature of glucose-induced insulin secretion by the pancreatic ß-cells.

The molecular mechanism of KATP channel-dependent glucose signaling has been well delineated (5); however, little is known about the molecular mechanism of the KATP channel-independent pathway. In previous studies, the role of ATP (6), GTP (7), glutamate (8), citrate (9), long-chain-acyl CoA (LC-CoA) (10), phospholipase A2 (11), nicotinamide adenine dinucleotide hydroxide shuttles (12), and protein kinases, especially protein kinases A and C (13) has been suggested but not without dispute (14, 15, 16, 17, 18). Nevertheless, substantial supportive data for the role of LC-CoA have been accumulated by us and others (10, 19, 20, 21, 22, 23, 24, 25, 26, 27). Namely, exposure of the ß-cells to a high concentration of glucose results in the elevation of cytosolic citrate, which is converted to malonyl CoA. Malonyl CoA suppresses carnitine palmitoyl transferase l, a rate-limiting enzyme for incorporation of LC-CoA into the mitochondria for oxidation (10, 19, 20, 21). Thus, glucose stimulation of the pancreatic ß-cell causes an accumulation of cytosolic LC-CoAs, which may function as signaling molecules to enhance insulin release (10, 19, 20, 21). Exogenous application of a micromolar concentration of free fatty acids (FFAs) at least partially mimics the KATP channel-independent effects of nutrients (22, 23), and an extracellular application of LC-CoA stimulates insulin release in permeabilized ß-cells by facilitating fusion of secretory granules (24). Furthermore, the enhancing effects of FFAs and LC-CoA on insulin release are inhibited by cerulenin, a protein acylation inhibitor (24, 25, 26). Cerulenin and tunicamycin, another class of acylation inhibitor, selectively attenuates nutrient stimulation of insulin release (24, 25, 26, 27). Protein acylation is known to play a key role in regulating cellular structure and functions (28, 29, 30). In general, protein palmitoylation is considered to facilitate protein-membrane interaction because of high affinity of the fatty acid toward the lipid bilayer (28, 29, 30). Reversibility of protein palmitoylation favors its role in signal transduction.

Based on these observations, we considered protein fatty acylation (28, 29, 30) as the key event in the KATP channel-independent glucose action. Accordingly, the present study was conducted to search for target protein(s) of acylation in rat pancreatic islets in which the KATP channel-independent glucose action is most prominent. We here describe palmitoylated 24-kDa proteins that are modulated by nutrients and acylation inhibitors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of pancreatic islets
Pancreatic islets were isolated from adult male Wistar rats weighing 250–450 g by collagenase dispersion as previously reported (31). For isolation and pooling of the islets, Krebs-Ringer bicarbonate (KRB) buffer supplemented with 0.1% fatty acid-free BSA containing (in mM) 129 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 5 NaHCO3, 5.5 glucose, and 10 HEPES/NaOH (pH 7.4) was used. KRB buffer containing 0.1% fatty acid-free BSA and the indicated concentrations of glucose was used for the experiments. KRB buffer containing 2.8 mM glucose was defined as basal KRB buffer in this study. The experimental protocols were approved by the Ethical Committee of the Institute for Molecular and Cellular Regulation, Gunma University.

Metabolic labeling of the islets with [3H]palmitic acid
Freshly isolated islets were picked up manually under the operating microscope with care to minimize contamination of the exocrine tissue, and 50–80 size-matched islets were placed into 5 ml RIA tubes. The islets were preincubated in 1 ml basal KRB buffer at 37 C for 30 min. After the preincubation, the rack (with all the tubes) was placed in ice-cold water and the following procedures were undertaken. Namely, 950 µl of the buffer was removed, and 130 µl labeling buffer and 20 µl basal KRB buffer with or without test substances were introduced. Incubation of a larger number of islets per indicated volume of buffer was associated with an obliteration of glucose-induced insulin secretion and therefore was avoided.

The labeling buffer was prepared as follows: first, 40 µl (200 µCi) [3H]palmitic acid (5 mCi/ml, 60 Ci/mmol in ethanol) was placed into a polystyrene tube, and ethanol was evaporated at room temperature under N2 stream. Then [3H]palmitic acid was reconstituted with 130 µl basal KRB buffer containing 0.2% ethanol and vortexed vigorously. When several labeling conditions were compared in parallel using one batch of the islets, labeling buffer for all conditions was prepared simultaneously in one tube to retain the specific activity constant among the labeling conditions. Solubilization and recovery of [3H]palmitic acid were ascertained by counting the radioactivity of the labeling buffer immediately before the labeling during each experiment. Finally, the total concentration of [3H]palmitic acid in the labeling buffer was 17 µM. Because most [3H]palmitic acid is bound to albumin in the solution, the free (nonalbumin-bound) form of [3H]palmitic acid was estimated from the molar ratio of FFAs and BSA as previously reported (32), as less than 0.1 µM. Obviously, this level of [3H]palmitic acid failed to augment any insulin release in these experiments (data not shown).

The labeling was performed by incubating the islets in the labeling buffer with and without test substances in every case for 1 h at 37 C with gentle shaking. At the end of the labeling, the supernatant was collected and kept at -20 C for later determination of insulin. The islets were washed twice with ice-cold BSA-free KRB buffer, centrifuged briefly (<1000 rpm), and dissolved in 10 µl 2x Laemmli sample buffer [125 mM Tris-HCl (pH 6.8), 4% sodium dodecyl sulfate (SDS), 20% glycerol] without reducing agents (33). Protein concentration was determined by bicinchonic acid protein assay method, and 25–50 µg of protein-equivalent lysate was loaded onto 10–20% gradient SDS-PAGE minigels or 12.5% SDS-PAGE gels. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was dried and then exposed to a BAS imaging plate. After 3–7 d of exposure, the plates were processed by a BAS 2000 system (Fuji Photo Film, Tokyo, Japan). In preliminary experiments, chloroform/methanol (2:1, vol/vol) treatment of the lysate, which removes noncovalently bound fatty acids, resulted in no detectable loss of labeling. We therefore omitted this procedure in the following experiments.

INS-1 cells were maintained in RPMI 1640 culture media as reported (34). After washing with PBS and preincubation in basal KRB buffer, the cells were labeled with the labeling buffer (1 mCi/ml [3H]palmitic acid, 2.8 glucose, see above) for 1 h, and whole-cell lysate was analyzed by SDS-PAGE.

When cerulenin or cycloheximide (CHX) was used, it was included from the beginning of preincubation and throughout the labeling period. All other test substances were included only during the labeling period.

Chemical analysis of the labeling
[3H]palmitic acid per se can bind to proteins via oxyester or thioester linkage (35), and [3H]myristic acid generated as a result of intracellular conversion of [3H]palmitic acid can bind to proteins through an amide bond (35). To ascertain that the labeling of the proteins by the tracer detected in the islet lysate was palmitoylation, which is a thioester bond, the following analytical procedures were undertaken (35). In this experiment, the labeling of the islets was performed in basal KRB buffer.

First, to distinguish amide and ester bonds, the membrane was treated with alkaline solutions: 0.1 M KOH in methanol (for 4 h) or alkaline 0.5 M hydroxylamine in 2-propanol/water (1:1, vol/vol, pH 9.0–11.0) (for 12 h) at room temperature. The amide bond is resistant and the ester bond is sensitive to alkaline treatments (35). Oxyester and thioester linkages were distinguished by the following method: The transferred membrane was treated with neutral 1 M hydroxylamine in 2-propanol/water (1:1, vol/vol, pH 7.4) for 10 h at room temperature and then rinsed four times with 200 ml 2-propanol/water (1:1, vol/vol). The thioester but not the oxyester bond was cleaved by the neutral hydroxylamine treatment (35). For the control of this procedure, treatment of the membrane with 1 M Tris in 2-propanol/water (1:1, vol/vol, pH 7.4) for 10 h at room temperature was also performed. To verify that proteins were not significantly removed by the above-mentioned procedures, membranes were stained with Coomassie Brilliant Blue after each treatment. In addition, dithiothreitol (DTT) sensitivity of the labeling was tested because the thioester bond is known to be sensitive to reducing agents (35). To this end, 25–200 mM DTT was added to the lysate and incubated at 37 C for 2 h before electrophoresis.

Pulse chasing of the proteins
To examine the time course of change in the labeling intensity of proteins, the same batch of islets was divided into 10 groups and labeled with [3H]palmitic acid simultaneously in basal KRB buffer. The islets were then chased by incubation in KRB buffer without [3H]palmitic acid containing 2.8 or 22.2 mM glucose for 0, 15, 30, 60, and 120 min at 37 C.

CHX treatment of the islets
The islets were preincubated in KRB buffer with and without 10 µM CHX for 1 h at 37 C. Subsequently, the labeling was performed for 1 h as described above in the presence of 2.8 mM glucose and in the continuous presence or absence of CHX. Accordingly, the islets were exposed to CHX for a total of 2 h, if it was present. It has been reported that protein synthesis of the islet cells is inhibited by more than 95% in such a procedure (36).

Immunoprecipitation
All of the following procedures were carried out at 4 C. Two hundred labeled islets were washed twice and rotated for 90 min in 100 µl of lysis buffer containing 20 mM HEPES (pH 7.4), 1% Triton X-100, 0.2% Nonidet P-40, 2 mM EDTA (pH 7.4), 2 mM EGTA, 10 mM sodium pyrophosphate, 0.1% SDS, 0.1% deoxycholic acid, 100 mM sodium fluoride, 2 mM sodium orthovanadate, and protease inhibitors (10 µg/ml aprotinin, 5 µg/ml leupeptin, 1.5 mM pepstatin, and 1 mM phenylmethylsulfonyl fluoride). The lysate was centrifuged for 15 min at 15,000 rpm. For the supernatants, 4 µg of antisynaptosome-associated protein of 25 kDa (SNAP-25) antibody (BR05) (37) and precipitation buffer containing 20 mM Tris (pH 7.4), 3 mM MgCl2, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl, 0.1% Triton X-100, 0.1% Nonidet P-40, 1 mM sodium orthovanadate, and 0.2 mM phenylmethylsulfonyl fluoride were added and incubated for 120 min with constant rotation. Thereafter, 30 µl Protein A Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) was added and incubated for 60 min. The immunoprecipitate was washed three times with the precipitation buffer, and protein bound to IgG was eluted from Protein A Sepharose with 15 µl 2x Laemmli sample buffer without reducing agents.

Two-dimensional gel electrophoresis
One hundred fifty to 200 labeled islets were washed three times in ice-cold BSA-free KRB buffer and dissolved with 40 µl of immobilized pH-gradient isoelectric focusing (IPG-IEF) lysis buffer containing 8 M urea, 0.5% Pharmalyte 3–10 (Amersham Pharmacia Biotech), 2.5 mM acetic acid, and 0.0025% orange G. The lysate was centrifuged for 5 min at 14,000 rpm at room temperature, and the supernatant was applied onto an immobiline DryStrip (pH 3-10NL, 13 cm, Amersham Pharmacia Biotech) for IPG-IEF (CoolPhoreStar IPG-IEF, Anatech, Tokyo, Japan). The voltage was 500 V during the initial 60 min, 1000 V during the following 60 min, and subsequently 3000 V for overnight at 15 C. After the IPG-IEF, the strip was equilibrated with 5 ml of SDS-equilibration buffer containing 6 M urea, 0.05 M Tris (pH 6.8), 2% (wt/vol) SDS, 25% (vol/vol) glycerol, and 0.0025% bromophenol blue for 20 min. Second dimensional separation was carried out by SDS-PAGE using vertical slab 12.5% gels (130 x 130 mm). Two SDS-PAGE gels were prepared in parallel: one was transferred to a PVDF membrane and exposed to a BAS plate for 2–4 wk for detection of radioactivity, and the other was used for visualization of protein spots by silver staining.

Measurement of insulin
Insulin was measured by RIA using commercially available kits (Eiken, Tokyo, Japan) with rat insulin as a standard (22, 23).

Materials
Fatty acid-free BSA, cerulenin, and CHX were obtained from Sigma (St. Louis, MO). [3H]palmitic acid (NET-043) was purchased from NEN Life Science Products (Boston, MA), reagents for protein determination by bicinchonic acid protein method from Pierce (Rockford, IL), 10–20% gradient SDS-PAGE minigels (Ready Gel) and 2D-silver staining kits from Daiichikagaku (Tokyo, Japan), PVDF membranes from Millipore (Bedford, MA), and the BAS imaging plate from Fuji Photo Film. Mannoheptulose was obtained from Funakoshi (Tokyo, Japan) and CBB-staining kits from Wako (Tokyo, Japan). Mouse anti-SNAP-25 monoclonal antibody (BR05) was a generous gift from Dr. Masami Takahashi (36) (University of Kitazato, Kanagawa, Japan). RPMI 1640 was purchased from Invitrogen (Grand Island, NY). Fetal bovine albumin was purchased from JRH Biosciences (Lenexa, KS). INS-1 cells were kind gift from Dr. Claes B. Wollheim (University of Geneva, Switzerland).

Statistical analysis
We quantified the change in the density of four major palmitoylated bands, i.e. the 24-kDa doublet band (indicated as "a" in Figs. 2–5GoGoGoGo), the 30-kDa band (indicated as "b" in Figs. 2–5GoGoGoGo), the 37–50 kDa broad-smear bands(indicated as "c" in Figs. 2–5GoGoGoGo), and the 70-kDa band (indicated as "d" in Figs. 2–5GoGoGoGo). Densitometry of the BAS imaging was performed by MacBAS version 2.3.1 (Fuji Photo Film). The labeling in the presence of 2.8 mM glucose was defined as the control, and the labeling in other conditions were regarded as the test. The density of the band in the test condition was divided by that in the control condition so that the ratio of the test/control was calculated for each protein band. An exception for this was the densitometric analysis in the pulse-chase experiment, in which the density at the beginning of the chase was taken as the denominator for each protein band, irrespective of the extracellular glucose concentration (see Results). Statistical analysis was performed by one-way ANOVA, with pairwise comparisons by Bonferroni/Dunn test (StatView, SAS Institute, Inc., Cary, NC). P < 0.05 was considered significant. Data are expressed as the mean ± SE.



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  		FIG. 2. Effects of sucrose and high glucose on the labeling of pancreatic islets with [3H]palmitic acid. Groups of islets were labeled in parallel with [3H]palmitic acid in the presence of 2.8 mM glucose or 2.8 mM glucose plus 22.2 mM sucrose (A), or 2.8, 11.1, 16.7, and 22.2 mM glucose (B). A and B, Radiolabeling of the whole-cell lysates. Representative results from two (A) or three (B) independent experiments with the same protocol are shown. Four labeling bands identified consistently and analyzed by densitometry were indicated as a–d. C, Insulin release during the 1-h labeling period (means ± SE) from the three experiments of B. Absolute value for basal insulin secretion was 34 + 2.2 pg/islet·60 min, which was low, compared with the values in regular insulin release experiments. This most likely is due to a large number of islets (n = 50) incubated in a small volume of buffer (200 µl). See Materials and Methods for details.

 


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  		FIG. 3. Effects of cerulenin on the labeling with [3H]palmitic acid. The islets were labeled with [3H]palmitic acid in the presence of test substances shown on the abscissa. A, Effects of cerulenin on the labeling in the presence of 22.2 mM glucose. B, Insulin release during the labeling period (n = 3, means + SE). Absolute value for basal insulin secretion was 42 + 2.4 pg/islet·60 min. C, Effects of cerulenin on the labeling in the presence of low and high concentration of glucose. In A and C, representative results from five independent experiments are shown. Four labeling bands identified consistently and analyzed by densitometry were indicated as a–d. See Materials and Methods for details.

 


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  		FIG. 4. Effects of mitochondrial fuels (A) and metabolic inhibition (B) on the labeling with [3H]palmitic acid. The labeling was performed in the presence of test substances indicated on the abscissa. Representative results from six (A) and five (B) independent experiments with the same protocol are shown. Four labeling bands identified consistently and analyzed by densitometry were indicated as a–d. See Materials and Methods for details.

 


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  		FIG. 5. Kinetics of turnover of the labeling with [3H]palmitic acid. A and B, The islets were labeled in the presence of 2.8 mM glucose and then chased with KRB buffer without [3H]palmitic acid containing 2.8 (A, left half) or 22.2 (A, right half) mM glucose for the indicated time intervals. Representative data from three independent experiments with the same protocol are shown in A. Quantification of change of the density of 24-kDa doublet during the chase period by densitometry is shown in B (means ± range from the three independent experiments). C, The islets were labeled with [3H]palmitic acid in the presence or absence of 10 µM CHX in 2.8 mM glucose. Four labeling bands identified consistently and analyzed by densitometry were indicated as a–d. See Materials and Methods for details.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Chemical analysis of the labeling
To identify the nature of protein labeling with added [3H]palmitic acid, we first examined whether the labeling was amide or ester bond. All of the labeling of the proteins was removed by the treatment of PVDF membrane with alkaline (Fig. 1AGo, b) or alkaline hydroxylamine (Fig. 1AGo, c), indicating that the labeling of the protein was via ester linkage. Next, we examined whether the ester linkage was oxyester or thioester linkage. Because the labeling was removed by neutral hydroxylamine (Fig. 1AGo, d) and DTT (Fig. 1BGo) treatment, it was judged to be thioester linkage. The results of the chemical analyses thus indicated that the labeling of proteins with [3H]palmitic acid was via thioester linkages, which is palmitoylation (35).



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  		FIG. 1. Chemical analysis of the labeling with [3H]palmitic acid. The islets were labeled with [3H]palmitic acid in the presence of 2.8 mM glucose. Labeled proteins were separated by the gradient SDS-PAGE gel and transferred onto a PVDF membrane. A, The membrane was cut into four pieces and soaked in four different reagents. A-a, 1 M Tris (pH 7.4) in 2-propanol/water; A-b, 0.1 M KOH/methanol; A-c, 0.5 M hydroxylamine in 2-propanol/water (pH 9.0–11.0); A-d, 1 M hydroxylamine in 2-propanol/water (pH 7.4). B, The lysate was incubated with increasing concentrations of DTT for 2 h at 37 C before electrophoresis. The arrow indicates the 24-kDa protein for which we focused the attention in the following series of experiments. Representative results from two independent experiments are shown for both A and B. See Materials and Methods for details.

 
Modulation of the palmitoylation by glucose
When the labeling was performed in basal KRB buffer (2.8 mM glucose), the palmitoylation of several bands occurred (Fig. 2AGo). Among them, the palmitoylation of the 24-kDa doublet band (indicated as band a), 30-kDa band (band b), 37- to 50-kDa broad-smear bands (band c), and 70-kDa band (band d) was consistently recognized. The palmitoylation was attenuated for all four of the bands when the labeling was performed in the presence of high concentrations (11.1, 16.7, and 22.2 mM) of glucose (Fig. 2BGo). Attenuation of the palmitoylation was not observed when the labeling was conducted in the presence of 22.2 mM sucrose, implying that the modulation of palmitoylation by high glucose was not simply an osmotic effect (Fig. 2AGo).

Quantitative analysis of the glucose effect was performed by densitometry using the data from 16 independent labeling experiments. In each of the 16 experiments, the same batch of islets were used, 2.8 and 22.2 mM glucose were included as the labeling conditions, and the cells were processed after labeling and the SDS-PAGE ran in parallel. The density of a certain band obtained during labeling in the presence of 22.2 mM glucose was divided by the density obtained during labeling in 2.8 mM glucose (see Materials and Methods). The ratio was calculated for each of the four major palmitoylated bands, and the values were 0.44 ± 0.06 for the 24-kDa doublet bands (band a), 0.66 ± 0.03 for the 30-kDa band (band b), 0.66 ± 0.04 for the 37- to 50-kDa broad-smear bands (band c), and 0.62 ± 0.07 for the 70-kDa band (band d). The ratio was significantly smaller (implying greater attenuation by high glucose) for the 24-kDa doublet (band a): P = 0.0047 vs. 30 kDa (band b), P = 0.0039 vs. 37–50 kDa (band c), and P = 0.0207 vs. 70 kDa (band d). Glucose stimulation of insulin release was clearly detected in the supernatant of the labeling buffer (Fig. 2CGo), implying that attenuation of the palmitoylation did occur in association with increased insulin secretion from the islets.

Recovery of cell-associated [3H]palmitic acid at the end of incubation was not significantly affected by exposure of the islets to high glucose (data not shown), indicating that a high concentration of glucose did not affect uptake of palmitic acid.

Effects of cerulenin, an acylation inhibitor, on the palmitoylation
We previously reported that cerulenin, a protein acylation inhibitor, selectively suppressed nutrient-induced insulin release and glucose-induced time-dependent potentiation (25, 26, 27). Therefore, the effects of cerulenin on the palmitoylation of the proteins were examined. Palmitoylation of the 24- and 70-kDa bands were clearly attenuated by 30 and 100 µg/ml cerulenin (Fig. 3AGo). But the palmitoylation of the 30-kDa bands and 37- to 50-kDa broad bands was less sensitive to cerulenin (Fig. 3AGo). The attenuation of the palmitoylation of the 24- and 70-kDa bands was associated with suppression of glucose stimulation of insulin release (Fig. 3BGo). It should be mentioned that the changes in the 24-kDa bands under 30 and 100 µg/ml cerulenin were statistically significant, compared with those of other bands (P < 0.0001, compared with the 30-, 37- to 50-, and 70-kDa bands, n = 5). Even when the labeling was performed in the basal KRB buffer with 2.8 mM glucose, cerulenin inhibited the palmitoylation of the 24-kDa doublets and 70-kDa bands (Fig. 3CGo) in the same manner. The effect of tunicamycin, which competitively inhibits protein acylation on the palmitoylation, was similar to that of cerulenin. Namely, an exposure to tunicamycin (30 and 100 µg/ml for 1 h) attenuated the palmitoylation of the 24-kDa doublet (data not shown).

Effects of mitochondrial fuels and mannoheptulose, a glucokinase inhibitor, on the palmitoylation
Fuel metabolism in the pancreatic ß-cell is indispensable for nutrient-induced insulin release (1, 2, 3, 4). Accordingly, dependency of the palmitoylation of the proteins on metabolic flux was examined by substitution of glucose with mitochondrial fuels and inhibition of glucose metabolism.

As a mitochondrial fuel, 20 mM {alpha}-ketoisocaproic acid (KIC) was employed. A combination of 10 mM glutamine and 2 mM leucine was also tested (38). Quite interestingly, 20 mM KIC selectively inhibited the palmitoylation of the 24-kDa doublet (2.8 mM glucose plus 20 mM KIC/2.8 mM glucose = 0.211 ± 0.021) but not that of other bands (at the same ratio as that of the 30-kDa band = 1.026 ± 0.156, 37- to 50-kDa bands = 1.146 ± 0.245, 70-kDa band = 1.064 ± 0.133. (Fig. 4AGo, n = 6). As a result, KIC attenuation of the palmitoylation of the 24-kDa doublet was significantly greater than that of other bands (P = 0.0037 vs. 30-kDa band, P = 0.0014 vs. 37- to 50-kDa bands, P = 0.0027 vs. 70-kDa band). A combination of glutamine and leucine also strongly suppressed the palmitoylation of the 24-kDa doublet (Fig. 4AGo).

Attenuation of the palmitoylation of the 24-kDa doublet by high glucose was partially blocked when mannoheptulose (20 mM), which inhibits glucokinase, the first and most rate-limiting enzyme of glycolysis, was included during the labeling of the islets with high glucose (Fig. 4BGo). Namely, palmitoylation of the 24-kDa doublets in the presence of 22.2 mM glucose was significantly weaker than that in the presence of 2.8 mM glucose (P = 0.0259), whereas palmitoylation of it in the presence of [22.2 mM glucose + mannoheptulose] was not significantly lower than that in the presence of 2.8 mM glucose (P = 0.2092). This was demonstrated only in the 24-kDa doublet band but not in other bands. The difference between [22.2 mM glucose] and [22.2 mM glucose + mannoheptulose] was not statistically significant (P = 0.2483), however.

Pulse chasing of the proteins by [3H]palmitic acid and the effect of CHX
To examine the fate and turnover of the labeling of the proteins, a pulse-chase experiment was conducted. The turnover rate (T1/2) of the palmitoylation of the 24-kDa doublet was significantly faster than that of other bands (Fig. 5AGo). The T1/2 (~45 min) was not significantly affected by the concentration of glucose during the chasing (Fig. 5BGo). Pretreatment of the islets with CHX tended to attenuate the palmitoylation of the 24-kDa doublet (Fig. 5CGo, 2.8 mM glucose plus CHX/2.8 mM glucose of 24 kDa = 0.728 ± 0.015, the same ratio of 30 kDa = 1.124 ± 0.015, 37–50 kDa = 1.205 ± 0.385, 70 kDa = 1.088 ± 0.093, the values are mean ± range, n = 2).

Immunoprecipitation with SNAP-25 antibody and the palmitoylated protein in INS-1 cells
Palmitoylation of SNAP-25 with [3H]palmitic acid was detected in the immunoprecipitate (Fig. 6AGo, middle lane), which was clearly distinct from that of the 24-kDa doublet (Fig. 6AGo, left lane). SNAP-25 was detected as an approximately 28-kDa protein both in total lysate (Fig. 6BGo, left lane) and immunoprecipitate (Fig. 6BGo, middle lane). Thus, SNAP-25 was palmitoylated in the islet by our experimental protocol, but it was not a component of the 24-kDa doublet.



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  		FIG. 6. Immunoprecipitation with anti-SNAP-25 antibody and labeling of INS-1 cells with [3H]palmitic acid. The islets were labeled in the presence of 2.8 mM glucose, and the lysate of labeled islets was immunoprecipitated with an anti-SNAP-25 antibody. INS-1 cells were labeled in the presence of 2.8 mM glucose, too. A, Total lysate (left lane) and the immunoprecipitate (middle lane) of the labeled islets and the total lysate of labeled INS-1 (right lane). B, Immunoblotting of the total lysate (left lane) and immunoprecipitate (middle lane) of labeled islets and total lysate of labeled INS-1 (right lane). SNAP-25 was detected as a 28-kDa protein as indicated. The 24-kDa doublet is indicated by an open arrow in A. Representative data from two independent experiments are shown. See Materials and Methods for details.

 
Palmitoylation of the 24-kDa doublet was not detected in INS-1 cells (Fig. 6AGo, right lane). Nevertheless, labeling of other proteins was clearly detected. A protein with a molecular mass of 28 kDa, most likely SNAP-25, was also palmitoylated (Fig. 6AGo, right lane).

Two-dimensional gel electrophoresis
By two-dimensional gel electrophoresis, the 24-kDa doublet (marked by the double-head arrow) was separated into several smears and spots around the pH 4–5 area (Fig. 7AGo, main rectangle). Due to scarcity of protein, however, protein(s) corresponding to the smear and spots could not be visualized by silver staining (Fig. 7BGo).



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  		FIG. 7. Two-dimensional gel electrophoresis of the labeled proteins. A, Radioactivity detected by the BAS system. The SDS-PAGE of the labeled islet lysate is shown at the left edge of A. B, Silver staining of the proteins in the gel electrophoresed in parallel with A. The arrow in A indicates the 24-kDa doublet. The arrow in B indicates the expected electrophoretic localization of the 24-kDa doublet in which protein is not detectable with this method. See Materials and Methods for details.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A seminal finding in the present study was the identification of the palmitoylated proteins, which were modulated by nutrients in several incubation conditions. In particular, preferential modulation of the palmitoylation of the 24-kDa acidic doublet by high glucose was intriguing. This glucose effect was mimicked by the mitochondrial fuels and blocked, albeit weakly, by inhibition of glucose metabolism by mannoheptulose. The palmitoylation of the 24-kDa doublet was also markedly attenuated by acylation inhibitors. Moreover, the palmitoylation of the 24-kDa proteins turned over significantly faster than that of other proteins.

Exogenously added [3H]palmitic acid enters the ß-cells and is converted to [3H]palmitoyl-CoA and eventually mixes with a cytosolic pool of palmitoyl-CoA. Therefore, the protein labeling takes place over a 1-h period, whereas the increase in cytosolic LC-CoA most likely occurs within minutes after glucose addition as suggested by the reported rapid increase in malonyl-CoA and diacylglycerol (10, 21, 39). Under this condition, the total level of protein palmitoylation is increased, but the labeling of the protein with [3H]palmitic acid is reduced due to lowering of the specific activity of [3H]palmitic acid in the cytosol.

We used radioactive palmitic acid as a tracer; therefore, only the rate, not the total level, of palmitoylation could be examined. With this experimental protocol, attenuation of the labeling of the protein by high glucose will occur if: 1) the total palmitoylation of the protein per se is suppressed, 2) lowering of the specific activity of the tracer by de novo accumulation of nonradioactive LC-CoA takes place, 3) the rate of depalmitoylation is enhanced, and/or 4) intracellular degradation (turnover) of the protein per se is enhanced. The first possibility is most unlikely because the data of previous insulin release experiments (19, 22, 23, 24, 25, 26, 27) strongly suggested that an increase, but not a decrease, in total palmitoylation is causally related to glucose stimulation of insulin release. We consider the second explanation most appropriate as mentioned above. However, nutrients, such as glucose and mitochondrial fuels, preferentially reduced labeling of the 24-kDa doublet. Therefore, it is most likely that there exists a nutrient-dependent, preferential modulation of palmitoylation for the 24-kDa doublet. An enhancement of depalmitoylation of the 24-kDa protein and/or degradation of the protein per se by these conditions might be causal, but T1/2 of the 24-kDa labeling was not enhanced by high glucose in our pulse-chase experiment. It should be mentioned that KIC per se may not be a true substrate for ATP production (40). However, transamination of KIC yields leucine, which favors anaplerotic influx by activating glutamate dehydrogenase (41) and consequently glutamine (from endogenous stores) metabolism and the production of the trichloroacetic acid cycle intermediate, {alpha}-ketoglutarate (40, 41). Increase in trichloroacetic acid cycle metabolism through the glutamate dehydrogenase activation would cause flux of citrate to the cytosol and eventually cause accumulation of LC-CoA in the cytosol, as in the case of glucose metabolism.

A relatively short (1 h) exposure to a high concentration of glucose, mitochondrial fuels, and acylation inhibitors all significantly attenuated the palmitoylation of the 24-Da doublet by the tracer. If the turnover of the 24-kDa doublet is significantly faster than that of other proteins, the labeling of it by exogenously added [3H]palmitic acid would have been preferentially attenuated on nonselective suppression of palmitoylation. The results of the CHX experiment and the pulse-chase experiment strongly supported such a possibility. Namely, CHX treatment, which was reported to cause a profound suppression of new protein synthesis (36), tended to cause attenuation of palmitoylation of the 24-kDa doublet, and the T1/2 of the labeling of the 24-kDa doublet was short, approximately 45 min. We could not directly examine the degradation rate of the 24-kDa protein because of its scarcity, however.

In INS-1 cells, we could not detect the labeling with [3H]palmitic acid of the 24-kDa protein by the current method, indicating expression of 24-kDa protein may be minimal, if any, in INS-1 cells. This might be causally related to a relatively small response to glucose (3-fold increase in the rate of insulin release on stimulation with 22.2 mM glucose in our hands) and absence of KATP-independent glucose action (42) in this cell line.

Protein acylation has been shown to play the key role in regulation of protein-membrane interaction, protein trafficking, and enzyme activity (28, 29, 30). Protein S acylation occurs on cysteine residues through reversible thioester linkages for which no clear consensus sequence has been identified. Palmitate is the most commonly found S-linked fatty acid, and this posttranslational modification is referred to as protein palmitoylation. A number of transmembrane, integral, or peripheral membrane proteins and cytoskeletal proteins are known to be palmitoylated (29, 30). GAP-43, SNAP-25, and {alpha}-subunit of heterotrimeric G proteins are typical examples. Through facilitation of the protein-membrane interaction, palmitoylation of the protein may dictate the localization of the molecule and thus modulate its signaling function. Relevancy of protein palmitoylation for dynamic cellular regulation is further implicated by the presence of acylation-deacylation cycle (29, 30).

In neuroendocrine cells, several proteins involved in exocytosis are palmitoylated: SNAP-25 (43, 44), cysteine-string protein (45, 46), synaptotagmin (43), and SNAP-23 (47). SNAP-25 is a target soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein involved in the regulated hormone release in neuroendocrine cells and insulin-secreting ß-cells (45). Palmitoylation of SNAP-25 is well established in endocrine cells other than ß-cells (43, 44, 47). Palmitoylated doublet protein found in the islet cells with the intriguing feature mentioned above was relatively close to 25 kDa. Thus, we thought SNAP-25 might be a component of the doublet, or, alternatively, it might be associated with the palmitoylated doublet. Palmitoylation of SNAP-25 in the pancreatic ß-cell was clearly demonstrated as reported (44); however, electrophoretic mobility of SNAP-25 was distinct from that of the 24-kDa doublet, implying that SNAP-25 is not a component of the 24-kDa doublet.

SNAP-25 was ruled out as a component of the 24-kDa doublet. Myosin light-chain and low-molecular-weight G proteins might be considered as candidates on the basis of molecular mass. However, isoelectric point of myosin light chain is clearly different from that of the 24-kDa protein (see http://us.expasy.org/ch2d/). Among the small G proteins, Ras family small G proteins (29, 30) and Rab/Ypt GTPase family Ara6 (48), Vac8p (49), and Rab38 (50) are palmitoylated. Vac8p is involved in a homotypic vacuole fusion reaction that requires dynamic palmitoylation of the protein during the priming step (49). Therefore, the intriguing possibility would be that novel Ras or Rab family G proteins regulated by palmitoylation are involved in exocytosis in pancreatic ß-cell.

A high concentration of glucose attenuated palmitoylation of not only the 24-kDa doublet but other bands as well; however, the mitochondrial fuels almost exclusively suppressed palmitoylation of the doublet. Such differential effects of glucose and mitochondrial fuels suggest that enhanced metabolic flux through the glycolytic pathway may be involved in suppression of palmitoylation of proteins in the islet cells.

Previous reports have shown that cerulenin can also act as an inhibitor of fatty acid, cholesterol, RNA, and protein synthesis (25). However, these possibilities are not considered responsible for the effect shown in this study because: 1) we showed this labeling as palmitoylation by chemical analyses; 2) activity of fatty acid synthase was very low in the pancreatic ß-cells (20); 3) involvement of inhibition of RNA and protein synthesis was excluded because of the acute (1 h) incubation conditions; and 4) other acylation inhibitor tunicamycin mimicked the effect of cerulenin on the palmitoylation in this study.

Cytosolic accumulation of LC-CoA in the rat islet ß-cell on exposure to high glucose has been demonstrated (39). An extracellular application of micromolar concentration of FFAs acutely stimulates insulin release (22, 23), and acylation inhibitors selectively inhibit nutrient-induced insulin release without impeding nutrient metabolism (25). As an extension of this line of evidence, we here demonstrated the presence of rapidly turning over palmitoylated proteins in the islet cells, which are acutely modulated by nutrients and acylation inhibitors. Thus, a link between protein palmitoylation and glucose stimulation of insulin release is strongly suggested. A relatively weak effect of mannoheptulose might deflate the hypothesis of glucose-induced palmitoylation being an important mechanism for insulin secretion. Potent inhibition of metabolism by the drug might have caused a nonspecific suppression of cellular function. At least, the observed effect of glucose on palmitoylation could never be attributed to an osmotic effect because nonmetabolizable sucrose did not show the same effect. Further studies are clearly needed to establish the nature and functional role of the palmitoylated proteins to definitively prove the legitimacy of the malonyl CoA hypothesis (21) in the nutrient stimulation of insulin secretion.


   Footnotes
 
Abbreviations: CHX, Cycloheximide; DTT, dithiothreitol; FFA, free fatty acid; IPG-IEF, immobilized pH-gradient isoelectric focusing; KATP, ATP-sensitive potassium; KIC, {alpha}-ketoisocaproic acid; KRB, Krebs-Ringer bicarbonate; LC-CoA, long-chain-acyl CoA; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SNAP-25, synaptosome-associated protein of 25 kDa; T1/2, turnover rate.

Received June 9, 2003.

Accepted for publication August 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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