Protein acylation in the inhibition of insulin secretion by norepinephrine, somatostatin, galanin, and PGE2

Haiying Cheng, Susanne G. Straub, and Geoffrey W. G. Sharp

Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Submitted 10 December 2002 ; accepted in final form 1 April 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin, and prostaglandin E2, act via specific receptors that activate pertussis toxin (PTX)-sensitive G proteins. Four inhibitory mechanisms are known: 1) activation of ATP-sensitive K channels and repolarization of the {beta}-cell; 2) inhibition of L-type Ca2+ channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a "distal" site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin, and polyunsaturated fatty acids, all of which suppress protein acyltransferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-hydroxypalmitate, and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin, and prostaglandin E2. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cAMP production. We also examined the effects of norepinephrine, 2-bromopalmitate, and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase, causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.

rat pancreatic islets; {beta}-cell; signaling; pertussis toxin; G proteins


INSULIN SECRETION is a complex process regulated by nutrients, such as glucose and amino acids, and hormonal and neural factors that provide stimulatory and inhibitory influences on the pancreatic {beta}-cells. The major physiological inhibitors, such as norepinephrine (NE), somatostatin, prostaglandin E2 (PGE2), and galanin, can inhibit insulin secretion that is stimulated by all of the nutrient and modulatory pathways. They interact with specific G protein-linked receptors at the plasma membrane, which activate pertussis toxin (PTX)-sensitive Gi and Go proteins that mediate the inhibition of insulin secretion (17, 27, 30).

The inhibitory pathways act in contradictory fashion on many of the same sites as the stimulatory pathways (30). For example, 1) inhibitors activate the ATP-sensitive K (KATP) channel, repolarize or hyperpolarize the cell membrane, and thus inhibit the action of glucose that depolarizes the membrane via closure of this channel; 2) inhibitors decrease the activity of the L-type Ca2+ channel by two means, indirectly by the activation of the KATP channel and hyperpolarization, and directly by inhibiting the channel itself; 3) inhibitors decrease the activity of adenylyl cyclase and lower cAMP levels, thus reducing the effect of cAMP to potentiate insulin secretion. However, the most dominant inhibitory mechanism (4) is at a "distal" site in stimulus-secretion coupling. This distal site is at a crucial, late step in exocytosis, beyond the elevation of intracellular Ca2+ and beyond the potentiating actions of cAMP and diacylglycerol. Information on the G protein interactions with these four major inhibitory sites is limited. It was reported that activation of KATP channels was due to {beta}{gamma}-subunits of Gi and/or Go proteins (43). The {alpha}-subunits of Gi-2 and Gi-3 mediate the decreased activity of adenylyl cyclase in the {beta}-cell (18).

In this study, we have taken advantage of two well-known inhibitors of protein acylation, 2-bromopalmitate (2-BrP) (8, 20, 36, 39) and cerulenin (11, 29, 31, 41), to investigate the distal inhibitory mechanisms of insulin secretion. By mediating protein-membrane and protein-protein interactions, protein acylation is thought to be important for cell signaling, e.g., in the T cell (25, 26, 34, 39) and {beta}-cell (6, 31, 41, 44). We found that inhibitors of protein acylation blocked the distal inhibitory effects of NE, somatostatin, galanin, and PGE2. The data suggest that the distal mechanism of inhibition of insulin secretion involves the acylation of a protein or proteins that are critical to the normal process of exocytosis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Isolation of pancreatic islets. Male Sprague-Dawley rats (250–400 g) were used. Immediately after CO2 asphyxiation, the pancreata were removed, and the islets were isolated by a collagenase digestion technique (16). Krebs-Ringer bicarbonate HEPES buffer (KRBH) containing (in mM) 129 NaCl, 5 NaHCO3, 4.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2.5 CaCl2, 2.8 glucose, 10 HEPES, and 0.1% BSA at pH 7.4 was used for isolation and selection of the islets. All procedures were approved by the Institutional Animal Care and Use Committee at Cornell University.

Measurements of insulin release. Insulin release was measured under static incubation conditions at 37°C with batches of five size-matched islets per tube. When Ca2+ was present in the buffer, islets were preincubated in regular KRBH buffer for 60 min. The islets were then incubated in the presence and absence of test agents (as we will describe) for 15 or 60 min. Under Ca2+-free conditions, the islets were washed three times with Ca2+-free KRBH buffer containing 1 mM EGTA (Ca2+-free KRBH-EGTA buffer) and preincubated in Ca2+-free KRBH-EGTA buffer for 60 min. They were then incubated in fresh Ca2+-free KRBH-EGTA buffer in the presence and absence of test agents for 60 min. At the end of the incubations, samples were taken and kept at -20°C until radioimmunoassayed for insulin using a charcoal separation method. Insulin secretion was expressed as fractional release, the percentage of the insulin content of the islets that was released over 15 or 60 min (15, 41). When exogenous free fatty acids (FFAs) and their analogs were used, the concentrations of the FFAs were estimated from the molar ratios of the fatty acids and FFA-free BSA, as reported in the literature (16, 18). In the last series of experiments (on the effects of arachidonic and eicosapentaenoic acids), the FFA-free BSA concentration was changed from 0.5 to 0.4%, because the new batch of BSA appeared to bind 2-BrP differently from the old batch, and the complete blockade of NE action by 2-BrP was obtained at 0.4%.

Palmitate oxidation measurement. Palmitate oxidation was measured as previously reported with minor modifications (5, 41). Briefly, groups of 15 islets (in duplicate) were incubated at 37°C for 1 h in 200 µl of KRB buffer, containing 0.2 µCi [1-14C]palmitate, 0.8 mM L-carnitine, and other components as indicated. At the end of the incubation, islet metabolism was terminated by adding 200 µl of 0.5 M HCl to the incubation mixture. 14CO2 was collected overnight in 300 µl of 1 M NaOH and measured by liquid scintillation spectrometry. One batch of islets was used for each experiment, so that all of the determinations were paired.

Measurement of palmitate incorporation into lipids. This procedure was performed as previously described with minor modification (1, 5). Briefly, groups of 30–50 islets were incubated at 37°C for 1 h in 200 µl of KRB buffer, containing 0.2 µCi [1-14C]palmitate, 0.8 mM L-carnitine, and other components as indicated. At the end of the incubations, the incubation media were removed, and the islets were washed twice with 1 ml of ice-cold PBS. Two hundred microliters of 0.2 M NaCl were added to the islet pellet, and the mixture was immediately frozen in liquid N2. After the islet pellet was thawed, 750 µl of chloroform-methanol (2:1) and 50 µl of 0.1 N KOH were added. After vigorous vortexing for 3 min, the phases were separated by centrifugation at 2,000 g for 20 min. The top aqueous layer was removed, and the bottom lipid-soluble layer was washed once with 200 µl of methanol-water-chloroform (48:47:3). Two hundred microliters of the lipid-soluble phase were added to 5 ml of BioSafe2 scintillation mixture, and incorporation of radiolabel into lipids was quantified by liquid scintillation spectrometry.

Measurement of cAMP. This measurement was performed as previously described with minor modification (9, 13, 40). Briefly, groups of 20 islets were preincubated at 37°C for 1 h in 200 µl of KRBH buffer, in the presence or absence of cerulenin as indicated. The islets were then incubated for 15 min in 200 µl of KRBH containing 5.6 mM Glc, 50 µM IBMX, 6 µM forskolin (Fsk), and 10 µM propranolol, with or without testing reagents as indicated. The incubation was terminated by addition of 300 µl of 0.2 N HCl. The tubes were then placed in boiling water for 15 min with occasional vortexing. cAMP was determined by the Biotrak cAMP enzymeimmunoassay (EIA) system.

Materials. Glucose, 2-BrP, palmitate, 16-hydroxy palmitate (16-OH palmitate), 2-hydroxy myristate (2-OH myristate), FFA-free BSA, NE, forskolin, 12-O-tetradecanoylphorbol 13-acetate (TPA), L-carnitine, somatostatin, galanin, and PGE2 were obtained from Sigma (St. Louis, MO). Cerulenin was purchased from Fluka (Milwaukee, WI). [1-14C]palmitate was obtained from New England Nuclear (Boston, MA). The Biotrak cAMP EIA system was from Amersham Pharmacia Biotech (Piscataway, NJ).

Statistical methods. All data are shown as means ± SE. Statistical significance was evaluated by two-way ANOVA or t-tests as appropriate. Differences were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effects of 2-BrP, palmitate, 16-OH palmitate, and 2-OH myristate on the inhibition of insulin secretion by NE. To "isolate" the distal inhibitory effect of NE and distinguish it from the effects of NE on the KATP and Ca2+ channels, the first studies were performed in the absence of extracellular Ca2+. In addition, a supermaximally effective concentration of Fsk was used (20) to eliminate any effect of NE via adenylyl cyclase. The results of the experiments are shown in Fig. 1. Under these Ca2+-free conditions, 11.1 mM glucose stimulated insulin secretion 2.6-fold (0.68 ± 0.12 vs. 2.43 ± 0.38% of content/h; {Delta} = 1.75 ± 0.35; P < 0.01) in the presence of 6 µM Fsk and 100 nM TPA, similar to data reported previously (14, 15). This glucose-stimulated insulin secretion was not altered by 20 µM 2-BrP (control, 2.43 ± 0.38 vs. 2-BrP, 2.49 ± 0.11% of content/h; {Delta} = -0.06 ± 0.04; P = 0.21) and was completely inhibited by 10 µM NE (2.43 ± 0.38 vs. 0.84 ± 0.24% of content/h; {Delta} = 1.59 ± 0.17; P < 0.01). However, in the presence of 20 µM 2-BrP, glucose-stimulated insulin secretion was unaffected by NE (control, 2.43 ± 0.38 vs. NE, 2.33 ± 0.38% of content/h; {Delta} = -0.10 ± 0.16; P = 0.57). The effect of 2-BrP to block the action of NE action was concentration dependent, with complete blockade at 20 µM.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1. Effects of 2-bromopalmitate (2-BrP) on inhibition of insulin secretion by norepinephrine (NE) under Ca2+-free conditions. Insulin secretion was evoked by 11.1 mM glucose (Glc) in the presence of 6 µM forskolin (Fsk) and 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA). 2-BrP and NE were present in the 60-min incubation period. Values are means ± SE; n = 4. *P < 0.05, **P < 0.005.

 

Experiments were next carried out under normal conditions in the presence of Ca2+. As shown in Fig. 2, insulin secretion was stimulated by 11.1 mM glucose, and this was not affected by 20 µM 2-BrP (control, 1.98 ± 0.41 vs. 2-BrP, 2.12 ± 0.47% of content/h; {Delta} = -0.14 ± 0.37; P = 0.73), a finding that is consistent with previous studies (32, 45). NE inhibited glucose-stimulated insulin secretion completely (1.98 ± 0.41 vs. 0.58 ± 0.09% of content/h; {Delta} = 1.40 ± 0.10; P < 0.001) in the absence of 2-BrP but incompletely (1.98 ± 0.41 vs. 1.01 ± 0.17% of content/h; {Delta} = 0.97 ± 0.06; P < 0.05) in the presence of 2-BrP. Thus the blocking effect of 2-BrP on the inhibitory action of NE in the presence of extracellular Ca2+ was less than that in the absence of extracellular Ca2+. Under Ca2+-free conditions, the only functional inhibitory mechanism for NE to inhibit insulin secretion is at the distal site, and this is completely blocked by 2-BrP. In the presence of extracellular Ca2+, the inhibitory effect of NE seen when the islets are exposed to 2-BrP could be exerted via activation of the KATP channels, reduced Ca2+ influx, and inhibition of adenylyl cyclase. These combined data suggest that only the distal inhibitory action of NE is blocked by 2-BrP.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 2. Effects of 2-BrP on the inhibition of insulin secretion by NE under normal conditions with Ca2+ present. Insulin secretion was evoked by 11.1 mM glucose. 2-BrP (20 µM) and NE were present in the 60-min incubation period. Values are means ± SE; n = 4. *P < 0.05.

 

To exclude the possibility that the action of 2-BrP was due to nonspecific effects rather than inhibition of protein acylation, we examined the effects of palmitate and 16-OH palmitate under the same Ca2+-free conditions as we tested 2-BrP. These two compounds are structurally similar to 2-BrP but do not block protein acylation (39) as does 2-BrP. Insulin secretion was not changed by either 20 µM palmitate (control, 2.64 ± 0.49 vs. palmitate, 2.72 ± 0.33% of content/h; {Delta} = -0.08 ± 0.39; P = 0.85) or 20 µM 16-OH palmitate (control, 2.29 ± 0.26 vs. 16-OH palmitate, 2.48 ± 0.38% of content/h; {Delta} = -0.19 ± 0.15; P = 0.21). It was inhibited equally by NE in the absence and presence of 20 µM palmitate (control, 0.74 ± 0.16 vs. palmitate, 0.51 ± 0.06% of content/h; {Delta} = 0.23 ± 0.11; P = 0.12), and in the absence and presence of 20 µM 16-OH palmitate (control, 0.78 ± 0.11 vs. 16-OH palmitate, 0.92 ± 0.12% of content/h; {Delta} = -0.14 ± 0.17; P = 0.31).

To distinguish between the two most common forms of protein acylation, palmitoylation and myristoylation, we examined the effect of 2-OH myristate, which blocks myristoylation but not palmitoylation (19). Under Ca2+-free conditions, insulin secretion was stimulated by 11.1 mM glucose in the presence of TPA and Fsk and was completely inhibited by NE (2.23 ± 0.37 vs. 0.79 ± 0.10% of content/h; {Delta} = 1.44 ± 0.43; P < 0.05). In the presence of 20 µM 2-OH myristate, glucose-stimulated secretion was not altered by 2-OH myristate (control, 2.23 ± 0.37 vs. 2-OH myristate, 2.58 ± 0.39% of content/h; {Delta} = -0.35 ± 0.40; P = 0.44) and was still completely inhibited by NE (2.58 ± 0.39 vs. 0.93 ± 0.18% of content/h; {Delta} = 1.65 ± 0.51; P < 0.05). Hence, NE inhibited glucose-stimulated insulin secretion to a similar extent in the absence and presence of 20 µM 2-OH myristate (0.79 ± 0.10 vs. 0.93 ± 0.18% of content/h; {Delta} = -0.14 ± 0.11; P = 0.23). Therefore, myristoylation does not appear to be involved in the mechanism of inhibition.

Effect of cerulenin to block the inhibition of insulin secretion by NE. The effects of cerulenin, a well-known inhibitor of protein acylation, are shown in Fig. 3. In this series of experiments, insulin secretion was evoked by a depolarizing concentration of 50 mM KCl in the presence of TPA and Fsk. We used KCl because nutrient-stimulated, but not non-nutrient-stimulated, insulin secretion is inhibited by cerulenin (41). Fsk was used to keep intracellular cAMP at supermaximal levels and, like TPA, to maximize the secretory response. In these experiments, cerulenin was included only in the preincubation period. In the absence of cerulenin, NE inhibited the stimulated insulin secretion by 96% (P < 0.001). Cerulenin blocked the inhibitory action of NE in a concentration-dependent manner. The IC50 for cerulenin to block the action of NE was 10 µg/ml (45 µM), and complete blockade was seen at 100 µg/ml (450 µM). In separate experiments, basal insulin secretion in the presence of 2.8 mM glucose was not significantly affected by 100 µg/ml cerulenin (control, 0.40 ± 0.08 vs. cerulenin, 0.48 ± 0.10% of content/15 min; {Delta} = -0.08 ± 0.04; P = 0.16), nor was insulin secretion stimulated by the combination of KCl, TPA, and forskolin (control, 0.96 ± 0.23 vs. cerulenin, 1.14 ± 0.25% of content/15 min; {Delta} = -0.18 ± 0.32; P = 0.63). The fact that 100 µg/ml cerulenin did not alter either basal or KCl-stimulated insulin secretion suggests that cerulenin affects neither the islet's ability to secrete insulin nor its membrane integrity to permit leakage of insulin. Under these experimental conditions, in the presence of 50 mM KCl and 6 µM Fsk, the effects of NE on the KATP channel to hyperpolarize the cell and to lower cAMP levels are bypassed. Therefore, the data presented so far with 2-BrP and cerulenin, two structurally different inhibitors of protein acylation, strongly suggest that protein acylation is involved in the distal inhibitory action of NE.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. Concentration dependence of effect of cerulenin to block inhibition of insulin secretion by NE. Insulin secretion was evoked by 50 mM KCl in the presence of 100 nM TPA and 6 µM Fsk for 15 min. Cerulenin was included only in the 60-min preincubation period and was not present in the incubation period. NE was present in the 15-min incubation period. Values are means ± SE; n = 7.

 

Effects of 2-BrP on the inhibition of insulin secretion by somatostatin, galanin, and PGE2 under Ca2+-free conditions. Three other physiological inhibitors of insulin secretion, somatostatin, galanin, and PGE2, were tested to see whether the effect of 2-BrP was specific for NE or whether it blocked the effects of other inhibitors also. As seen in Fig. 4, under Ca2+-free conditions, 1 µM somatostatin, 100 nM galanin, and 10 µM PGE2 inhibited glucose-stimulated insulin secretion by 73% (P < 0.01), 61% (P < 0.01), and 66% (P < 0.01), respectively. In contrast, in the presence of 20 µM 2-BrP, these inhibitors had no effect on insulin secretion; their inhibitory actions were completely blocked. Thus the effect of 2-BrP is a general effect against several inhibitors.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4. Effects of 2-BrP on inhibition of insulin secretion by somatostatin, galanin, and PGE2 under Ca2+-free conditions. Insulin secretion was evoked by 11.1 mM glucose in the presence of 6 µM Fsk and 100 nM TPA. 2-BrP (20 µM) was present in the 60-min incubation period. 1 µM Somatostatin, 100 nM galanin, and 10 µM PGE2 were present during the last 10 min of the 60-min preincubation and in the 60-min test incubation. Values are means ± SE; n = 6. *P < 0.01.

 

Effects of NE, 2-BrP, and cerulenin on palmitate metabolism. It has been reported that NE reduced the oxidation of palmitate as well as the incorporation of glucose into phospholipids and neutral lipids in the presence of palmitate through an {alpha}2-adrenergic mechanism (35). 2-BrP and cerulenin have also been reported to affect lipid metabolism (11, 17). Consequently, we examined the effects of NE, 2-BrP, and cerulenin on lipid metabolism to determine whether a change in metabolism was a contributory factor in the inhibitory effects of 2-BrP and cerulenin on the action of NE. As expected, and as shown in Table 1, palmitate oxidation was significantly lower in the presence of 11.1 mM glucose than in the presence of 2.8 mM glucose (0.38 ± 0.03 vs. 0.51 ± 0.04 pmol · islet-1 · h-1; {Delta} = -0.13 ± 0.02; P < 0.01). Palmitate oxidation was not significantly affected by NE at either 2.8 mM or 11.1 mM glucose. Similarly, NE did not significantly change [1-14C]palmitate incorporation into cellular lipids at either 2.8 mM or 11.1 mM glucose. Glucose at 11.1 mM increased [1-14C]palmitate incorporation into lipid by 20% (P < 0.001) relative to incorporation in the presence of 2.8 mM glucose. Furthermore, both 2-BrP and cerulenin decreased 11.1 mM glucose-induced palmitate oxidation by 66% in the absence and presence of NE. However, the effects of 2-BrP and cerulenin on [1-14C]palmitate incorporation were different. Cerulenin had no effect, whereas 2-BrP increased 11.1 mM glucose-induced palmitate esterification.


View this table:
[in this window]
[in a new window]
 
Table 1. Effects of norepinephrine, 2-BrP, and cerulenin on palmitate oxidation and incorporation into lipids

 

Effects of NE, 2-BrP, and cerulenin on cAMP production. The data so far demonstrated that the distal site of NE action was blocked by 2-BrP and cerulenin. However, it was not clear whether 2-BrP and cerulenin acted at any of the other sites of NE action. Therefore, we examined their effects on cAMP production of islets. As shown in Table 2, 10 µM NE significantly decreased Fsk-stimulated cAMP production from 580 ± 57 to 275 ± 52 fmol · islet-1 · 15 min-1, a 53% inhibition (P < 0.01). More importantly, in the presence of 20 µM 2-BrP or 100 µg/ml cerulenin, NE was still able to suppress cAMP production (Table 2). This demonstrates that neither 2-BrP nor cerulenin blocks the binding of NE to the {alpha}2-adrenergic receptor, receptor activation of related G proteins, and G protein inhibition of adenylyl cyclase.


View this table:
[in this window]
[in a new window]
 
Table 2. Effects of norepinephrine, 2-BrP, and cerulenin on cAMP production

 

Effect of 2-BrP on the inhibition of insulin secretion by high concentrations of NE. On the basis of these data on cAMP production, the displacement of NE from the {alpha}2-adrenergic receptor appears not to contribute to the blocking effect of 2-BrP on NE action. To strengthen this point, we further tested the effect of 2-BrP in the presence of a very high NE concentration (100 µM) under Ca2+-free conditions. As shown in Fig. 5, insulin secretion was stimulated by 11.1 mM glucose in the presence of TPA and Fsk, and this was not affected by 2-BrP (control, 3.65 ± 0.45 vs. 2-BrP, 3.18 ± 0.35% of content/h; {Delta} = -0.47 ± 0.34; P = 0.27). This stimulated insulin secretion was completely inhibited by NE at both 10 µM (3.65 ± 0.45 vs. 0.73 ± 0.08% of content/h; {Delta} = 2.92 ± 0.52; P < 0.01) and 100 µM (3.65 ± 0.45 vs. 0.86 ± 0.19% of content/h; {Delta} = 2.78 ± 0.62; P < 0.01). However, in the presence of 2-BrP, insulin secretion was not affected by NE at either 10 µM (3.65 ± 0.45 vs. 2.89 ± 0.21% of content/h; {Delta} = 0.76 ± 0.48; P = 0.40) or 100 µM (3.65 ± 0.45 vs. 3.44 ± 0.62% of content/h; {Delta} = 0.21 ± 0.99; P = 0.73). Therefore, increasing the NE concentration, even to 100 µM, did not reinstate its inhibitory action on insulin secretion.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Effects of 2-BrP or etomoxir on inhibition of insulin secretion by NE under Ca2+-free conditions. Insulin secretion was evoked by 11.1 mM glucose in the presence of 6 µM Fsk and 100 nM TPA. 2-BrP, etomoxir, and NE were present in the 60-min incubation period. Values are means ± SE; n = 4. *P < 0.01.

 

Effects of etomoxir and polyunsaturated fatty acids on the inhibition of insulin secretion. 2-BrP has been used extensively to suppress carnitine palmitoyltransferase I (CPT I) activity. To determine whether the effects of 2-BrP to block the inhibitory effect of NE could be related to this suppression of CPT I activity, we examined the effects of another CPT I inhibitor, etomoxir (45). In paired experiments shown in Fig. 5, etomoxir was used under exactly the same conditions as was 2-BrP. In contrast to 2-BrP, insulin secretion was inhibited by NE to a similar extent in the absence and presence of etomoxir (0.73 ± 0.08 vs. 1.06 ± 0.17% of content/h; {Delta} = -0.33 ± 0.14; P = 0.10). Hence, inhibition of CPT I activity is not a contributory factor in the effects of 2-BrP on NE action.

In further control experiments, we asked whether polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, could mimic the effect of 2-BrP on NE-mediated inhibition of insulin secretion. Both arachidonic acid and eicosapentaenoic acid interfere with T cell signal transduction, an action that is thought to be due to the inhibition of protein palmitoylation (39). Under Ca2+-free conditions, NE failed to inhibit insulin secretion in the presence of 0.5 mM arachidonic acid or eicosapentaenoic acid (data not shown).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
In the work presented here, 2-BrP and cerulenin, both of which inhibit protein acyl transferase (39, 41), blocked the distal effect of NE to inhibit insulin secretion. In contrast, control compounds such as palmitate, 16-OH palmitate, and etomoxir (which do not block protein acylation) had no effect on the action of NE. These data suggest that protein acylation is involved in the distal inhibitory action of NE. Furthermore, 2-BrP also blocked the distal inhibitory actions of somatostatin, galanin, and PGE2. Therefore, protein acylation appears to be generally involved in the distal inhibition of insulin secretion and is not specific to a particular inhibitor.

Inhibition of protein acylation by 2-BrP and cerulenin is considered responsible for the effects shown in this study. The reasons for this follow. 1) If the suppression of CPT I activity (23) were the reason for 2-BrP to block the inhibitory action of NE, etomoxir would be expected to mimic this effect, because etomoxir is also a CPT I inhibitor. Instead, etomoxir had no effect on the action of NE. Additionally, cerulenin is not a CPT I inhibitor. Furthermore, whereas both 20 µM 2-BrP and 100 µg/ml cerulenin inhibited palmitate oxidation of 11.1 mM glucose, only 2-BrP significantly increased 11.1 mM glucose-induced palmitate incorporation into lipids. Hence, the different influences on palmitate metabolism by 2-BrP and cerulenin suggest that palmitate metabolism has no significant role in the effects of 2-BrP and cerulenin on the action of NE. 2) The similarities between the concentration-response characteristics of cerulenin to block the action of NE in this study and the concentration-response characteristics for the inhibition of protein acylation in other reports (11, 29, 41) suggest that cerulenin is functioning under our conditions as an inhibitor of protein acylation. 3) The short incubation time of our experimental conditions precludes the long-term effects of cerulenin and 2-BrP. These include the effects of cerulenin on apoptosis, cell growth, DNA, RNA, and protein synthesis (10, 11, 21, 22, 29) and the effects of 2-BrP on the levels of insulin and uncoupling protein-2 mRNA (3, 38).

Because of the fast, reversible nature of inhibition of insulin secretion by most physiological inhibitors, the rapidly reversible palmitoylation reaction would be more important for modulating this function (7) than the relatively slower myristoylation reaction. This is in accord with our observation that 2-OH myristate, which blocks myristoylation but not palmitoylation (19), had no influence on the inhibitory action of NE.

There is indirect evidence to indicate that NE inactivates long-chain acyl-CoA synthetase and other enzymes of glycerolipid synthesis (35). However, in the present study, we did not find any effects of NE on palmitate oxidation. Nor did we find any effects on esterification at low or high concentrations of glucose. Moreover, 2-BrP was still able to increase [1-14C]palmitate incorporation into cellular lipid at 11.1 mM glucose even in the presence of NE. Thus our data provide no evidence for an effect of NE on palmitate metabolism under our experimental conditions.

With respect to the other actions of NE in the {beta}-cell, neither 2-BrP nor cerulenin affected the action of NE to decrease cAMP production. This indicates that inhibitors of protein acylation do not block the interaction of NE at the {alpha}2-adrenergic receptor, related G protein activation, or inhibition of adenylyl cyclase. This further excludes the possibility that the effects of 2-BrP and cerulenin are due to nonspecific effects.

In a study using capacitance measurements in mouse pancreatic {beta}-cells, activation of calcineurin was proposed as the distal mechanism for NE, galanin, and somatostatin to inhibit insulin secretion (24). Calcineurin is a Ca2+- and calmodulin-dependent protein phosphatase (42). However, NE-induced inhibition of insulin secretion was unaffected by blockade of calcineurin in INS-1 (12), {beta}HC-9, and HIT cell lines (L. M. Shen and G. W. G. Sharp, unpublished observations). Moreover, under stringent Ca2+-free conditions that presumably exclude the Ca2+-dependent activity of calcineurin, NE completely blocks stimulated insulin secretion (13). Additionally, under these Ca2+-free conditions, 2-BrP still effectively blocked the distal inhibitory effects of NE, somatostatin, galanin, and PGE2. Calcineurin does not appear to be involved in the mechanism of the distal inhibition of insulin secretion. PTX-sensitive Gi and Go proteins can exert their inhibitory effects on insulin secretion via two possible mechanisms: 1) direct action of the Gi and Go subunits ({alpha} and/or {beta}{gamma}) on proteins critical for exocytosis; 2) activation of an enzyme or protein mediator of the inhibition by the {alpha}- and/or {beta}{gamma}-subunits. Relevant to the latter, our data suggest that distal inhibition of insulin secretion is achieved by activation of a specific protein acyltransferase.

It is known that, regarding inhibition of insulin secretion, {alpha}2-adrenergic receptor, Gi{alpha}1 and Go{alpha}, SNAP-25, and synaptotagmin are palmitoylated, whereas Gi{alpha}1 and Go{alpha} are myristoylated (2, 4, 28, 33, 37). Finally, on the basis of our finding that inhibitors of protein acylation blocked the distal site of inhibitors of insulin secretion, we propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyltransferase that results in acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of essential and precise interactions of the exocytotic machinery would block insulin secretion. It should be noted that protein acyltransferase would be a novel target for G protein activation.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42063 and a New York State Bridge Grant (to G. W. G. Sharp), by a Career Development Award from the Juvenile Diabetes Association (to S. G. Straub), and by a Predoctoral Fellowship from the Pharmaceutical and Research Manufacturers Association (to H. Cheng).


    FOOTNOTES
 

Address for reprint requests and other correspondence: G. W. G. Sharp, Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853-6401 (E-mail: gws2{at}cornell.edu).

Submitted 10 December 2002

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

  1. Antinozzi PA, Segall L, Prentki M, McGarry JD, and Newgard CB. Molecular or pharmacologic perturbation of the link between glucose and lipid metabolism is without effect on glucose-stimulated insulin secretion. A re-evaluation of the long-chain acyl-CoA hypothesis. J Biol Chem 273: 16146–16154, 1998.[Abstract/Free Full Text]
  2. Bhamre S, Wang HY, and Friedman E. Serotonin-mediated palmitoylation and depalmitoylation of G alpha proteins in rat brain cortical membranes. J Pharmacol Exp Ther 286: 1482–1489, 1998.[Abstract/Free Full Text]
  3. Briaud I, Harmon JS, Kelpe CL, Segu VB, and Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes 50: 315–321, 2001.[Abstract/Free Full Text]
  4. Chen CA and Manning DR. Regulation of G proteins by covalent modification. Oncogene 20: 1643–1652, 2001.[ISI][Medline]
  5. Chen S, Ogawa A, Ohneda M, Unger RH, Foster DW, and McGarry JD. More direct evidence for a malonyl-CoA-carnitine palmitoyltransferase I interaction as a key event in pancreatic beta-cell signaling. Diabetes 43: 878–883, 1994.[Abstract]
  6. Deeney JT, Gromada J, Hoy M, Olsen HL, Rhodes CJ, Prentki M, Berggren PO, and Corkey BE. Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells). J Biol Chem 275: 9363–9368, 2000.[Abstract/Free Full Text]
  7. Dunphy JT and Linder ME. Signalling functions of protein palmitoylation. Biochim Biophys Acta 1436: 245–261, 1998.[ISI][Medline]
  8. El-Husseini AD, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-Campbell C, Aguilera-Moreno A, Nicoll RA, and Bredt DS. Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108: 849–863, 2002.[ISI][Medline]
  9. El-Mansoury AM and Morgan NG. Activation of protein kinase C modulates alpha2-adrenergic signalling in rat pancreatic islets. Cell Signal 10: 637–643, 1998.[ISI][Medline]
  10. Heath RJ, White SW, and Rock CO. Lipid biosynthesis as a target for antibacterial agents. Prog Lipid Res 40: 467–497, 2001.[ISI][Medline]
  11. Jochen AL, Hays J, and Mick G. Inhibitory effects of cerulenin on protein palmitoylation and insulin internalization in rat adipocytes. Biochim Biophys Acta 1259: 65–72, 1995.[ISI][Medline]
  12. Kampermann J, Herbst M, and Ullrich S. Effects of adrenaline and tolbutamide on insulin secretion in INS-1 cells under voltage control. Cell Physiol Biochem 10: 81–90, 2000.[ISI][Medline]
  13. Komatsu M, McDermott AM, Gillison SL, and Sharp GW. Time course of action of pertussis toxin to block the inhibition of stimulated insulin release by norepinephrine. Endocrinology 136: 1857–1863, 1995.[Abstract]
  14. Komatsu M, Schermerhorn T, Aizawa T, and Sharp GW. Glucose stimulation of insulin release in the absence of extracellular Ca2+ and in the absence of any increase in intracellular Ca2+ in rat pancreatic islets. Proc Natl Acad Sci USA 92: 10728–10732, 1995.[Abstract]
  15. Komatsu M, Schermerhorn T, Noda M, Straub SG, Aizawa T, and Sharp GW. Augmentation of insulin release by glucose in the absence of extracellular Ca2+: new insights into stimulus-secretion coupling. Diabetes 46: 1928–1938, 1997.[Abstract]
  16. Lacy PE and Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 16: 35–39, 1967.[ISI][Medline]
  17. McDermott AM and Sharp GW. Inhibition of insulin secretion: a fail-safe system. Cell Signal 5: 229–234, 1993.[ISI][Medline]
  18. McDermott AM and Sharp GW. Gi2 and Gi3 proteins mediate the inhibition of adenylyl cyclase by galanin in the RINm5F cell. Diabetes 44: 453–459, 1995.[Abstract]
  19. Paige LA, Zheng GQ, DeFrees SA, Cassady JM, and Geahlen RL. Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA: protein N-myristoyltransferase. Biochemistry 29: 10566–10573, 1990.[ISI][Medline]
  20. Percherancier Y, Planchenault T, Valenzuela-Fernandez A, Virelizier JL, Arenzana-Seisdedos F, and Bachelerie F. Palmitoylation-dependent control of degradation, life span, and membrane expression of the CCR5 receptor. J Biol Chem 276: 31936–31944, 2001.[Abstract/Free Full Text]
  21. Pizer ES, Chrest FJ, DiGiuseppe JA, and Han WF. Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines. Cancer Res 58: 4611–4615, 1998.[Abstract]
  22. Pizer ES, Wood FD, Pasternack GR, and Kuhajda FP. Fatty acid synthase (FAS): a target for cytotoxic antimetabolites in HL60 promyelocytic leukemia cells. Cancer Res 56: 745–751, 1996.[Abstract]
  23. Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, and Corkey BE. Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267: 5802–5810, 1992.[Abstract/Free Full Text]
  24. Renstrom E, Ding WG, Bokvist K, and Rorsman P. Neuro-transmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin. Neuron 17: 513–522, 1996.[ISI][Medline]
  25. Resh MD. Regulation of cellular signalling by fatty acid acylation and prenylation of signal transduction proteins. Cell Signal 8: 403–412, 1996.[ISI][Medline]
  26. Resh MD. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451: 1–16, 1999.[ISI][Medline]
  27. Robertson RP, Seaquist ER, and Walseth TF. G proteins and modulation of insulin secretion. Diabetes 40: 1–6, 1991.[Abstract]
  28. Rose JJ, Taylor JB, Shi J, Cockett MI, Jones PG, and Hepler JR. RGS7 is palmitoylated and exists as biochemically distinct forms. J Neurochem 75: 2103–2112, 2000.[ISI][Medline]
  29. Schlesinger MJ and Malfer C. Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation. J Biol Chem 257: 9887–9890, 1982.[Abstract/Free Full Text]
  30. Sharp GW. Mechanisms of inhibition of insulin release. Am J Physiol Cell Physiol 271: C1781–C1799, 1996.[Abstract/Free Full Text]
  31. Straub SG, Yajima H, Komatsu M, Aizawa T, and Sharp GW. The effects of cerulenin, an inhibitor of protein acylation, on the two phases of glucose-stimulated insulin secretion. Diabetes 51, Suppl 1: S91–S95, 2002.
  32. Tamarit-Rodriguez J, Vara E, and Tamarit J. Starvation-induced secretory changes of insulin, somatostatin, and glucagon and their modification by 2-bromostearate. Horm Metab Res 16: 115–119, 1984.[ISI][Medline]
  33. Tu Y, Wang J, and Ross EM. Inhibition of brain Gz GAP and other RGS proteins by palmitoylation of G protein alpha subunits. Science 278: 1132–1135, 1997.[Abstract/Free Full Text]
  34. Van't Hof W and Resh MD. Targeting proteins to plasma membrane and membrane microdomains by N-terminal myristoylation and palmitoylation. Methods Enzymol 327: 317–330, 2000.[ISI][Medline]
  35. Vara E and Tamarit-Rodriguez J. Norepinephrine inhibits islet lipid metabolism, 45Ca2+ uptake, and insulin secretion. Am J Physiol Endocrinol Metab 257: E923–E929, 1989.[Abstract/Free Full Text]
  36. Varner AS, De Vos ML, Creaser SP, Peterson BR, and Smith CD. A fluorescence-based high performance liquid chromatographic method for the characterization of palmitoyl acyl transferase activity. Anal Biochem 308: 160–167, 2002.[ISI][Medline]
  37. Veit M, Sollner TH, and Rothman JE. Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett 385: 119–123, 1996.[ISI][Medline]
  38. Viguerie-Bascands N, Saulnier-Blache JS, Dandine M, Dauzats M, Daviaud D, and Langin D. Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes. Biochem Biophys Res Commun 256: 138–141, 1999.[ISI][Medline]
  39. Webb Y, Hermida-Matsumoto L, and Resh MD. Inhibition of protein palmitoylation, raft localization, and T cell signaling by 2-bromopalmitate and polyunsaturated fatty acids. J Biol Chem 275: 261–270, 2000.[Abstract/Free Full Text]
  40. Yajima H, Komatsu M, Sato Y, Yamada S, Yamauchi K, Sharp GW, Aizawa T, and Hashizume K. Norepinephrine inhibits glucose-stimulated, Ca2+-independent insulin release independently from its action on adenylyl cyclase. Endocr J 48: 647–654, 2001.[ISI][Medline]
  41. Yajima H, Komatsu M, Yamada S, Straub SG, Kaneko T, Sato Y, Yamauchi K, Hashizume K, Sharp GW, and Aizawa T. Cerulenin, an inhibitor of protein acylation, selectively attenuates nutrient stimulation of insulin release: a study in rat pancreatic islets. Diabetes 49: 712–717, 2000.[Abstract]
  42. Yakel JL. Calcineurin regulation of synaptic function: from ion channels to transmitter release and gene transcription. Trends Pharmacol Sci 18: 124–134, 1997.[ISI][Medline]
  43. Yamada M, Inanobe A, and Kurachi Y. G protein regulation of potassium ion channels. Pharmacol Rev 50: 723–760, 1998.[Abstract/Free Full Text]
  44. Yamada S, Komatsu M, Aizawa T, Sato Y, Yajima H, Yada T, Hashiguchi S, Yamauchi K, and Hashizume K. Time-dependent potentiation of the beta-cell is a Ca2+-independent phenomenon. J Endocrinol 172: 345–354, 2002.[Abstract/Free Full Text]
  45. Zhou YP, Priestman DA, Randle PJ, and Grill VE. Fasting and decreased B cell sensitivity: important role for fatty acid-induced inhibition of PDH activity. Am J Physiol Endocrinol Metab 270: E988–E994, 1996.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
285/2/E287    most recent
00535.2002v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (6)
Google Scholar
Articles by Cheng, H.
Articles by Sharp, G. W. G.
Articles citing this Article
PubMed
PubMed Citation
Articles by Cheng, H.
Articles by Sharp, G. W. G.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2003 by the American Physiological Society.