Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells

Rengasamy Palanivel, Rajakrishnan Veluthakal, and Anjaneyulu Kowluru

Department of Pharmaceutical Sciences, Wayne State University, and {beta} Cell Biochemistry Laboratory, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48202

Submitted 29 December 2003 ; accepted in final form 11 February 2004


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 ABSTRACT
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Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrient-induced insulin secretion from isolated {beta}-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated {beta}-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phosphoenolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion.

glucose metabolism; insulin-secreting cells


THE PHOSPHORYLATION STATUS of proteins is regulated by the balance of activities of protein kinases and phosphatases, which induce the addition and removal of phosphoprotein phosphate, respectively (8, 42). Although several previous studies were focused on the identification and characterization of protein kinases in islets (4, 11, 16), little information is available on the localization and regulation of phosphoprotein phosphatases in the pancreatic {beta}-cell. With use of relatively specific inhibitors of protein phosphatase function, recent studies (1, 2, 23, 31, 36, 40) have suggested important roles for various phosphoprotein phosphatases, such as protein phosphatase 2A (PP2A), in the pancreatic {beta}-cell function. For example, okadaic acid (OKA), a selective inhibitor of protein phosphatases, stimulates basal and cAMP-stimulated insulin secretion from electrically permeabilized islets (31). There is also evidence to indicate inhibition by OKA of glucose, glyceraldehyde, and KCl-stimulated insulin secretion from the {beta}-cell (2, 40). These findings thus implicate protein (de)phosphorylation in insulin secretion. In addition to such modulatory roles, protein phosphatase activation has also been reported to be involved in the intermediary metabolism of multiple cell types (8, 42, 43). For example, acetyl-CoA carboxylase (ACC), an enzyme involved in fatty acid metabolism, is regulated by phosphorylation (inactive) and dephosphorylation (active); a PP2A-like activity has been implicated in the dephosphorylation and thereby activation of ACC (8, 42, 43). In this context, we recently characterized a glutamate- and magnesium-activated protein phosphatase in normal rat islets and clonal {beta}-cells that catalyzed the dephosphorylation and activation of ACC (18).

The COOH-terminal sequence of PP2Ac (Thr-Pro-Asp-Tyr-Phe-Leu) is highly conserved from yeast to humans, suggesting that posttranslational modification at the COOH-terminal amino acid residues (e.g., leucine) may provide a regulatory mechanism for PP2A activity (42). A growing body of experimental evidence in multiple cell types, including our own in the islet {beta}-cell, indicates that PP2Ac is subjected to posttranslational modifications, such as phosphorylation and carboxylmethylation (CML) (8, 12, 23, 45, 46). It has been suggested that the CML of PP2Ac promotes not only its interaction with other subunits of the PP2A but also its association with regulatory proteins (6, 41, 42, 44). Furthermore, we (23) and others (12) have reported that the CML of PP2Ac increases its catalytic activity. It has also been shown that OKA, a selective blocker (but not norokadaone, its inactive analog) of PP2A activity, also inhibits the CML of PP2Ac (23), suggesting a close relationship between the CML of PP2Ac and its catalytic activity (12, 23). All these data support a formulation that the CML of PP2Ac leads to its functional activation of this enzyme, presumably via assembly of the holoenzyme.

In this context, recent studies by Sjoholm et al. (38) have shown direct inhibition by glucose and its metabolites of protein phosphatase activities in the islet {beta}-cell. They also demonstrated inhibition by membrane-depolarizing concentrations of KCl of PP2A-like activity in insulin-secreting (INS) cells (37). The present study was undertaken to examine whether inhibitory effects of glucose (and its metabolites) or calcium are mediated via their effects on the CML of PP2Ac. We present evidence in support of such a formulation in insulin-secreting, glucose-responsive INS-1 cells, implicating the CML of PP2Ac with a critical regulatory role in physiological insulin secretion.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
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Materials. S-adenosyl-L-[methyl-3H]methionine (SAM) was purchased from New England Nuclear (Boston, MA). Glucose 6-phosphate, glucose-1,6-bisphosphate, fructose 6-phosphate, fructose-1,6-bisphosphate, {beta}-glycerophosphate, 2-phosphoglyceric acid, 3-phosphoglyceric acid, pyruvate, phosphoenolpyruvate (PEP), succinate, citrate, isocitrate, {alpha}-ketoglutarate, oxaloacetate, malate, fumarate, diazoxide, and mannoheptulose were purchased from Sigma (St. Louis, MO). Polyclonal antiserum directed against the methylated PP2Ac was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-mouse second antibody was purchased from BD Transduction Laboratories (Lexington, KY). All other reagents used were of highest purity available.

Sources of {beta}-cells and isolation of subcellular fractions. INS-1 cells were generously provided by Dr. Chris Newgard (Duke University Medical Center, Durham, NC). They were cultured in RPMI 1640 medium supplemented with 1 mM sodium pyruvate, 50 µM 2-mercaptoethanol, and 10 mM HEPES at pH 7.4. The medium was changed twice weekly, and cells were trypsinized and subcloned weekly. Total particulate and cytosolic fractions from INS cells were isolated by a single-step centrifugation of homogenates at 105,000 g for 60 min (Beckman Ultima TL-100), as we previously described in Refs. 21 and 22.

Quantitation of CML of PP2Ac in intact cells and cell-free preparations. This was determined by two mutually complementary methods. In the first, the carboxylmethylated PP2Ac was identified by Western blotting with an antiserum directed against the methylated form of PP2Ac. This approach enabled us to determine the abundance of carboxylmethylated PP2Ac in intact INS-1 cells and cytosolic fractions. In the second approach, we quantitated the CML of PP2Ac using [3H]SAM as the methyl donor, followed by determination of the degree of CML via the vapor-phase equilibration assay (see below for additional details).

For the first approach, INS-1-intact cells were incubated in an isotonic Krebs-Ringer bicarbonate medium in a metabolic incubator at 37°C (under an atmosphere of 95% O2-5% CO2) in the presence of glucose, mannoheptulose, diazoxide, or KCl, as we will indicate in the text. The cells (~600 x 106) were washed twice with cold PBS and scraped into isolation buffer (pH 7.4) containing (in mM) 225 mannitol, 75 sucrose, 20 HEPES, and 1 EDTA. A mixture of protease cocktail (Roche, Indianapolis, IN) was added to the homogenization medium. The cells were disrupted by sonication (2 x 10 s) on ice. For separation of cytosolic fractions, the homogenates were centrifuged for 60 min at 105,000 g (as before). Proteins in the cytosolic fraction from control and treated cells (30 µg) were separated by 10% SDS-PAGE. The separated proteins were transferred onto a nitrocellulose membrane by use of a semi-dry electrophoretic transfer unit (Bio-Rad Laboratories, Hercules, CA). Nitrocellulose filters were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% defatted dry milk (Bio-Rad Laboratories) for overnight, washed with TBST (2 x 15 min), and probed with primary antibody directed against the carboxylmethylated form of PP2Ac (anti-methyl PP2Ac; 1:1,000). Nitrocellulose membrane was again washed with TBST (2 x 15 min) and incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution in TBST). After a final wash with TBST (2 x 15 min), the immune complex was visualized by use of the enhanced chemiluminescence (ECL plus) kit (Amersham Biosciences, Piscataway, NJ).

To examine the effects of metabolites of glucose or divalent metals on the CML of PP2Ac, the cytosolic fractions from untreated cells were treated appropriately with divalent cations or metabolites of glucose, as indicated in the text. Relative abundance of the methylated PP2Ac was determined by Western blotting, as described above.

For the second approach, the reaction mixture (100 µl) consisted of 100 mM Tris·HCl (pH 7.5), 1 mM EDTA, 4 mM DTT, and the {beta}-cell cytosolic protein (25–30 µg). The reaction was initiated by the addition of [methyl-3H]SAM (100 µCi/ml; 0.7 µM) and continued for 60 min at 37°C, as indicated in the text. Glucose metabolites or divalent cations were present in concentrations as indicated (see results and Figs. 18). The reaction was terminated by the addition of SDS-PAGE sample buffer, the labeled proteins were separated by SDS-PAGE, and the degree of methylation was quantitated by vapor-phase equilibration assay, as will describe.



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Fig. 1. Inhibition by glycolytic intermediates (A) and phosphoenolpyruvate (PEP, B and C) of the carboxylmethylation (CML) of catalytic subunit of protein phosphatase 2A (PP2Ac) in insulin-secreting (INS)-1 cells. A: INS cell cytosolic fractions were incubated in the presence of a fixed concentration (2 mM) of various glucose and fructose metabolites. CML of PP2Ac was carried out, as described in MATERIALS AND METHODS, with S-adenosyl-L-[methyl-3H]methionine ([3H]SAM) as the methyl donor. Labeled proteins were separated by SDS-PAGE, and the degree of CML of PP2Ac was determined by vapor-phase equilibration assay. Data are expressed as base-labile methanol released as a percentage of control (mean ± SE of 9 individual determinations). **P < 0.01 and *P < 0.001, respectively, vs. control. B and C: INS cell cytosolic proteins were carboxylmethylated with [3H]SAM in the presence of varying concentrations (0–2 mM) of PEP (B) and a fixed concentration (2 mM) of PEP at different incubation times (C). CML of PP2A was carried out as in A. F-1,6P2, fructose 1,6-bisphosphate; 3PGA [PDB] , 3-phosphoglyceric acid; F-6-P, fructose 6-phosphate; G-6-P, glucose 6-phosphate; 2PGA, 2-phosphoglyceric acid; G-1,6P2, glucose-1,6-bisphosphate; {beta}GP, {beta}-glycerophosphate. Values are expressed as base-labile methanol released as a percentage of control (means ± SE). Data are representative of 3 independent experiments. **P < 0.01 and *P < 0.001, respectively, vs. control.

 


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Fig. 8. Dose- and time-dependent inhibition of CML of PP2Ac by calcium in INS-1 cells. An INS cell cytosolic fraction was incubated in the presence of varying concentrations (0–50 µM) of calcium for 60 min (A) or of a fixed concentration (50 µM) of calcium for different time intervals (B). CML of PP2Ac was carried out as described in MATERIALS AND METHODS with [3H]SAM as methyl donor. Labeled proteins were separated by SDS-PAGE, and degree of CML of PP2Ac was determined by vapor-phase equilibration assay. CML data are expressed as a percentage of control (means ± SE of 3 determinations in each case). **P < 0.01 and *P < 0.001, respectively, vs. control.

 
Vapor-phase equilibration assay. The {alpha}-carboxyl methyl group on the leucine 309 residue of modified PP2Ac is base labile (12, 26, 45, 46). To quantify this labeling, individual lanes of dried gels were sliced (3–5 mm) and placed in 1.5-ml Eppendorf tubes (without caps) containing 350–500 µl of 1 N NaOH. Tubes were then placed in 20-ml scintillation vials containing 5 ml of scintillant (Ultima Gold, Packard Instrument, Meriden, CT). The vials were then capped and left at 37°C overnight to facilitate the base-catalyzed hydrolysis of methyl esters and the equilibration of released [3H]methanol. After the incubation, the tubes were gently removed from the vials, and the sides of the tubes were rinsed (into the vials) with an additional 2 ml of scintillant; the radioactivity was quantitated by scintillation spectrometry (28).

Other methods. The protein concentration was determined by a dye-binding method described previously (24), with BSA as a standard. SDS-PAGE (10% acrylamide gels) was carried out as previously described (19, 21). Each experiment was repeated at least three times, and the statistical significance of differences was determined by the Student's t-test. A P value <0.05 was considered significant.


    RESULTS
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Glycolytic and tricarboxylic acid cycle intermediates of glucose metabolism inhibit the CML of PP2Ac in INS-1 cell lysates. It is well established that the intracellular concentrations of glucose metabolites [glycolytic as well as the tricarboxylic acid (TCA) cycle] vary over a large concentration range. Several earlier studies have reported concentrations of these metabolites in submillimolar or millimolar range (38, and references therein). On the basis of this information, in the initial studies we examined the effects of glycolytic and TCA cycle intermediates (2 mM each) on the CML of PP2Ac in INS-1 cell lysates. The degree of CML of PP2Ac was quantitated using [3H]SAM as the methyl donor in the absence or presence of metabolites of glucose. Data in Fig. 1A indicate that, of all the metabolites tested, PEP was the most potent. Greater than 60% inhibition of the CML of PP2Ac was noticed in the presence of PEP. Modest, but significant, inhibition of the CML of PP2Ac was also demonstrable in the presence of fructose-1,6-diphosphate, 3-phosphoglycerate, fructose 6-phosphate, and glucose 6-phosphate (Fig. 1A). Other metabolites had no measurable effects. These data establish a differential responsiveness of the CML of PP2Ac to metabolic intermediates of glucose. Data in Fig. 1B demonstrate a concentration-dependent inhibition of the CML of PP2Ac by PEP. Half-maximal inhibition was observed at PEP concentrations <500 µM. Time course experiments for PEP inhibition of the CML of PP2Ac indicated that half-maximal inhibition was demonstrable within 20 min of incubation (Fig. 1C). Together, data in Fig. 1, A, B, and C, indicate specific inhibitory effects by glucose metabolites of the CML of PP2Ac.

Next, we verified the effects of TCA cycle intermediates on the CML of PP2Ac in INS-1 cell cytosol, with [3H]SAM as the methyl donor. Data in Fig. 2A demonstrate that citrate (to a larger degree) and oxalate, malate, and isocitrate (to a lesser degree) inhibited the CML of PP2Ac. Nearly 60% inhibition of the CML of PP2Ac was noticed in the presence of 2 mM citrate. In a manner akin to PEP, half-maximal inhibition of CML of PP2Ac was observed at citrate concentrations <500 µM (Fig. 2B). Time course experiments revealed that half-maximal inhibition of CML of PP2Ac by citrate was seen within 30 min of incubation (Fig. 2C). Together, these data (Figs. 1 and 2) indicated differential, but specific, inhibitory effects by glucose metabolites on the CML of PP2Ac in INS-1 cells.



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Fig. 2. Inhibition by tricarboxylic acid (TCA) cycle metabolites (A) and citrate (B and C) of the CML of PP2Ac in INS-1 cells. A: CML of PP2Ac was quantitated in the INS cell cytosolic fraction in the presence of a fixed concentration (2 mM) of TCA cycle metabolites. CTR, citrate; OXA, oxalate; IsoCTR, isocitrate; {alpha}KG, {alpha}-ketoglutarate. CML of PP2Ac was carried out as described in Fig. 1, with [3H]SAM as methyl donor. Labeled proteins were separated by SDS-PAGE (see details in MATERIALS AND METHODS). Data are expressed as base-labile methanol released as a percentage of control (means ± SE of 3 separate experiments carried out in triplicate). **P < 0.01 and *P < 0.001, respectively, vs. control. B and C: cytosolic proteins were carboxylmethylated using [3H]SAM in the presence of varying concentrations (0–2 mM) of citrate (B) and a fixed concentration (2 mM) of citrate (C). CML assay was carried out as described in MATERIALS AND METHODS. CML data are expressed as base-labile methanol released as a percentage of control (means ± SE of 3 determinations in each case). **P < 0.01 and *P < 0.001, respectively, vs. control.

 
Exposure of INS-1 cells to glucose leads to inhibition of the CML of PP2Ac. Data described in Figs. 1 and 2 prompted us to investigate whether incubation of intact cells to glucose leads to inhibition of the CML of PP2Ac. As described in MATERIALS AND METHODS, intact INS cells were incubated in the presence of no, submaximal (5 mM), or high concentrations (10 or 25 mM) of glucose, and relative degree of abundance of methylated PP2Ac was determined in cell lysates by use of an antiserum directed against the methylated PP2Ac (Fig. 3). These data suggested a significant increase in the CML of PP2Ac in cells incubated in the presence of submaximal (basal) concentrations of glucose. Compatible with data described in Figs. 1 and 2, incubation of these cells in high (10 or 25 mM) glucose for 30 min also led to marked inhibition of the CML of PP2Ac.



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Fig. 3. Inhibition by glucose of CML of PP2Ac in INS-1 cells. Intact INS cells were incubated in the presence of various concentrations (0–25 mM) of glucose for 30 min. Cytosolic fractions were isolated by centrifugation (see MATERIALS AND METHODS). Proteins were separated by SDS-PAGE (10%) and were transferred onto a nitrocellulose membrane, which was then probed using an antiserum directed against methylated PP2Ac (36 kDa). The degree of CML of PP2Ac was quantitated by densitometry. Data are representative of 3 individual experiments, and values are expressed as a percentage of control (means ± SE). **P < 0.001 and *P < 0.01 vs. control. A representative blot is shown at top.

 
Mannoheptulose, an inhibitor of glucose metabolism, prevents glucose-mediated inhibition of CML of PP2Ac. It is well established that mannoheptulose (MH), in a competitive manner, inhibits phosphorylation, metabolism, and functional effects of glucose in the pancreatic {beta}-cell (32). To further substantiate the inhibitory effects of glucose (Fig. 3) on the CML of PP2Ac subsequent to its metabolism (Figs. 1 and 2), we studied the effect of MH on glucose-induced inhibition of the CML of PP2Ac. For this purpose, intact INS-1 cells were incubated with a high concentration of glucose (25 mM) in either the absence or presence of MH (30 mM), and the relative degree of abundance of methylated PP2Ac was determined in cytosolic fractions using an antiserum directed against the methylated PP2Ac. The experimental data described in Fig. 4 revealed that the inhibitory effect of glucose on the CML of PP2Ac was completely prevented by MH, suggesting that glucose-induced inhibitory effects on the CML of PP2Ac are largely due to its metabolism, thus compatible with data described in Figs. 1 and 2.



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Fig. 4. Protection by mannoheptulose (MH) of glucose-mediated inhibition of CML of PP2Ac in INS-1 cells. Intact INS cells were incubated in the presence or absence of MH (30 mM) or glucose (25 mM). Cytosolic fractions from each condition were isolated (see details in MATERIALS AND METHODS), and proteins were separated by SDS-PAGE (10%). Separated proteins were transferred onto a nitrocellulose membrane, which was then probed with antiserum directed against methylated PP2Ac. Relative abundance of the carboxylmethylated PP2Ac (top) was quantitated by densitometry. Values are expressed as a percentage of control (means ± SE of 3 separate experiments). *P < 0.001 vs. control.

 
Diazoxide fails to prevent glucose-mediated inhibition of the CML of PP2Ac. Data described in Figs. 13 prompted us to further investigate potential roles of glucose metabolism (i.e., generation of metabolic intermediates) in the regulation of the CML of PP2Ac. This was accomplished by use of diazoxide, which is known to inhibit membrane depolarization and the influx of extracellular calcium without significantly affecting metabolism of glucose (10, 15). Data in Fig. 5 indicate that coprovision of diazoxide (250 µM) alongside a stimulatory concentration of glucose had no demonstrable effect on glucose-mediated inhibition of the CML of PP2Ac. These data further support our hypothesis of metabolic regulation by glucose of the CML of PP2Ac in the islet {beta}-cell.



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Fig. 5. Diazoxide fails to prevent glucose-mediated inhibition of CML of PP2Ac in INS-1 cells. Intact INS cells were incubated in the presence or absence of diazoxide (250 µM) or glucose (25 mM). After incubation, cytosolic fractions were isolated (see MATERIALS AND METHODS), and proteins were separated by SDS-PAGE (10%). Separated proteins were transferred onto a nitrocellulose membrane, which was then probed with antiserum directed against methylated PP2Ac. Relative abundance of carboxylmethylated PP2Ac (top) was quantitated by densitometry. Values are expressed as means ± variance from 2 experiments.

 
A membrane-depolarizing concentration of KCl inhibits the CML of PP2Ac. The next series of experiments were carried out to examine whether a membrane-depolarizing concentration of KCl and the ensuing increase in intracellular concentration of calcium exert inhibitory effects on the CML of PP2Ac. Data in Fig. 6 indicate that significant inhibition in the CML of PP2Ac was demonstrable within 5 min of exposure to membrane-depolarizing concentration (30 mM) of KCl. Effects of KCl appeared to be time dependent, since >70% inhibition in the CML of PP2Ac was demonstrable after 30 min of incubation with KCl.



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Fig. 6. Inhibition of CML of PP2Ac by a membrane-depolarizing concentration of KCl in INS-1 cells: time course. An intact INS cell was incubated in the presence of a membrane-depolarizing concentration (30 mM) of KCl for different time intervals (0–30 min). Cytosolic fractions from each condition were isolated, and proteins were separated by SDS-PAGE (10%) and transferred onto a nitrocellulose membrane, which was then probed with antiserum directed against methylated PP2Ac. Relative abundance of carboxylmethylated PP2Ac (top) was quantitated by densitometry. Data are representative of 3 individual experiments, and values are expressed as percentage of control (means ± SE). **P < 0.01 and *P < 0.001, respectively, vs. control.

 
To determine the specificity of calcium-mediated effects, we then studied the effects of divalent cations on the CML of PP2Ac. For this purpose, effects of micomolar concentrations of calcium, magnesium, zinc, or manganese were examined on the CML of PP2Ac in INS cell lysates. Data in Fig. 7 indicate that >70% inhibition of the CML of PP2Ac was demonstrable in the presence of 50 µM calcium. A modest, but significant, inhibition of CML of PP2Ac was also demonstrable in the presence of manganese (Fig. 7). Other divalent cations elicited no measurable effects on the CML of PP2Ac. Such inhibitory effects of calcium on the CML of PP2Ac were also verified by vapor-phase equilibration assay, with [3H]SAM as the methyl donor. Data in Fig. 8A suggest a concentration-dependent inhibition by calcium of the CML of PP2Ac. Nearly 50% inhibition was demonstrable in the presence of 10 µM calcium. These data are compatible with those observations described in Fig. 6. However, the degree of inhibition by calcium of the CML of PP2Ac appeared to be greater when [3H]SAM was used as the substrate compared with a Western blot approach that uses the antiserum directed against the methylated form of PP2Ac (Fig. 6). This may be due to the fact that the vapor-phase equilibration assay is relatively more sensitive than the Western blot technique. Along these lines, the inhibitory effects of calcium appear to be time dependent (Fig. 8B), as >80% inhibition in the CML of PP2Ac was detected in the presence of 50 µM calcium within 60 min of incubation. Taken together, these data indicate that, in addition to intermediates of glucose metabolism, calcium also appears to facilitate inhibition of the CML of PP2Ac, thereby retaining the candidate proteins in their phosphorylated state, which may be required for exocytotic secretion of insulin.



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Fig. 7. Specific inhibitory effects of calcium on CML of PP2Ac in INS-1 cells. A cytosolic fraction from untreated INS cells was incubated in the presence of a fixed concentration (50 µM) of various divalent cations. CML of PP2Ac was quantitated as described in Fig. 5. Data are representative of 3 determinations in each case, and values are expressed as percentage of control (means ± SE). **P < 0.01 and *P < 0. 001, respectively, vs. control.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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One of the principal objectives of the current study was to examine the metabolic effects of glucose on the CML of PP2Ac. Our data indicated that 1) specific metabolites of glucose (glycolytic as well as TCA cycle intermediates) inhibit the CML of PP2Ac in INS cell lysates; 2) stimulatory concentrations of glucose or a membrane-depolarizing concentration of KCl can also inhibit the CML of PP2Ac in intact INS-1; 3) inhibitory effect of glucose on the CML of PP2Ac is prevented by coprovision of MH, an inhibitor of glucose metabolism; and 4) inhibitory effect of glucose on the CML of PP2Ac is not prevented by diazoxide. These data raise an interesting possibility, that metabolites of glucose and calcium could directly regulate (de)phosphorylation of specific {beta}-cell proteins that might be relevant to insulin secretion.

Several original studies by us (20) and those of others (3, 11, 16, 17, 25, 29, 30, 35, 37, 38, 47) have indicated stimulation by insulin secretagogues of phosphorylation of {beta}-cell proteins, implying that such an event is critical for insulin secretion. As indicated above, net phosphorylation status of a given protein is determined by the efficiency and concerted actions of protein kinases as well as protein phosphatases. The present study provides the first evidence to suggest metabolic regulation by glucose of the CML of PP2Ac. These findings could have direct relevance to the functional regulation of this protein phosphatase in the {beta}-cell for the following reasons.

First, emerging evidence indicates that CML of PP2Ac is necessary for the assembly of the holoenzyme, since it has been demonstrated in multiple cell types that the CML of PP2Ac increases the affinity of the regulatory B subunit to the A and C subunit complexes (6, 41, 42, 44). Second, it has been shown in earlier studies, including our own in the {beta}-cell, that the CML of PP2Ac results in a significant increase in the catalytic activity of PP2A (12, 23). Third, using specific inhibitors of the carboxyl methyl esterase of PP2Ac (e.g., ebelactone), we have been able to demonstrate reversible inhibition of glucose- or {alpha}-ketoisocaproate-induced insulin secretion from normal rat islets (23). Finally, OKA, a specific inhibitor of the CML of PP2Ac (2, 40), markedly inhibits nutrient-induced insulin secretion from isolated {beta}-cells. These data provided important clues with regard to potential regulation of insulin secretion in a metabolic cascade involving the CML of PP2Ac.

In support of these data are several recent studies by Sjoholm and coworkers (33–38), which demonstrated significant inhibition of PP2A activity by various insulin secretagogues as well as metabolites of glucose. Using {beta}-cell homogenates, these investigators described evidence for inhibition of PP2A activity by metabolites of glucose (38). Minor variations and differences between our findings and those of Sjoholm (33) on the magnitude of effects by these modulators may, in part, be due to the fact that these studies employed 10 mM concentrations of these metabolites as opposed to the 2 mM concentrations that we employed in the current studies.

In the above context, it is important to note that the intracellular concentrations of glucose metabolites (glycolytic as well as the TCA cycle) vary over a large concentration range. Several earlier studies have reported concentrations of these metabolites in submillimolar or millimolar range. A recent publication by Sjoholm et al. (38) discussed these points in a detailed fashion. Our data indicated that half-maximal inhibition of the CML of PP2Ac was demonstrable at 500 µM concentration of PEP or citrate. Indeed, published evidence indicates that such concentrations of PEP or citrate are attained in the stimulated {beta}-cell. For example, original studies by Sugden and Ashcroft (39) reported PEP levels as high as 1.2 mM in a glucose-stimulated (rat) islet. Likewise, studies by Boquist (5) estimated PEP concentrations at 0.89 mM in a glucose-stimulated (mouse) islet (recalculated on the basis of mean islet volume on 1 nl, per Sugden and Ashcroft; see Ref. 39). In the same study, Boquist reported citrate levels as high as 1.82 mM in a mouse islet stimulated by high glucose. These findings further support our current observations of regulation by glucose metabolites of the CML of PP2Ac and suggest that they are physiologically relevant. Compatible with our findings of potential regulatory effects of PEP and 3-phosphoglycerate are observations by Pek et al. (29), who demonstrated a significant increase in protein phosphorylation in the presence of 3-phosphoglycerate and PEP in isolated {beta}-cells. Taken together, our current data provide additional evidence to support the viewpoint that glucose and calcium could contribute toward the net phosphorylation of islet proteins. Additional studies are needed to determine the identity of the phosphoprotein substrates whose dephosphorylation is catalyzed by PP2A in the islet {beta}-cell, culminating in insulin secretion.

We also observed that KCl induced significant inhibition of the CML of PP2Ac, implicating a membrane depolarization-associated increase in the intracellular calcium as regulator of the CML of PP2Ac. Our data in broken cell preparations with exogenous calcium (Figs. 7 and 8) also support a regulatory role for calcium in the regulation of the CML of PP2Ac. However, it should be kept in mind that several earlier studies in isolated {beta}-cells (13, 14, 27) have reported intracellular calcium concentrations in a stimulated {beta}-cell to be ~200–400 nM. Even when the fact is considered that there may be spatial differences in calcium concentrations within specific sites in the {beta}-cell (e.g., just underneath the plasma membrane and/or near the endoplasmic reticulum stores), it is unlikely that intracellular concentrations of calcium would reach those high levels. Therefore, although the KCl data are convincing and implicate a potential role for an increase in the intracellular calcium in the regulation of CML of PP2Ac, caution is warranted in interpreting findings derived from broken cells and exogenous calcium.

Our original studies on the CML of PP2Ac in insulin-secreting cells have provided important clues with regard to the potential contribution of CML of PP2Ac to the catalytic activation of this protein (23). In these studies, we reported that OKA, in low nM concentrations a specific inhibitor of PP2A, inhibited the CML of PP2Ac in a dose-dependent manner in INS-1 cell cytosol. Maximal inhibition (nearly 70%) was obtained in the presence of 50 nM OKA. Under those conditions, we observed a 70% inhibition of the catalytic activity of PP2A in cognate fractions from INS-1 cells. On the basis of this information, we estimate >60% inhibition of the catalytic function of PP2A in INS-1 cells in the presence of maximal inhibitory concentrations of either PEP or citrate; this estimate is compatible with observations of Sjoholm et al. (38) on the inhibitory effects of glucose metabolites on protein phosphatase activity in {beta}-cells.

On the basis of our current findings and the available information to date, we propose the following model for potential regulation by glucose of the CML of PP2A and subsequent insulin secretion (Fig. 9). In support of this model, we report that specific glucose metabolites, generated via glycolysis and the TCA cycle, inhibit the CML of PP2Ac. Such an inhibition in the CML of PP2Ac could be brought about by either an increase in the methyl transferase activity or a decrease in the methyl esterase activity, or both. In either case, inhibition of the CML leads to a significant decrease in the association of various subunits of PP2A (e.g., regulatory, structural, and the catalytic subunits), leading to potential decrements in the catalytic function of the enzyme. Our formulation of metabolic effects of glucose on the CML of PP2Ac gains further support from our observations that MH, an inhibitor of glucose metabolism, prevented glucose-mediated inhibition of the CML of PP2Ac. Our data also suggest that an increase in intracellular calcium could contribute toward the inhibition of CML of PP2Ac. As indicated above, it remains to be seen whether glucose and/or calcium exerts direct stimulatory effects on the carboxylmethyl esterase activity or inhibitory effects on carboxylmethyl transferase activity. Our data using [3H]SAM tend to support the formulation that they could mediate inhibition of the methyl transferase activity. In either case, it is expected that under such conditions of decreased methylated state of PP2Ac, significant alterations in the interaction of structural, regulatory, and catalytic subunits ensue, resulting in inhibition of the catalytic function of the enzyme. This, in turn, would retain putative signaling proteins in the phosphorylated state, which may be essential for exocytotic secretion of insulin.



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Fig. 9. A proposed model for involvement of PP2A in insulin secretion: potential regulation by glucose and calcium. We propose that CML of PP2Ac represents a key regulatory step in the sequence of events leading to insulin secretion. Relative degree of abundance of PP2Ac represents a reflection of the concerted actions of its carboxylmethyl transferase and carboxylmethyl esterase activities. In our current experimental paradigm, it appears that glucose and calcium specifically reduce the CML of PP2Ac, leading to significant alterations in the subunit association of PP2Ac and thereby decreasing the holoenzyme assembly. There is ample evidence in the literature to suggest that CML of PP2Ac facilitates its interaction with other subunits and promotes the formation of the holoenzyme (see text). As shown by us and others previously, inhibition of CML of PP2Ac could also lead to inhibition of the catalytic function of PP2A (12, 23). This formulation gains further support from recent studies (38) indicating significant inhibition by metabolites of glucose of PP2A activity in insulin-secreting cells. We propose, further, that such an inhibition in PP2A activity results in retention of putative exocytotic proteins in their phosphorylated state, which may be necessary for insulin secretion. It is important to point out that several earlier studies from us and others (9, 16, 17, 20, 25, 30, 37, 47) have indicated glucose-mediated increases in phosphorylation of several signaling proteins in the islet {beta}-cell. Together, our current findings and extant observations in this area indicate rather dual effects by nutrient secretagogues, such as glucose, on the phosphorylation of candidate proteins, namely, stimulation of kinases as well as inhibition of respective phosphoprotein phosphatases. Additional studies are needed to identify the relevant proteins whose net phosphorylation status is controlled by insulin secretagogues.

 
In conclusion, our current studies provide first evidence for modulation of posttranslational modification of PP2Ac by glucose and calcium in isolated {beta}-cells. These findings, along with recent data on potential direct effects of these modulators on the catalytic function of PP2A (38), provide novel insights into potential negative modulation by glucose of the {beta}-cell protein phosphatases. The current as well as earlier findings of potential dual regulatory effects of glucose on islet protein phosphorylation, namely, stimulation of protein kinases as well as inhibition of protein phosphatase function, further strengthen the original, long-held hypothesis that protein phosphorylation is critical for glucose-stimulated insulin secretion.


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These studies were supported by grants (to A. Kowluru) from the National Institute of Diabetes and Digestive and Kidney Disorders (DK-56005), Department of Veterans Affairs (Merit Review and REAP awards), and the American Diabetes Association. A. Kowluru is the recipient of a Research Career Scientist award from the Department of Veterans Affairs.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Kowluru, Dept. of Pharmaceutical Sciences, Wayne State Univ., 259 Mack Ave., Detroit, MI 48202 (E-mail:akowluru{at}med.wayne.edu).

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.


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