Glyceraldehyde phosphate: An insulin secretagogue with possible effects on inositol phosphate formation in pancreatic islets

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 26Y, No. 1, February 15, pp. 194-200.1989

Glyceraldehyde Phosphate: An Insulin Secretagogue with Possible on lnositol Phosphate Formation in Pancreatic Islets MICHAEL Department

J. MACDONALD,’ of Pediatrics,

ROBERT J. MERTZ, AND RAJENDRA

University

of

Wisconsin

Medical

School, Madison,

Wisconsin

Effects

S. RANA 53706

Received June 20,1988, and in revised form October 12,1988

The insulinotropic action of glucose, the most potent physiologic insulin secretagogue, involves its metabolism. However, no glucose metabolite has ever been identified as a key intermediate. We tested the abilities of a number of glucose metabolites to stimulate insulin release from pancreatic islets. Of all of these metabolites, glyceraldehyde 3-phosphate was the most potent insulin secretagogue. In numerous experiments over 3 years, insulin release by 4 mM glyceraldehyde phosphate ranged from 50 to 200% of that initiated by 16.7 MM glucose-a near-maximal insulin stimulus. At concentrations of 1 and 4 mM, glyceraldehyde phosphate was even more potent than the known secretagogues glucose and glyceraldehyde. Glucose metabolites were also tested for their ability to stimulate inositol tris-, bis-, and monophosphate formation by permeabilized islets. Only glyceraldehyde phosphate stimulated inositol phosphate formation and this stimulation occurred at concentrations of glyceraldehyde phosphate which could be present in the /3 cell under physiologic conditions (K0.5 = 25 FM). The current results are consistent with the idea that glyceraldehyde phosphate is a key insulinotropic glucose metabolite that might act directly (or rather directly via a receptor) on the phospholipase C that forms inositol trisphosphate in the plasma membrane. o 1989AcademicPress, I~C.

The p cell of the pancreatic islet may be unique in that the primary stimulus for excitation, i.e., insulin release, involves metabolism of a secretagogue rather than only the interaction of an agonist (the secretagogue) with a receptor. Because stimulus-secretion coupling for glucose, the most potent physiologic insulin secretagogue, involves aerobic glycolysis (l-8), we tested a number of glucose metabolites for their abilities to stimulate insulin release from pancreatic islets. Even though the rationale behind this approach was oversimplified because metabolism-induced insulin release is probably the complex aggregate of a number of processes, we reasoned that if a sufficient intracellular concentration of a particularly important intermedi-

ate was achieved, it might initiate insulin release. Of all of the glucose metabolites tested, glyceraldehyde phosphate was the only metabolite that consistently stimulated insulin release at concentrations that were lower than those at which the electrically neutral (and presumably more permeable) secretagogues glucose or glyceraldehyde, stimulated insulin release. Glucose metabolites, such as 2,3-diphosphoglycerate and fructose 1,6-bisphosphate have been shown to inhibit inositol trisphosphate breakdown and to stimulate inositol trisphosphate-induced calcium release in permeabilized pancreatic islets (9, 10) suggesting that inhibition of IPs2 dega Abbreviations used: EGTA, ethylene glycol bi@aminoethyl ether) N&‘-acetic acid; IP, inositol phosphate; IPa, inositol bisphosphate; IPa, inositol trisphosphate; PIPa, phosphatidylinositol 4,5-bisphosphate; TCA, trichloroacetic acid.

i To whom correspondence should be addressed at: University of Wisconsin Medical School, 1300 University Avenue, Room 3459, Madison, WI 53706. 0003-Y861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

194

INSULIN

AND

INOSITOL

radation may be involved in glucose-induced insulin release. However, the physiologic importance of inhibition of IPB breakdown depends upon the concomitant generation of IP3. It is known that glucose, presumably via a metabolic signal, stimulates IP3 formation in islets (Refs. (11) and (12) and unpublished data). With this in mind, various glucose metabolites were tested for their ability to stimulate inositol phosphate formation in permeabilized pancreatic islets. Only glyceraldehyde phosphate stimulated the formation of inositol phosphates at micromolar concentrations. Thus, glucose metabolism in addition to generating metabolites that inhibit IP3 degredation, produces glyceraldehyde phosphate, an intermediate that stimulates IP3 formation. Both of these effects of glycolytic products may be important in the transduction of the secretory signal in the /3cell. Part of this work has been published in preliminary form (13). EXPERIMENTAL

PROCEDURES

General procedures. Standard procedures were used to isolate pancreatic islets from well-fed Sprague-Dawley rats (4, 7,14,15), to incubate islets for studying insulin release (five islets in 2 ml KrebsRinger bicarbonate buffer, pH 7.4, containing 0.5% bovine serum albumin; l-h incubation period) (4, ‘7) and to assay insulin (16). D-Glyceraldehyde 3-phosphate concentrations were measured enzymatically (17) and glyceraldehyde plus glyceraldehyde phosphate concentrations were also measured chemically (18) using glyceraldehyde as a standard. Tests of statistical significance were calculated with raw data from within an experimental set and on pooled data with Student’s t test. When pooled data were reported, the tests with intraexperimental data and pooled data were always in agreement. All chemicals were from the Sigma Chemical Co. Concentrations of glyceraldehyde and glyceraldehyde phosphate refer to the D isomer. In preparations of DL-glyceraldehyde, the D isomer was equal to that of the L isomer. In preparations of glyceraldehyde phosphate, the concentration of the D isomer was established with the enzymatic assay. Three different commercial types of glyceraldehyde 3-phosphate were used; the free acid (consisting of 50% D isomer, catalog No. G5251), the D isomer exclusively (catalog No. G-8007), and an equal mixture of D and L isomers (catalog No. G-5376). The second and third types were prepared by

PHOSPHATES

IN ISLETS

195

hydrolysis of the diethyl diacetal salt in the presence of Dowex 50 resin. Measurement of formation of inositol phosphates. Islets (1500-2000) were cultured for 2 days at 37°C in 5 ml of inositol-free RPM1 1640 (K. C. Biologicals, Kansas City, MO) containing 10% dialyzed fetal calf serum and 100 &i of myo-[3H]inositol (15 Ci/mmol) (Amersham). In experiments requiring intact islets, islets were washed five times in Krebs-Ringer bicarbonate buffer (pH 7.3) and incubated in 180 ~1 of the Krebs-Ringer buffer for 30 min at 37°C. LiCl and test agents were then added in a volume of 20 ~1 of KrebsRinger buffer to give a final concentration of 10 mM LiCl and desired concentrations of test agents. Metabolism was stopped after 10 min by the addition of 200 ~1 of 15% TCA. In experiments requiring permeabilized islets, islets were washed and incubated for 30 min in Krebs-Ringer buffer as described above. The islets were then washed, resuspended in 180 gl of 100 mM KCl, 2 mM MgCl, , 20 @g/ml saponin, 200 fiM EGTA (free Ca” = (5 X lo-* M), and 50 mM Tris chloride, pH 7.2 (TKM buffer). After 5 min, LiCl and test agents were added in a volume of 20 ~1 of TKM buffer to give 10 mM LiCl and the desired concentrations of test agents. The incubation period was continued for 10 min at 37°C and then the reaction was stopped by adding 200 ~115% TCA. Inositol phosphates were separated (19) and radioactivity in the inositol phosphates was determined as previously described (10). The pH of each test agent was always adjusted to exactly 7.2 before it was added to the medium. In Table II and Fig. 1, “IP,” refers to the sum of inositol 1,4,5trisphosphate and inositol1,3,4-trisphosphate. RESULTS

Insulin release. Table I summarizes results of individual insulin release studies with various intermediates in experiments performed over 3 years. Because the microunits of insulin released by standard concentrations of known secretagogues, such as glucose, varies between experiments and because not all conditions can be tested within a single experiment, results from within individual experiments were compared with the insulin released by a standard concentration (16.7 mM) of glucose-usually a near-maximal insulin stimulus. Results of individual experiments are presented to demonstrate that despite the typical variability in insulin response between experiments, certain compounds consistently initiated high insulin release. One millimolar glyceraldehyde phosphate was as potent or more potent

196

MAC DONALD,

MERTZ, TABLE

SUMMARYOFINSULINRELEASEEXPERIMENTS Addition GIucose,5.5 mM Glucose, 16.7 mM (control) Glucose 6-phosphate, 10 mM Fructose &phosphate, 10 mM Fructose 1,6-bisphosphate, 10 mM Glyceraldehyde 3-phosphate, 1 mM Glyceraldehyde phosphate, 4 mM Dihydroxyacetone phosphate, 1 mM Dihydroxyacetone phosphate, 4 mM Dihydroxyacetone phosphate, 10 mM Dihydroxyacetone phosphate, 20 mM Glycerol 3-phosphate, 10 mM 2,3-Diphosphoglycerate, 4 mM 2,3-Diphosphoglycerate, 10 mM 3-Phosphoglycerate, 10 mM 2-Phosphoglycerate, 10 mM Phosphoenolpyruvate, 10 mM Pyruvate, 10 mM Glyceraldehyde, 1 mM Glyceraldehyde, 4 mM Glyceraldehyde, 10 mM Glyceraldehyde, 20 mM Dihydroxyacetone, 1 mM Dihydroxyacetone, 4 mM Dihydroxyacetone, 10 mM Dihydroxyacetone, 20 mM Methylglyoxal, 8 mM

AND

RANA

I

WITHGLYCOLYTICANDOTHERINTERMEDIATES Relative

insulin release (percentage

of control)’

25 f 5 (5), 16 f 6 (5), 21 k 4 (6), 32 f 5 (4), 0 + 0 (5), 21 f 3 (4), 24 -1-6 (5) 100 6 _t 8 (5), l_+ 3 (5) 3 + 6 (5), 2 + 4 (5) 0 + 0 (5), 3 +- 6 (5) 22 f 10 (5), 24 + 5 (3), 42 + 11(5), 64 + 5 (5), 29 f 14 (5), 20 f 0 (3) 112 f 28 (5), 54 f 0 (5), 62 I17 (4), 56 f 16 (5), 52 k 12 (5), 192 f 34 (5), 217 f 15 (5), 51 +- 2 (5) 9 2~10 (4), 11 F 8 (5), 0 + 0 (3), 3 + 6 (4) 9 +- 10 (4), 10 f 4 (5), 5 + 4 (3). 6 + 4 (4), 0 F 2 (5), 6 k 4 (5), 9 k 2 (5) 23 f 10 (3) 34 + 11 (3), 32 f 11 (5) 0 + 0 (5), 4 _’ ll(5) 3 2 3 (5) 5 t 6 (5) 0 f 0 (5), 0 f 0 (6) 0 f 0 (5) 0 f 0 (5), 3 + 6 (5), 1 f 3 (5) 0 * 0 (5), 0 i 0 (4) 0 t 0 (5), 5 _+4 (5), 9 f 2 (5), Ok 0 (5), 6 + 6 (5), 0 k 0 (5) 18 f 11 (5), 12 k 3 (5), 30 + 6 (5), 30 _+5 (5), 134 f 1’7 (5), 57 f 18 (3), 41 t 5 (5) 112 ?z41(5) 104 + 9 (5) I? 2 (5), 4 * 5 (3) 3 _‘3 (3), 9 t 5 (3), 18 f 13 (4), 12 +- 8 (4) 7 t 8 (3), 21 t 16 (5) 27 + 4 (5) 4 f 5 (10)

Note. Insulin release is expressed as a percentage of that induced by a standard concentration of glucose (16.7 mM). Seven different conditions, including 16.7 mM glucose, were separately tested within one experiment so that most compounds were tested simultaneously against glyceraldehyde phosphate. Each entry is from an individual experiment and is followed by parentheses indicating the number of replicate incubations of a condition. Insulin released in the absence of an addition averaged 16 f 6 rU/5 islets/h and insulin released in the presence of 16.7 mM glucose ranged from 156 f 14 to 286 2 39 @U/5 islets/h. The insulin released in the absence of an addition was subtracted from the insulin released in the presence of 16.7 mM glucose. This value was defined as 100%. Data are expressed as the means f SD. a Insulin release of 18% of the standard (16.7 mM glucose) or higher was significantly greater than that in the absence of an addition with a P value of at least ~0.05.

than 5.5 mM glucose, and 4 mM glyceraldehyde phosphate ranged from one-half to twice as potent as 16.7 mM glucose. Dihydroxyacetone phosphate was slightly stimulatory at 10 and 20 m&q as was dihydroxyacetone at 20 mM. Glyceraldehyde, as is well known, was stimulatory at 4, 10, and 20 mM. However, in contrast to glyceralde-

hyde phosphate, glyceraldehyde was not stimulatory at 1 mM. To rule out the possibility that the failure of the above intermediates to stimulate insulin release was not due to their lacking a fuel function, some of the intermediates were incubated with a threshold concentration (5.5 mM) of glucose as

INSULIN

AND

INOSITOL

a source of fuel (4). In four separate experiments glyceraldehyde phosphate (4 mM) was the only intermediate that strongly potentiated glucose-induced insulin release. Glyceraldehyde phosphate increased insulin release two- to threefold and 4 mM dihydroxyacetone phosphate potentiated insulin release by about 50%. Other phosphorylated intermediates (2,3diphosphoglycerate, 2- and 3-phosphoglycerate, phosphoenolpyruvate, and glycerol 3-phosphate) at concentrations of 10 or 20 mM were without effect (data not shown). Glyceraldehyde phosphate is a somewhat labile compound. Because glyceraldehyde phosphate stimulated insulin release at a lower concentration than glyceraldehyde, glyceraldehyde phosphate’s insulinotropic effect could not have been due to its breakdown to a known secretagogue, such as glyceraldehyde. Glyceraldehyde phosphate, in fact, decomposes at neutral pH to methylglyoxal(20). Neither methylglyoxal (Table I) nor ethanol (data not shown), which is released in the hydrolysis of glyceraldehyde phosphate diacetal to glyceraldehyde phosphate, stimulated insulin release. Enzymatic measurements showed that under the conditions of the insulin release experiments (37°C in KrebsRinger bicarbonate buffer, pH ‘7.4), less than one-third of the glyceraldehyde phosphate was degraded by the end of the l-h incubation period. Various commercial preparations of glyceraldehyde phosphate were tested over a 3-year period using islets from rats of different sizes (150 to 500 g) and both sexes, including retired breeders. All conditions gave similar results. If a contaminant in the preparations was responsible for insulin release, its concentration in all preparations was directly proportional to that of D-glyceraldehyde phosphate in numerous different preparations. The D isomer of glyceraldehyde phosphate is the physiologically occurring isomer. In both the insulin release experiments and the experiments of inositol phosphate formation (see below), only the D isomer of glyceraldehyde phosphate appeared to be the active isomer. No greater effects were observed with a concentration

PHOSPHATES

IN ISLETS

197

of a racemic mixture of D and L isomer equal to twice that of the D isomer alone. Formation of inositol phosphates. Glyceraldehyde phosphate at 4 MM stimulated IP, IP2, and IP3 formation in intact islets to the same extent as 20 mM glucose (Table II). Various other glycolytic intermediates were tested to measure their effects on inositol phosphate formation by permeabilized islets. Of all of the intermediates of the glycolytic pathway between glucose 6phosphate and pyruvate, only glyceraldehyde phosphate increased inositol tris-, bis-, and monophosphate formation. Since the unbound calcium concentration in the incubation mixture was buffered at 0.05 pM or lower, this effect probably did not require calcium. The concentration of Dglyceraldehyde phosphate that stimulated half-maximal formation was about 25 pM (Fig. l), whereas 2 mM concentrations of the other intermediates were without effect (Table II). DISCUSSION

The reason that glyceraldehyde phosphate is the only glycolytic intermediate that is an insulin secretagogue (Table I) might be because it is the last metabolite of glucose metabolism that is situated at a branchpoint that can feed all of the pathways necessary for stimulus-secretion coupling. Glyceraldehyde phosphate can supply substrate to the glycerol phosphate shuttle which appears to be important for carbohydrate-induced insulin release (7). The oxidation of glyceraldehyde phosphate by NAD is a major supplier of NADH to fulfill the raison d’etre of the glycerol phosphate shuttle; and the handling of reducing equivalents (possibly beyond the mere necessity of preventing a high NADH/NAD ratio from inhibiting glycolysis) appears to be important for insulin release for yet unknown reasons (5,7, 8,21-23). In addition, glyceraldehyde phosphate can be converted to 2,3-diphosphoglycerate and fructose 1,6-bisphosphate which have been demonstrated to inhibit IPB degradation (9) and potentiate IP,-induced calcium release from detergent-permeabilized islets (10).

198

MAC DONALD,

MERTZ,

AND

RANA

TABLE II

EFFECTOFVARIOUSGLUCOSEMETABOLITESONTHEFORMATION OFINOSITOLPHOSPHATES BYPERMEABILIZEDANDINTACTPANCREATICISLETS Inositol Addition

IP Permeabilized

None (18) Glucose (3) Glucose 6-phosphate (4) Fructose 6-phosphate (4) Fructose 1,6-bisphosphate

phosphate formation

(2)

Glyceraldehyde phosphate (6) Dihydroxyacetone phosphate (4) 3-Phosphoglycerate (4) Phosphoenolpyruvate (4) Glycerol 3-phosphate (3) Glyceraldehyde (4)

IP2

(% of control) IP3

islets

IOO? 6 97 -c 10 98+- 5 93* 5 9Ok 8

lOOk 8 101 + 10 102k 7 92 +- 10 92-c 11

loo+ill? 105 + 94? 84+

8 11 11 7 10

144 +- 15* lOOk 4 88_‘10 100a 7 lOOk 6 99 +- 22

144 f 34” 101 f 6 104 +- 6 99+ 6 102* 5 95 f 15

137 + 103k 112 + 94k 101 f 97+

24” 5 16 8 2 15

lOOk 7 123+ 6” 140+- 3b

lOOk 9 136 f 19’ 131k 6b

Intact islets None (7) Glucose (6) Glyceraldehyde

phosphate

(4)

loo-+ 7 144 + 17” 141 t 11*

Note. In experiments with permeabilized islets the concentration of each compound was 2 mM except for glyceraldehyde phosphate which was 0.5 mM. In experiments with intact islets the concentrations of glucose and glyceraldehyde phosphate were 20 and 4 mM, respectively. Results are expressed as a percentage of the control (no addition equals lOO%, means + SD) with the number of observations in parentheses. a P < 0.01. ‘P < 0.001 versus no addition.

Glyceraldehyde phosphate can also be metabolized to pyruvate which can be metabolized intramitochondrially. Pyruvate metabolism is essential for carbohydrateinduced insulin release, but it is not sufficient since pyruvate does not stimulate insulin release (discussed previously in Refs. (7,24)). The reason that pyruvate does not stimulate insulin release might be because glycolysis cannot be reversed from pyruvate to glyceraldehyde phosphate or to any other key intermediate in the triosephosphate segment of the glycolytic pathway. Pancreatic islets lack phosphoenoZpyruvate carboxykinase (24) which catalyzes one of the two essential steps in the conversion of pyruvate to phosphoenolpyruvate. However, even if the p cell could convert pyruvate to phosphoenolpyruvate, the NAD/NADH ratio in the /3cell does not fa-

vor the reverse of the glyceraldehyde phosphate dehydrogenase reaction (25-27). This may explain why intermediates besides pyruvate that occur distal to glyceraldehyde phosphate in the glycolytic pathway, also do not stimulate insulin release (Table I). If glyceraldehyde phosphate is the last metabolite in the glycolytic pathway that can feed all pathways necessary for insulin release, then why do glycolytic intermediates proximal to the triosephosphates, such as glucose 6-phosphate, fructose 6phosphate, and fructose 1,6-bisphosphate, not also stimulate insulin release? The reason may be that these intermediates are not taken up in amounts sufficient to generate an adequate glyceraldehyde phosphate concentration. Besides possible differences in permeability or the possibil-

INSULIN

AND

INOSITOL

1000 4

0 50 100 GLYCERALDEHYDE-PHOSPHATE

+M)

FIG. 1. Effect of glyceraldehyde phosphate on inosito1 phosphate formation by permeabilized pancreatic islets. Conditions were as described under Experimental Procedures. Results are the means f SEM and the number of observations for each point are in parentheses. All points for 25 pM and higher concentrations of glyceraldehyde phosphate were significantly different from the control (no glyceraldehyde phosphate) at the P < 0.001 level (except IP3 for which P < 0.05). The radioactivity recovered in the inositol phosphate (IP), inositol bisphosphate (IP,), and inosito1 trisphosphate (IP,) fractions in the absence of glyceraldehyde phosphate were 144.4, 15.2, and 12.8 X lo3 dpm/lOO islets/l0 min, respectively.

ity of glyceraldehyde phosphate’s occupying a strategic position in ,B cell metabolism, a major effect of glyceraldehyde phosphate may involve a direct pharmacologic-like stimulation of a key reaction in stimulus-secretion coupling. One such re-

PHOSPHATES

IN ISLETS

199

action might be that catalyzed by the plasma membrane phospholipase C that forms IPS . Permeabilized islets, which should have fairly intact plasma membranes but contain insufficient amounts of cytosolic enzymes, such as glycolytic enzymes, as well as cofactors for these enzyme reactions, were used to test for a direct effect of the various glucose metabolites on the formation of inositol phosphates. Only glyceraldehyde phosphate stimulated IP3, IP2, and IP formation in the permeabilized islets (Table II). The concentration of D-glyceraldehyde phosphate that caused half-maximal inositol phosphate formation was about 25 PM (Fig. l), which is a concentration of glyceraldehyde phosphate that is probably attained during glucose-induced insulin release (27-30). The percentage stimulation of IPs, IP2, and IP formation above that of the control in the presence of glyceraldehyde phosphate was low (2550% above the control) compared to the stimulation observed in the presence of pharmacologic agents such as carbachol(l0). However, the stimulation was comparable to that caused by glucose in intact islets (Table II) and it is known that glucose-stimulated inositol phosphate formation is low relative to that of pharmacologic agents (11,12,31). The results of the current study show that glyceraldehyde phosphate is a potent insulin secretagogue. Its effects on inositol phosphate formation raise the speculation that besides being a possible strategic intermediate of glucose metabolism, glyceraldehyde phosphate has a more direct, nonmetabolic, pharmacologic effect in signal transduction, such as assisting in the stimulation of IP3 formation by interacting with a PIP2 phosphodiesterase system on the inside of the plasma membrane either directly or via a guanine nucleotidelike receptor known to be involved in the activation of this phosphodiesterase. It is unlikely that glyceraldehyde phosphate stimulates phospholipase C by increasing cytosolic calcium since the stimulation occurred in the permeabilized cells in the presence of a calcium-EGTA buffer. ACKNOWLEDGMENTS The authors thank Dr. Henry A. Lardy and Dr. Lowell E. Hokin for helpful discussions. This work

200

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MERTZ,

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