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Assessment of the Nutritional Value of Glycerol-1,2,3-tris(methylsuccinate) in Fed and Starved Rats
Molecular Genetics and Metabolism 67, 254 –260 (1999) Article ID mgme.1999.2869, available online at http://www.idealibrary.com on
Assessment of the Nutritional Value of Glycerol-1,2,3tris(methylsuccinate) in Fed and Starved Rats L. Ladrie`re,* F. Bjo¨rkling,† and W. J. Malaisse* ,1 *Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium; and †Leo Pharmaceutical Products, Ballerup, Denmark Received January 29, 1999, and in revised form April 13, 1999
the treatment of non-insulin-dependent diabetes mellitus. The use of succinic esters in such a perspective is often limited, however, by the high amounts required to achieve significant stimulation of insulin secretion in vivo and, hence, by their nutritional value in extrapancreatic tissues, e.g., their role as gluconeogenic precursors in hepatocytes (3,4). In the present study, the nutritional value of G3MS was assessed by investigating its effects upon metabolic, hormonal, and enzymatic variables in both fed and starved rats. For this purpose, the ester was infused for 3 days at a rate of 1.2 mmol z day 21 z g body wt 21. Such an infusion rate corresponds to a total daily supply of G3MS representing more than 15 times the amount of the ester (0.07 mmol z g body wt 21) previously found to stimulate insulin release and to enhance the insulinotropic action of gliquidone and repaglinide when injected intravenously over 30 s in fed anesthetized rats (2).
The nutritional value of glycerol-1,2,3-tris(methylsuccinate), a novel ester of succinic acid with high insulinotropic efficiency both in vitro and in vivo, was assessed in both fed and starved rats. The infusion of the ester, given in a daily amount (1.2 mmol z g body wt 21) well in excess of what could result from its repeated intravenous administration as an insulinotropic agent in non-insulin-dependent diabetes (0.07 mmol z g body wt 21 for each administration), failed to prevent the fall in body weight, liver and muscle glycogen contents, and plasma D-glucose or insulin concentration, as well as the increase in plasma free fatty acid and b-hydroxybutyrate concentrations caused by starvation. The sole indications that the ester may serve, to a limited extent, as an alternative nutrient in starved rats consisted in a somewhat higher weight of both liver and paraovarian adipose tissue and somewhat higher activity of liver glucokinase in rats receiving the ester than in animals infused with saline. The low nutritional value of this ester thus answers the objection of its possible role as an extrapancreatic nutrient or gluconeogenic precursor in the perspective of its use as an insulinotropic tool in type 2 diabetes. © 1999 Academic Press Key Words: glycerol-1,2,3-tris(methylsuccinate); insulin secretion; starvation; non-insulin-dependent diabetes.
MATERIALS AND METHODS Glycerol-1,2,3-tris(methylsuccinate) was prepared by direct esterification of glycerol by monomethyl succinic acid ester chloride (5). The experimental design and analytical procedures used to assess its nutritional value were the same as those used in prior studies (6,7). Briefly, a catheter was introduced, under anesthesia with ketamine (125 mg/g body wt given intraperitoneally), in the right heart of female Wistar rats. During the following 2–3 days, the animals were given free access to food (KM-04-k12; Pavan Service, Oud Turnhout, Belgium) and tap water. They were then starved or not
Glycerol-1,2,3-tris(methylsuccinate) (G3MS) is a novel ester of succinic acid with high insulinotropic efficiency both in vitro (1) and in vivo (2). It was proposed, therefore, as a new insulinotropic tool for 1 To whom correspondence should be addressed at Laboratory of Experimental Medicine, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium. Fax: 132-2-5556239.
254 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
TABLE 1 Liver Weight, Protein Content, and Glycogen Content Rats:
Fed Nil
Saline
Saline 1 G3MS
Nil
Saline
6.55 6 0.30 3.42 6 0.13
7.68 6 0.31 3.84 6 0.11
8.26 6 0.31 4.10 6 0.14
4.67 6 0.09 2.66 6 0.10
5.25 6 0.20 2.95 6 0.08
5.70 6 0.20 3.18 6 0.08
(8) (8)
224 6 10
218 6 19
199 6 8
223 6 15
232 6 14
198 6 11
(8)
211.6 6 15.2
141.9 6 7.0
114.9 6 4.9
7.3 6 0.9
9.9 6 1.7
10.1 6 2.9
(8)
Infusion: Wet weight g % of body wt Protein content mg/g wet wt Glycogen content mmol of glucose residue/g wet wt
Starved
for 3 days and simultaneously infused with saline (NaCl 0.9%; Baxter, Lessines, Belgium) containing, when required, G3MS (10.0 mM) and delivered at a rate of 120 ml z day 21 z g body wt 21. Blood samples were collected in conscious rats at about 9 AM from the severed tip of the tail. On the last day, blood was also collected upon decapitation of the rats about 5 min after the tail blood sampling for the measurement of plasma D-glucose, insulin, free fatty acids, and b-hydroxybutyrate concentration. The liver and paraovarian fat weight, liver and gastrocnemius protein and glycogen contents, and the activity of glucokinase in liver homogenates were also measured. All results are expressed as mean values (6SEM) together with the number of individual observations. The statistical significance of differences between mean values was assessed by Student’s t test. RESULTS Body Weight Before surgery, the body weight of the rats averaged 204 6 2 g (n 5 32). The rats lost only 1 to 2 g (n 5 26) over the 2- to 3-day period between surgery and onset of the G3MS infusion. Over the ensuing 3 days, fed rats lost 5 6 1 and 10 6 2 g (n 5 8 in both cases) when infused with saline alone or saline containing G3MS, respectively. Over the same period of 3 days, starved rats lost 31 6 1 g (n 5 8) when not infused, 23 6 2 g (n 5 8) when infused with saline alone, and 25 6 2 g (n 5 8) when infused with saline containing G3MS. Liver Weight and Protein and Glycogen Contents As shown in Table 1, the liver wet weight, when expressed relative to paired body weight, was much
Saline 1 G3MS (n)
lower (P , 0.001) in starved rats than in fed rats. In both cases, it was higher (P , 0.05) in saline-infused animals than in noninfused rats. It was even further increased (P , 0.02) in rats infused with saline containing G3MS, the values recorded in these animals averaging 107.9 6 2.2% (n 5 16) of the mean corresponding value (100.0 6 1.9%; n 5 16) found in rats in the same nutritional state (fed or starved) infused with saline alone. Incidentally, the presence of G3MS in saline also increased (P , 0.02) the wet weight of paraovarian adipose tissue in both fed and starved rats, the values recorded in the animals receiving the ester averaging 125.1 6 5.9% (n 5 16) of the mean corresponding value (100.0 6 8.0%; n 5 16) found in rats with the same nutritional status (fed or starved) and infused with saline alone. The protein content of the liver was not significantly different in fed and starved rats, animals infused with saline or not, and rats receiving saline alone or saline and G3MS. The glycogen content of the liver was dramatically lower in starved rats than in fed rats (Table 1). In the fed rats, it was lower (P , 0.001) in saline-infused rats than in control animals and further decreased (P , 0.01) when G3MS was present in the saline infusion. These changes are in fair agreement with the effects of saline alone and saline together with G3MS on body weight, both series of data suggesting a lesser food intake in saline-infused rats than in control animals and a further decrease of food intake due to the administration of G3MS. In the starved rats, however, the liver glycogen content was not lower in salineinfused animals than in control noninfused rats and in animals receiving G3MS than in rats infused with saline alone.
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TABLE 2 Gastrocnemius Protein and Glycogen Contents Rats:
Fed
Infusion: Protein content mg/g wet wt Glycogen content mmol of glucose residue/g wet wt
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
135.3 6 8.3
115.1 6 6.5
124.0 6 10.6
123.6 6 8.9
153.3 6 12.2
125.0 6 8.4
(8)
13.5 6 1.0
13.0 6 1.3
15.8 6 2.1
4.8 6 0.9
5.8 6 0.8
6.3 6 1.0
(8)
Muscle Protein and Glycogen Contents The protein content of the gastrocnemius, like that of the liver, was not affected significantly by starvation, saline infusion, or G3MS administration (Table 2). The glycogen content of the gastrocnemius was lower (P , 0.001) in starved rats than in fed rats. Whether in fed or starved animals, it was not significantly different in control rats, saline-infused animals, and those receiving both saline and G3MS. In both fed and starved rats, the administration of G3MS tended to increase the muscle glycogen content, but this difference failed to achieve statistical significance. Plasma Concentration of Free Fatty Acids and bHydroxybutyrate In blood samples collected from the severed tip of the tail, the plasma free fatty acid concentration was lower (P , 0.001) in fed rats (643 6 38 mM; n 5 48) than in starved animals (1471 6 129 mM; n 5 7). In the fed rats, it failed to be affected significantly by the infusion of saline alone or saline together with G3MS, the paired difference between the last (Day 3) and first (Day 0) measurements averaging, respectively, 134 6 92 and 1131 6 62 mM (n 5 8 in both cases). In the starved rats, such a paired difference averaged 1607 6 161 mM (n 5 7; P , 0.01)
Saline 1 G3MS (n)
in the control noninfused animals, 1526 6 57 mM (n 5 8; P , 0.001) in the saline-infused rats, and 1570 6 67 mM (n 5 8; P , 0.001) after infusion of saline and G3MS. The latter three mean values were not significantly different from one another. The handling of the rats at the time of the last bleeding from the tip of the tail and the subsequent killing of the animals by decapitation failed to affect significantly the plasma free fatty acid concentrations in fed rats, with a mean value for the paired difference between the measurements made at and shortly before sacrifice of 227 6 60 mM (n 5 20). In the starved rats, however, such a paired difference averaged 2376 6 64 mM (n 5 23; P , 0.001). These two mean values refer, in each case, to control (noninfused) rats as well as animals infused with saline alone or together with G3MS, the results being comparable under these three experimental conditions (Table 3). In the blood samples obtained from the tail, the plasma concentration of b-hydroxybutyrate was also lower (P , 0.001) in fed rats (142 6 7 mM; n 5 47) than in starved rats (1525 6 309 mM; n 5 8). In the fed rats, no significant change was observed after the 3 days of infusion of saline alone or together with G3MS, with mean paired differences between Day 3 and Day 0 of 118 6 12 and 18 6 6 mM (n 5 8 in both cases), respectively (Table 4). In the starved rats,
TABLE 3 Plasma Free Fatty Acid Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
763 6 65 (8) 839 6 158 (6)
613 6 105 (8) 591 6 68 (8) 503 6 73 (8) 647 6 59 (8) 520 6 50 (7)
517 6 97 (8) 545 6 86 (8) 639 6 77 (7) 649 6 49 (8) 574 6 57 (7)
809 6 94 (8) 1219 6 55 (8) 1579 6 69 (8) 1471 6 129 (7) 1080 6 67 (8)
516 6 76 (8) 938 6 34 (8) 1119 6 99 (8) 1041 6 89 (8) 658 6 58 (8)
639 6 81 (8) 1029 6 60 (8) 1279 6 79 (8) 1222 6 88 (8) 835 6 90 (8)
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GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
TABLE 4 Plasma b-Hydroxybutyrate Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
154 6 8 (8) 277 6 85 (6)
164 6 24 (8) 179 6 22 (8) 173 6 18 (8) 182 6 22 (8) 238 6 30 (8)
91 6 5 (8) 110 6 8 (8) 125 6 27 (8) 99 6 5 (8) 167 6 10 (8)
187 6 9 (8) 817 6 63 (8) 1108 6 191 (8) 1525 6 309 (8) 1360 6 314 (8)
129 6 10 (8) 580 6 72 (8) 1130 6 134 (8) 1516 6 186 (8) 1425 6 226 (8)
127 6 10 (7) 508 6 62 (8) 1268 6 222 (8) 1595 6 301 (8) 1451 6 296 (8)
such paired differences averaged 11339 6 317 mM (n 5 8; P , 0.005) in the control noninfused animals, 11387 6 180 mM (n 5 8; P , 0.001) in the salineinfused rats and, 11512 6 337 mM (n 5 7; P , 0.005) after infusion of saline together with G3MS. These three mean values are not significantly different from one another. Pooling together the results obtained in control (noninfused) rats and animals infused with saline, whether alone or together with G3MS, the paired difference between the measurements made at killing and shortly before sacrifice (see above) averaged 178 6 27 mM (n 5 21; P , 0.01) in fed rats, as distinct (P , 0.005) from 2133 6 61 mM (n 5 24; P , 0.05) in starved animals. Plasma Concentrations of D-Glucose and Insulin Over 3 days of starvation, the plasma concentration of D-glucose, as measured in samples obtained from the tail of the rats, decreased (P , 0.001) from 6.81 6 0.12 mM (n 5 48) in fed animals to 4.55 6 0.41 mM (n 5 8) in starved rats (Table 5). In the fed rats, it failed to be affected after 3 days of saline infusion, with or without G3MS, the paired difference between Day 3 and Day 0 averaging 20.06 6 0.35 mM (n 5 8) in animals infused with saline alone and 20.07 6 0.17 mM (n 5 8) in rats infused
with saline containing G3MS. In the starved rats, such a paired difference amounted to 22.65 6 0.49 mM (n 5 8; P , 0.005) in the control animals (no infusion), 22.88 6 0.27 mM (n 5 8; P , 0.001) in saline-infused rats, and 22.21 6 0.29 mM (n 5 8; P , 0.001) in animals receiving both saline and G3MS. These three mean values were not significantly different from one another. The handling of the rats at the end of the experiments (see above) always increased the plasma concentration of D-glucose. Such an increment averaged 2.12 6 0.23 mM (n 5 21; P , 0.001) in fed rats and 2.28 6 0.18 mM (n 5 24; P , 0.001) in starved rats, these two mean values being comparable to one another (P . 0.5). In the samples collected from the severed tip of the tail, the plasma concentration of insulin was lower (P , 0.001) in starved rats (7.5 6 1.5 mU/ml; n 5 8) than in fed animals (34.0 6 1.9 mU/ml; n 5 48). It decreased significantly during infusion of saline, with or without G3MS, in fed rats, the paired difference between Day 3 and Day 0 averaging 211.0 6 2.8 mU/ml (n 5 8; P , 0.01) in the animals receiving saline alone and 27.4 6 2.8 mU/ml (n 5 8; P , 0.05) in those receiving saline and G3MS (Table 6). These two mean values are not significantly different from one another. In the starved rats, the
TABLE 5 Plasma D-Glucose Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
7.29 6 0.28 (8) 9.74 6 0.23 (6)
6.93 6 0.21 (8) 7.24 6 0.26 (8) 6.74 6 0.20 (8) 6.87 6 0.25 (8) 8.58 6 0.49 (8)
6.38 6 0.14 (8) 6.60 6 0.42 (8) 6.30 6 0.26 (8) 6.31 6 0.07 (8) 8.68 6 0.44 (7)
7.20 6 0.26 (8) 5.05 6 0.10 (8) 4.27 6 0.10 (8) 4.55 6 0.41 (8) 6.87 6 0.37 (8)
6.94 6 0.44 (8) 4.88 6 0.64 (8) 4.15 6 0.49 (8) 4.06 6 0.38 (8) 6.51 6 0.38 (8)
6.12 6 0.12 (8) 4.72 6 0.13 (8) 4.23 6 0.19 (8) 3.91 6 0.25 (8) 5.96 6 0.52 (8)
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TABLE 6 Plasma Insulin Concentration (mU/ml) Rats:
Fed Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
27.8 6 5.4 (8) 27.3 6 2.2 (6)
31.3 6 3.7 (8) 26.8 6 2.3 (8) 23.4 6 3.6 (8) 20.3 6 2.4 (8) 20.8 6 4.4 (7)
30.4 6 1.7 (8) 24.4 6 1.5 (8) 23.4 6 1.8 (8) 22.9 6 2.1 (8) 33.0 6 5.6 (7)
26.3 6 2.4 (8) 8.5 6 1.7 (8) 4.6 6 1.4 (8) 7.5 6 1.5 (8) 8.6 6 2.1 (8)
39.8 6 2.8 (8) 10.0 6 1.9 (8) 5.3 6 1.1 (8) 4.1 6 1.3 (8) 7.2 6 1.8 (8)
48.5 6 5.8 (8) 8.2 6 0.8 (8) 5.7 6 1.3 (8) 4.9 6 0.9 (8) 5.3 6 1.6 (8)
Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Starved
paired difference in plasma insulin concentration at Day 3 minus Day 0 averaged 218.8 6 1.7 mU/ml (n 5 8; P , 0.001) in the control animals (no infusion), 235.7 6 3.4 mU/ml (n 5 8; P , 0.001) in the rats infused with saline alone, and 243.5 6 6.2 mU/ml (n 5 8; P , 0.001) after infusion of both saline and G3MS. Such paired differences were thus more marked (P , 0.05 or less) in starved rats than in fed rats. In the former animals, the starvation-induced decrease in plasma insulin concentration was higher (P , 0.005 or less) in the infused rats than in the control animals, but not significantly different (P . 0.2) after infusion of saline alone or together with G3MS. The increase in plasma D-glucose concentration at the end of the experiments (see above) failed to be associated with any significant increase in plasma insulin concentration. Thus, the paired difference between the measurements make at sacrifice and shortly before averaged in fed and starved rats, respectively, 12.7 6 3.7 mU/ml (n 5 20; P . 0.4) and 11.5 6 1.1 mU/ml (n 5 24; P . 0.15). Glucokinase Activity in Liver Homogenates It was previously documented that certain esters of succinic acid, when infused in starved rats, oppose the effect of starvation to decrease the activity of liver glucokinase (6 – 8). In the present experiments,
the activity of the glucokinase was measured, therefore, in liver homogenates from either fed or starved rats, saline-infused or control (no infusion) animals, and rats infused with saline alone or both saline and G3MS (Table 7). At a low concentration of D-glucose (1.0 mM), the activity of glucokinase was judged from measurements made in the concomitant presence of 3.0 mM D-glucose 6-phosphate, used to inhibit the low K m hexokinase isoenzyme, and 1.0 mM D-fructose 1-phosphate, used to relieve glucokinase from any inhibitory action mediated, at the intervention of its regulatory protein, by D-fructose 6-phosphate, which is rapidly generated in tissue homogenates from exogenous D-glucose 6-phosphate. In fed rats, the activity of glucokinase (always expressed as pmol z min 21 z mg wet wt 21), at the low concentration of D-glucose, was not vastly different in control animals (104 6 4; n 5 8), saline-infused rats (94 6 4; n 5 8), and animals infused with both saline and G3MS (106 6 4; n 5 8). In starved animals, the values averaged 81 6 3, 63 6 3, and 91 6 2 (n 5 8 in all cases) in control, saline-infused, and G3MS-infused rats. The activity of glucokinase was thus higher (P , 0.001) in fed than starved animals. In the latter rats, it seemed slightly decreased by saline infusion (P , 0.005), the infusion of G3MS apparently protecting (P , 0.001) against such a decrease.
TABLE 7 Glucokinase Activity in Liver Homogenates (pmol z min 21 z mg wet wt 21) Rats:
Fed
Starved
Infusion:
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
(n)
1.0 mM D-glucose a 10.0 mM D-glucose a
104 6 4 1,045 6 40
94 6 4 885 6 55
106 6 4 868 6 46
81 6 3 730 6 42
63 6 3 485 6 31
91 6 2 618 6 30
(8) (8)
a
All measurements were made in the presence of 3.0 mM D-glucose 6-phosphate and 1.0 mM D-fructose 1-phosphate.
259
GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
Whether in fed or starved rats, the reaction velocity for D-glucose phosphorylation, at the low concentration of D-glucose, was much lower in the presence of the exogenous hexose esters than in their absence. It averaged, in fed and starved rats, respectively, 33.4 6 2.9 and 26.1 6 2.4% (n 5 24 in both cases). As expected, such a relative value was thus lower in the starved rats with low glucokinase activity. Such was the case (P , 0.001 in all cases) in control (noninfused), saline-infused, and G3MS-infused rats, the reaction velocity in the presence of the exogenous esters relative to that found in their absence averaging, in the starved rats, 77.9 6 2.0% (n 5 24; P , 0.001) of that found in fed rats (100.0 6 2.0%; n 5 24) treated under the same conditions (i.e., not infused, infused with saline, or infused with both saline and G3MS). At a higher concentration of D-glucose (10.0 mM), the activity of glucokinase in fed rats, as judged from the measurements made in the presence of exogenous D-glucose 6-phosphate and D-fructose 1-phosphate and expressed as pmol z min 21 z mg wet wt 21, was somewhat higher (P , 0.05 or less) in control (noninfused) animals (1045 6 40; n 5 8) than in either saline-infused rats (885 6 55) or after infusion of both saline and G3MS (868 6 46). In starved animals, the value found in control (noninfused) rats (730 6 42) was again lower (P , 0.001) than that found in fed rats but higher (P , 0.001) than that recorded in saline-infused starved animals (485 6 31). The administration of G3MS increased (P , 0.01) the activity of glucokinase to 618 6 30 in the saline-infused starved animals, such a value remaining lower (P , 0.05), however, than that found in the control starved rats. At variance with the situation found at 1.0 mM D-glucose (see above), the rate of D-glucose phosphorylation at a 10.0 mM concentration of the hexose was comparable in the absence and presence of exogenous D-glucose 6-phosphate and D-fructose 1-phosphate, as expected from the low fractional contribution of the low K m hexokinase isoenzyme to the overall reaction velocity. Thus, the results recorded in the presence of the exogenous phosphate esters, when expressed relative to those found in the absence of such esters, averaged 86.3 6 3.5 and 100.9 6 6.0% (n 5 24 in both cases) in fed and starved rats, respectively. The latter two mean values were not significantly different from one another.
DISCUSSION The present results reveal that the infusion of G3MS, given in a daily amount (1.2 mmol z g body wt 21) largely in excess of the cumulative value that would result from repeated intravenous administrations of the ester to stimulate insulin release or potentiate the B-cell secretory response to hypoglycemic sulfonylureas or meglitinide analogs (0.07 mmol z g body wt 21 for each administration), only displays minimal nutritional value, whether in fed or starved rats. Thus, the infusion of the ester failed to prevent the fall in body weight and liver glycogen content seen in saline-infused rats with either free access to food or starved for 3 days. It also failed to affect, in either fed or starved rats, the muscle glycogen content, which was decreased by starvation, the plasma concentrations of free fatty acids and b-hydroxybutyrate, which both increased during starvation, and the plasma concentrations of D-glucose and insulin, which both decreased in the starved rats. The sole indications that G3MS may have served, to a limited extent, as an alternative nutrient in starved rats consisted in a somewhat higher weight of both liver and paraovarian adipose tissue and a somewhat higher activity of liver glucokinase in the rats receiving the ester than in the animals infused with saline alone. Incidentally, the present study also indicates that, in both fed and starved rats, the handling of rats at the time of the last bleeding from the tip of the tail and, shortly thereafter, at the time of killing by decapitation results in an increase in plasma D-glucose concentration, not associated with any increase in plasma insulin concentration. In the starved rats, a decrease in both plasma free fatty acid and b-hydroxybutyrate concentrations was also observed, while the concentration of the ketone body increased in fed rats at the same time. Such changes were already reported and their determinism discussed in prior publications (9 –11). No significant effect of G3MS upon these changes could be detected in the present study. In conclusion, it is proposed that the low nutritional value of G3MS, given in a daily amount well in excess of what could result from its repeated administration as an insulinotropic agent in noninsulin-dependent diabetes, answers the potential objection of its extrapancreatic role as an alternative nutrient or gluconeogenic precursor in such a therapeutic perspective.
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glycerol-1,2,3-trimethylsuccinate in rat pancreatic islets. Biochem Biophys Res Commun 236:26 –28, 1997.
ACKNOWLEDGMENTS This study was supported by a Concerted Research Action (94/99-183) of the French Community of Belgium and a grant (3.4513.94) from the Belgian Foundation for Scientific Medical Research. We are grateful to N. Bolaky for technical assistance and C. Demesmaeker for secretarial help.
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Ladrie`re L, Zhang T-M, Malaisse WJ. Effects of succinic acid dimethyl ester infusion on metabolic, hormonal and enzymatic variables in starved rats. J Parenteral Enteral Nutr 20:251–256, 1996.
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Ladrie`re L, Malaisse WJ. Nutritional value of succinic acid monoethyl ester in starvation. Ann Nutr Metab 41:118 –125, 1997.
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Eizirik DL, Welsh N, Sener A, Malaisse WJ. Protective action of succinic cid monomethyl ester against the impairment of glucose-stimulated insulin release caused by glucopenia or starvation: Metabolic determinants. Biochem Med Metab Biol 53:34 – 45, 1994.
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Laghmich A, Ladrie`re L, Malaisse-Lagae F, Malaisse WJ. Pancreatic islet responsiveness to D-glucose after repeated administration of repaglinide. Eur J Pharmacol 348:265– 270, 1998.
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Laghmich A, Ladrie`re L, Malaisse-Lagae F, Malaisse WJ. Long-term effects of glibenclamide and nateglinide upon pancreatic islet function in normal and diabetic rats. Submitted for publication.
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Ladrie`re L, Malaisse WJ. Effects of the dimethyl ester of succinic acid upon the metabolic and hormonal response to exercise in fed and starved rats. Submitted for publication.
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Assessment of the Nutritional Value of Glycerol-1,2,3tris(methylsuccinate) in Fed and Starved Rats L. Ladrie`re,* F. Bjo¨rkling,† and W. J. Malaisse* ,1 *Laboratory of Experimental Medicine, Brussels Free University, Brussels, Belgium; and †Leo Pharmaceutical Products, Ballerup, Denmark Received January 29, 1999, and in revised form April 13, 1999
the treatment of non-insulin-dependent diabetes mellitus. The use of succinic esters in such a perspective is often limited, however, by the high amounts required to achieve significant stimulation of insulin secretion in vivo and, hence, by their nutritional value in extrapancreatic tissues, e.g., their role as gluconeogenic precursors in hepatocytes (3,4). In the present study, the nutritional value of G3MS was assessed by investigating its effects upon metabolic, hormonal, and enzymatic variables in both fed and starved rats. For this purpose, the ester was infused for 3 days at a rate of 1.2 mmol z day 21 z g body wt 21. Such an infusion rate corresponds to a total daily supply of G3MS representing more than 15 times the amount of the ester (0.07 mmol z g body wt 21) previously found to stimulate insulin release and to enhance the insulinotropic action of gliquidone and repaglinide when injected intravenously over 30 s in fed anesthetized rats (2).
The nutritional value of glycerol-1,2,3-tris(methylsuccinate), a novel ester of succinic acid with high insulinotropic efficiency both in vitro and in vivo, was assessed in both fed and starved rats. The infusion of the ester, given in a daily amount (1.2 mmol z g body wt 21) well in excess of what could result from its repeated intravenous administration as an insulinotropic agent in non-insulin-dependent diabetes (0.07 mmol z g body wt 21 for each administration), failed to prevent the fall in body weight, liver and muscle glycogen contents, and plasma D-glucose or insulin concentration, as well as the increase in plasma free fatty acid and b-hydroxybutyrate concentrations caused by starvation. The sole indications that the ester may serve, to a limited extent, as an alternative nutrient in starved rats consisted in a somewhat higher weight of both liver and paraovarian adipose tissue and somewhat higher activity of liver glucokinase in rats receiving the ester than in animals infused with saline. The low nutritional value of this ester thus answers the objection of its possible role as an extrapancreatic nutrient or gluconeogenic precursor in the perspective of its use as an insulinotropic tool in type 2 diabetes. © 1999 Academic Press Key Words: glycerol-1,2,3-tris(methylsuccinate); insulin secretion; starvation; non-insulin-dependent diabetes.
MATERIALS AND METHODS Glycerol-1,2,3-tris(methylsuccinate) was prepared by direct esterification of glycerol by monomethyl succinic acid ester chloride (5). The experimental design and analytical procedures used to assess its nutritional value were the same as those used in prior studies (6,7). Briefly, a catheter was introduced, under anesthesia with ketamine (125 mg/g body wt given intraperitoneally), in the right heart of female Wistar rats. During the following 2–3 days, the animals were given free access to food (KM-04-k12; Pavan Service, Oud Turnhout, Belgium) and tap water. They were then starved or not
Glycerol-1,2,3-tris(methylsuccinate) (G3MS) is a novel ester of succinic acid with high insulinotropic efficiency both in vitro (1) and in vivo (2). It was proposed, therefore, as a new insulinotropic tool for 1 To whom correspondence should be addressed at Laboratory of Experimental Medicine, Brussels Free University, 808 Route de Lennik, B-1070 Brussels, Belgium. Fax: 132-2-5556239.
254 1096-7192/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
TABLE 1 Liver Weight, Protein Content, and Glycogen Content Rats:
Fed Nil
Saline
Saline 1 G3MS
Nil
Saline
6.55 6 0.30 3.42 6 0.13
7.68 6 0.31 3.84 6 0.11
8.26 6 0.31 4.10 6 0.14
4.67 6 0.09 2.66 6 0.10
5.25 6 0.20 2.95 6 0.08
5.70 6 0.20 3.18 6 0.08
(8) (8)
224 6 10
218 6 19
199 6 8
223 6 15
232 6 14
198 6 11
(8)
211.6 6 15.2
141.9 6 7.0
114.9 6 4.9
7.3 6 0.9
9.9 6 1.7
10.1 6 2.9
(8)
Infusion: Wet weight g % of body wt Protein content mg/g wet wt Glycogen content mmol of glucose residue/g wet wt
Starved
for 3 days and simultaneously infused with saline (NaCl 0.9%; Baxter, Lessines, Belgium) containing, when required, G3MS (10.0 mM) and delivered at a rate of 120 ml z day 21 z g body wt 21. Blood samples were collected in conscious rats at about 9 AM from the severed tip of the tail. On the last day, blood was also collected upon decapitation of the rats about 5 min after the tail blood sampling for the measurement of plasma D-glucose, insulin, free fatty acids, and b-hydroxybutyrate concentration. The liver and paraovarian fat weight, liver and gastrocnemius protein and glycogen contents, and the activity of glucokinase in liver homogenates were also measured. All results are expressed as mean values (6SEM) together with the number of individual observations. The statistical significance of differences between mean values was assessed by Student’s t test. RESULTS Body Weight Before surgery, the body weight of the rats averaged 204 6 2 g (n 5 32). The rats lost only 1 to 2 g (n 5 26) over the 2- to 3-day period between surgery and onset of the G3MS infusion. Over the ensuing 3 days, fed rats lost 5 6 1 and 10 6 2 g (n 5 8 in both cases) when infused with saline alone or saline containing G3MS, respectively. Over the same period of 3 days, starved rats lost 31 6 1 g (n 5 8) when not infused, 23 6 2 g (n 5 8) when infused with saline alone, and 25 6 2 g (n 5 8) when infused with saline containing G3MS. Liver Weight and Protein and Glycogen Contents As shown in Table 1, the liver wet weight, when expressed relative to paired body weight, was much
Saline 1 G3MS (n)
lower (P , 0.001) in starved rats than in fed rats. In both cases, it was higher (P , 0.05) in saline-infused animals than in noninfused rats. It was even further increased (P , 0.02) in rats infused with saline containing G3MS, the values recorded in these animals averaging 107.9 6 2.2% (n 5 16) of the mean corresponding value (100.0 6 1.9%; n 5 16) found in rats in the same nutritional state (fed or starved) infused with saline alone. Incidentally, the presence of G3MS in saline also increased (P , 0.02) the wet weight of paraovarian adipose tissue in both fed and starved rats, the values recorded in the animals receiving the ester averaging 125.1 6 5.9% (n 5 16) of the mean corresponding value (100.0 6 8.0%; n 5 16) found in rats with the same nutritional status (fed or starved) and infused with saline alone. The protein content of the liver was not significantly different in fed and starved rats, animals infused with saline or not, and rats receiving saline alone or saline and G3MS. The glycogen content of the liver was dramatically lower in starved rats than in fed rats (Table 1). In the fed rats, it was lower (P , 0.001) in saline-infused rats than in control animals and further decreased (P , 0.01) when G3MS was present in the saline infusion. These changes are in fair agreement with the effects of saline alone and saline together with G3MS on body weight, both series of data suggesting a lesser food intake in saline-infused rats than in control animals and a further decrease of food intake due to the administration of G3MS. In the starved rats, however, the liver glycogen content was not lower in salineinfused animals than in control noninfused rats and in animals receiving G3MS than in rats infused with saline alone.
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TABLE 2 Gastrocnemius Protein and Glycogen Contents Rats:
Fed
Infusion: Protein content mg/g wet wt Glycogen content mmol of glucose residue/g wet wt
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
135.3 6 8.3
115.1 6 6.5
124.0 6 10.6
123.6 6 8.9
153.3 6 12.2
125.0 6 8.4
(8)
13.5 6 1.0
13.0 6 1.3
15.8 6 2.1
4.8 6 0.9
5.8 6 0.8
6.3 6 1.0
(8)
Muscle Protein and Glycogen Contents The protein content of the gastrocnemius, like that of the liver, was not affected significantly by starvation, saline infusion, or G3MS administration (Table 2). The glycogen content of the gastrocnemius was lower (P , 0.001) in starved rats than in fed rats. Whether in fed or starved animals, it was not significantly different in control rats, saline-infused animals, and those receiving both saline and G3MS. In both fed and starved rats, the administration of G3MS tended to increase the muscle glycogen content, but this difference failed to achieve statistical significance. Plasma Concentration of Free Fatty Acids and bHydroxybutyrate In blood samples collected from the severed tip of the tail, the plasma free fatty acid concentration was lower (P , 0.001) in fed rats (643 6 38 mM; n 5 48) than in starved animals (1471 6 129 mM; n 5 7). In the fed rats, it failed to be affected significantly by the infusion of saline alone or saline together with G3MS, the paired difference between the last (Day 3) and first (Day 0) measurements averaging, respectively, 134 6 92 and 1131 6 62 mM (n 5 8 in both cases). In the starved rats, such a paired difference averaged 1607 6 161 mM (n 5 7; P , 0.01)
Saline 1 G3MS (n)
in the control noninfused animals, 1526 6 57 mM (n 5 8; P , 0.001) in the saline-infused rats, and 1570 6 67 mM (n 5 8; P , 0.001) after infusion of saline and G3MS. The latter three mean values were not significantly different from one another. The handling of the rats at the time of the last bleeding from the tip of the tail and the subsequent killing of the animals by decapitation failed to affect significantly the plasma free fatty acid concentrations in fed rats, with a mean value for the paired difference between the measurements made at and shortly before sacrifice of 227 6 60 mM (n 5 20). In the starved rats, however, such a paired difference averaged 2376 6 64 mM (n 5 23; P , 0.001). These two mean values refer, in each case, to control (noninfused) rats as well as animals infused with saline alone or together with G3MS, the results being comparable under these three experimental conditions (Table 3). In the blood samples obtained from the tail, the plasma concentration of b-hydroxybutyrate was also lower (P , 0.001) in fed rats (142 6 7 mM; n 5 47) than in starved rats (1525 6 309 mM; n 5 8). In the fed rats, no significant change was observed after the 3 days of infusion of saline alone or together with G3MS, with mean paired differences between Day 3 and Day 0 of 118 6 12 and 18 6 6 mM (n 5 8 in both cases), respectively (Table 4). In the starved rats,
TABLE 3 Plasma Free Fatty Acid Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
763 6 65 (8) 839 6 158 (6)
613 6 105 (8) 591 6 68 (8) 503 6 73 (8) 647 6 59 (8) 520 6 50 (7)
517 6 97 (8) 545 6 86 (8) 639 6 77 (7) 649 6 49 (8) 574 6 57 (7)
809 6 94 (8) 1219 6 55 (8) 1579 6 69 (8) 1471 6 129 (7) 1080 6 67 (8)
516 6 76 (8) 938 6 34 (8) 1119 6 99 (8) 1041 6 89 (8) 658 6 58 (8)
639 6 81 (8) 1029 6 60 (8) 1279 6 79 (8) 1222 6 88 (8) 835 6 90 (8)
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GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
TABLE 4 Plasma b-Hydroxybutyrate Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
154 6 8 (8) 277 6 85 (6)
164 6 24 (8) 179 6 22 (8) 173 6 18 (8) 182 6 22 (8) 238 6 30 (8)
91 6 5 (8) 110 6 8 (8) 125 6 27 (8) 99 6 5 (8) 167 6 10 (8)
187 6 9 (8) 817 6 63 (8) 1108 6 191 (8) 1525 6 309 (8) 1360 6 314 (8)
129 6 10 (8) 580 6 72 (8) 1130 6 134 (8) 1516 6 186 (8) 1425 6 226 (8)
127 6 10 (7) 508 6 62 (8) 1268 6 222 (8) 1595 6 301 (8) 1451 6 296 (8)
such paired differences averaged 11339 6 317 mM (n 5 8; P , 0.005) in the control noninfused animals, 11387 6 180 mM (n 5 8; P , 0.001) in the salineinfused rats and, 11512 6 337 mM (n 5 7; P , 0.005) after infusion of saline together with G3MS. These three mean values are not significantly different from one another. Pooling together the results obtained in control (noninfused) rats and animals infused with saline, whether alone or together with G3MS, the paired difference between the measurements made at killing and shortly before sacrifice (see above) averaged 178 6 27 mM (n 5 21; P , 0.01) in fed rats, as distinct (P , 0.005) from 2133 6 61 mM (n 5 24; P , 0.05) in starved animals. Plasma Concentrations of D-Glucose and Insulin Over 3 days of starvation, the plasma concentration of D-glucose, as measured in samples obtained from the tail of the rats, decreased (P , 0.001) from 6.81 6 0.12 mM (n 5 48) in fed animals to 4.55 6 0.41 mM (n 5 8) in starved rats (Table 5). In the fed rats, it failed to be affected after 3 days of saline infusion, with or without G3MS, the paired difference between Day 3 and Day 0 averaging 20.06 6 0.35 mM (n 5 8) in animals infused with saline alone and 20.07 6 0.17 mM (n 5 8) in rats infused
with saline containing G3MS. In the starved rats, such a paired difference amounted to 22.65 6 0.49 mM (n 5 8; P , 0.005) in the control animals (no infusion), 22.88 6 0.27 mM (n 5 8; P , 0.001) in saline-infused rats, and 22.21 6 0.29 mM (n 5 8; P , 0.001) in animals receiving both saline and G3MS. These three mean values were not significantly different from one another. The handling of the rats at the end of the experiments (see above) always increased the plasma concentration of D-glucose. Such an increment averaged 2.12 6 0.23 mM (n 5 21; P , 0.001) in fed rats and 2.28 6 0.18 mM (n 5 24; P , 0.001) in starved rats, these two mean values being comparable to one another (P . 0.5). In the samples collected from the severed tip of the tail, the plasma concentration of insulin was lower (P , 0.001) in starved rats (7.5 6 1.5 mU/ml; n 5 8) than in fed animals (34.0 6 1.9 mU/ml; n 5 48). It decreased significantly during infusion of saline, with or without G3MS, in fed rats, the paired difference between Day 3 and Day 0 averaging 211.0 6 2.8 mU/ml (n 5 8; P , 0.01) in the animals receiving saline alone and 27.4 6 2.8 mU/ml (n 5 8; P , 0.05) in those receiving saline and G3MS (Table 6). These two mean values are not significantly different from one another. In the starved rats, the
TABLE 5 Plasma D-Glucose Concentration (mM) Rats: Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Fed
Starved
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
7.29 6 0.28 (8) 9.74 6 0.23 (6)
6.93 6 0.21 (8) 7.24 6 0.26 (8) 6.74 6 0.20 (8) 6.87 6 0.25 (8) 8.58 6 0.49 (8)
6.38 6 0.14 (8) 6.60 6 0.42 (8) 6.30 6 0.26 (8) 6.31 6 0.07 (8) 8.68 6 0.44 (7)
7.20 6 0.26 (8) 5.05 6 0.10 (8) 4.27 6 0.10 (8) 4.55 6 0.41 (8) 6.87 6 0.37 (8)
6.94 6 0.44 (8) 4.88 6 0.64 (8) 4.15 6 0.49 (8) 4.06 6 0.38 (8) 6.51 6 0.38 (8)
6.12 6 0.12 (8) 4.72 6 0.13 (8) 4.23 6 0.19 (8) 3.91 6 0.25 (8) 5.96 6 0.52 (8)
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TABLE 6 Plasma Insulin Concentration (mU/ml) Rats:
Fed Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
27.8 6 5.4 (8) 27.3 6 2.2 (6)
31.3 6 3.7 (8) 26.8 6 2.3 (8) 23.4 6 3.6 (8) 20.3 6 2.4 (8) 20.8 6 4.4 (7)
30.4 6 1.7 (8) 24.4 6 1.5 (8) 23.4 6 1.8 (8) 22.9 6 2.1 (8) 33.0 6 5.6 (7)
26.3 6 2.4 (8) 8.5 6 1.7 (8) 4.6 6 1.4 (8) 7.5 6 1.5 (8) 8.6 6 2.1 (8)
39.8 6 2.8 (8) 10.0 6 1.9 (8) 5.3 6 1.1 (8) 4.1 6 1.3 (8) 7.2 6 1.8 (8)
48.5 6 5.8 (8) 8.2 6 0.8 (8) 5.7 6 1.3 (8) 4.9 6 0.9 (8) 5.3 6 1.6 (8)
Infusion: Day 0 Day 1 Day 2 Day 3 At sacrifice
Starved
paired difference in plasma insulin concentration at Day 3 minus Day 0 averaged 218.8 6 1.7 mU/ml (n 5 8; P , 0.001) in the control animals (no infusion), 235.7 6 3.4 mU/ml (n 5 8; P , 0.001) in the rats infused with saline alone, and 243.5 6 6.2 mU/ml (n 5 8; P , 0.001) after infusion of both saline and G3MS. Such paired differences were thus more marked (P , 0.05 or less) in starved rats than in fed rats. In the former animals, the starvation-induced decrease in plasma insulin concentration was higher (P , 0.005 or less) in the infused rats than in the control animals, but not significantly different (P . 0.2) after infusion of saline alone or together with G3MS. The increase in plasma D-glucose concentration at the end of the experiments (see above) failed to be associated with any significant increase in plasma insulin concentration. Thus, the paired difference between the measurements make at sacrifice and shortly before averaged in fed and starved rats, respectively, 12.7 6 3.7 mU/ml (n 5 20; P . 0.4) and 11.5 6 1.1 mU/ml (n 5 24; P . 0.15). Glucokinase Activity in Liver Homogenates It was previously documented that certain esters of succinic acid, when infused in starved rats, oppose the effect of starvation to decrease the activity of liver glucokinase (6 – 8). In the present experiments,
the activity of the glucokinase was measured, therefore, in liver homogenates from either fed or starved rats, saline-infused or control (no infusion) animals, and rats infused with saline alone or both saline and G3MS (Table 7). At a low concentration of D-glucose (1.0 mM), the activity of glucokinase was judged from measurements made in the concomitant presence of 3.0 mM D-glucose 6-phosphate, used to inhibit the low K m hexokinase isoenzyme, and 1.0 mM D-fructose 1-phosphate, used to relieve glucokinase from any inhibitory action mediated, at the intervention of its regulatory protein, by D-fructose 6-phosphate, which is rapidly generated in tissue homogenates from exogenous D-glucose 6-phosphate. In fed rats, the activity of glucokinase (always expressed as pmol z min 21 z mg wet wt 21), at the low concentration of D-glucose, was not vastly different in control animals (104 6 4; n 5 8), saline-infused rats (94 6 4; n 5 8), and animals infused with both saline and G3MS (106 6 4; n 5 8). In starved animals, the values averaged 81 6 3, 63 6 3, and 91 6 2 (n 5 8 in all cases) in control, saline-infused, and G3MS-infused rats. The activity of glucokinase was thus higher (P , 0.001) in fed than starved animals. In the latter rats, it seemed slightly decreased by saline infusion (P , 0.005), the infusion of G3MS apparently protecting (P , 0.001) against such a decrease.
TABLE 7 Glucokinase Activity in Liver Homogenates (pmol z min 21 z mg wet wt 21) Rats:
Fed
Starved
Infusion:
Nil
Saline
Saline 1 G3MS
Nil
Saline
Saline 1 G3MS
(n)
1.0 mM D-glucose a 10.0 mM D-glucose a
104 6 4 1,045 6 40
94 6 4 885 6 55
106 6 4 868 6 46
81 6 3 730 6 42
63 6 3 485 6 31
91 6 2 618 6 30
(8) (8)
a
All measurements were made in the presence of 3.0 mM D-glucose 6-phosphate and 1.0 mM D-fructose 1-phosphate.
259
GLYCEROL-1,2,3-TRIS(METHYLSUCCINATE)
Whether in fed or starved rats, the reaction velocity for D-glucose phosphorylation, at the low concentration of D-glucose, was much lower in the presence of the exogenous hexose esters than in their absence. It averaged, in fed and starved rats, respectively, 33.4 6 2.9 and 26.1 6 2.4% (n 5 24 in both cases). As expected, such a relative value was thus lower in the starved rats with low glucokinase activity. Such was the case (P , 0.001 in all cases) in control (noninfused), saline-infused, and G3MS-infused rats, the reaction velocity in the presence of the exogenous esters relative to that found in their absence averaging, in the starved rats, 77.9 6 2.0% (n 5 24; P , 0.001) of that found in fed rats (100.0 6 2.0%; n 5 24) treated under the same conditions (i.e., not infused, infused with saline, or infused with both saline and G3MS). At a higher concentration of D-glucose (10.0 mM), the activity of glucokinase in fed rats, as judged from the measurements made in the presence of exogenous D-glucose 6-phosphate and D-fructose 1-phosphate and expressed as pmol z min 21 z mg wet wt 21, was somewhat higher (P , 0.05 or less) in control (noninfused) animals (1045 6 40; n 5 8) than in either saline-infused rats (885 6 55) or after infusion of both saline and G3MS (868 6 46). In starved animals, the value found in control (noninfused) rats (730 6 42) was again lower (P , 0.001) than that found in fed rats but higher (P , 0.001) than that recorded in saline-infused starved animals (485 6 31). The administration of G3MS increased (P , 0.01) the activity of glucokinase to 618 6 30 in the saline-infused starved animals, such a value remaining lower (P , 0.05), however, than that found in the control starved rats. At variance with the situation found at 1.0 mM D-glucose (see above), the rate of D-glucose phosphorylation at a 10.0 mM concentration of the hexose was comparable in the absence and presence of exogenous D-glucose 6-phosphate and D-fructose 1-phosphate, as expected from the low fractional contribution of the low K m hexokinase isoenzyme to the overall reaction velocity. Thus, the results recorded in the presence of the exogenous phosphate esters, when expressed relative to those found in the absence of such esters, averaged 86.3 6 3.5 and 100.9 6 6.0% (n 5 24 in both cases) in fed and starved rats, respectively. The latter two mean values were not significantly different from one another.
DISCUSSION The present results reveal that the infusion of G3MS, given in a daily amount (1.2 mmol z g body wt 21) largely in excess of the cumulative value that would result from repeated intravenous administrations of the ester to stimulate insulin release or potentiate the B-cell secretory response to hypoglycemic sulfonylureas or meglitinide analogs (0.07 mmol z g body wt 21 for each administration), only displays minimal nutritional value, whether in fed or starved rats. Thus, the infusion of the ester failed to prevent the fall in body weight and liver glycogen content seen in saline-infused rats with either free access to food or starved for 3 days. It also failed to affect, in either fed or starved rats, the muscle glycogen content, which was decreased by starvation, the plasma concentrations of free fatty acids and b-hydroxybutyrate, which both increased during starvation, and the plasma concentrations of D-glucose and insulin, which both decreased in the starved rats. The sole indications that G3MS may have served, to a limited extent, as an alternative nutrient in starved rats consisted in a somewhat higher weight of both liver and paraovarian adipose tissue and a somewhat higher activity of liver glucokinase in the rats receiving the ester than in the animals infused with saline alone. Incidentally, the present study also indicates that, in both fed and starved rats, the handling of rats at the time of the last bleeding from the tip of the tail and, shortly thereafter, at the time of killing by decapitation results in an increase in plasma D-glucose concentration, not associated with any increase in plasma insulin concentration. In the starved rats, a decrease in both plasma free fatty acid and b-hydroxybutyrate concentrations was also observed, while the concentration of the ketone body increased in fed rats at the same time. Such changes were already reported and their determinism discussed in prior publications (9 –11). No significant effect of G3MS upon these changes could be detected in the present study. In conclusion, it is proposed that the low nutritional value of G3MS, given in a daily amount well in excess of what could result from its repeated administration as an insulinotropic agent in noninsulin-dependent diabetes, answers the potential objection of its extrapancreatic role as an alternative nutrient or gluconeogenic precursor in such a therapeutic perspective.
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glycerol-1,2,3-trimethylsuccinate in rat pancreatic islets. Biochem Biophys Res Commun 236:26 –28, 1997.
ACKNOWLEDGMENTS This study was supported by a Concerted Research Action (94/99-183) of the French Community of Belgium and a grant (3.4513.94) from the Belgian Foundation for Scientific Medical Research. We are grateful to N. Bolaky for technical assistance and C. Demesmaeker for secretarial help.
6.
Ladrie`re L, Zhang T-M, Malaisse WJ. Effects of succinic acid dimethyl ester infusion on metabolic, hormonal and enzymatic variables in starved rats. J Parenteral Enteral Nutr 20:251–256, 1996.
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Ladrie`re L, Malaisse WJ. Nutritional value of succinic acid monoethyl ester in starvation. Ann Nutr Metab 41:118 –125, 1997.
8.
Eizirik DL, Welsh N, Sener A, Malaisse WJ. Protective action of succinic cid monomethyl ester against the impairment of glucose-stimulated insulin release caused by glucopenia or starvation: Metabolic determinants. Biochem Med Metab Biol 53:34 – 45, 1994.
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Laghmich A, Ladrie`re L, Malaisse-Lagae F, Malaisse WJ. Pancreatic islet responsiveness to D-glucose after repeated administration of repaglinide. Eur J Pharmacol 348:265– 270, 1998.
10.
Laghmich A, Ladrie`re L, Malaisse-Lagae F, Malaisse WJ. Long-term effects of glibenclamide and nateglinide upon pancreatic islet function in normal and diabetic rats. Submitted for publication.
11.
Ladrie`re L, Malaisse WJ. Effects of the dimethyl ester of succinic acid upon the metabolic and hormonal response to exercise in fed and starved rats. Submitted for publication.
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