Effect of sulfonylureas on hepatic glycogen metabolism: Activation of glycogen phosphorylase

Effect of Sulfonylureas on Hepatic Glycogen Metabolism: Activation of Glycogen Phospborylase M. Mojena, M.L. Marcos, L. Monge, and J.E. Feliu In hepatocytas isolated from fad rats, both tolbutamida and glipizida caused a dose-dependent activation of glycogan phosphorylasa, possibly by a Ca2+-mediated mechanism. Maximal affects (about twofold) ware already obtained when drugs ware used at 0.5 mmol/L, the calculated concentrations of tolbutamida and glipizide responsible for the half-maximal affects being 60 and 30 fimol/L, respectively. The activation of glycogan phosphorylasa caused the mobilization of glycogan and increased the cellular concentration of haxosa 6phosphatas (glucose 6phosphata plus fructose &phosphate) and that of fructose 2.6bisphosphata. Under the influence of sulfonyluraas, glucose formation was slightly stimulated while the rata of L-lactate production was more markedly incremented, indicating that sulfonyluraas canalize the metabolic flux coming from glycogan mainly to the glycolytic pathway. These results suggest that a glycoganolytic action of sulfonyluraas could collaborate to raise hapatic fructose 2,6-bisphosphata concentration in the fad animal. (D 1999 by Grune & Stratton, Inc.

I

N THE LAST FEW YEARS, several reports have demonstrated that sulfonylureas may increase the concentration of fructose 2,6-bisphosphate (F-2,6-P*) in perfused rat livers’*’ and isolated rat hepatocytes.3s4 Moreover, it has been shown that tolbutamide significantly raises the content of this regulatory metabolite in isolated rat soleus muscle.’ In liver preparations the relative efficacy of different sulfonylureas incrementing F-2,6-P* levels was closely associated to their known hypoglycemic potency.‘” In agreement with the physiologic role of F-2,6-P2 in the regulation of hepatic glucose metabolism,6m* it has been established that in isolated rat hepatocytes the rise in F-2,6-Pz caused by sulfonylureas is closely correlated with an acceleration of glycolysis and a reduction of gluconeogenesis.3V4” However, the biochemical mechanism by which sulfonylureas increase F-2,6-P, levels has not been completely clarified. Furthermore, it has been reported that the addition of tolbutamide to suspensions of isolated rat hepatocytes affects the activity of the interconvertible enzyme 6phosphofructo 2-kinase/fructose 2,6_bisphosphatase by stimulating the kinase and blocking the phosphatase.‘*” However, this does not seem to be the only mechanism responsible for the rise of the cellular F-2,6-P* content. Our group found that glipizide and tolbutamide can increase the concentration of both fructose 6-phosphate and glucose 6-phosphate either in hepatocytes isolated from 24-hour fasted4 or from normally fed rats (results reported in this paper). In this latter situation, a possible glycogenolytic

From Servicio de Endocrinologia Experimental. Clinica Puerta de Hierro. Universidad Autbnoma de Madrid, Madrid, Spain. Supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social and from the Comisibn Asesora de Investigacibn Cientifca y Ticnica. Spain. Dr Mojena is a postdoctoral fellow of the P.F.P.I., Ministerio de Educacibn y Ciencia. Spain. Presented in part in abstract form at the 22nd Annual Meeting of the European Association for the Study of Diabetes, Rome, September 1986. Address reprint requests to J.E. Feliu, MD. Servicio de Endocrinologia Experimental, Clinica Puerta de Hierro. San Martin de Porres. 4. 28035 Madrid, Spain. o I989 by Grune &i Stratton, Inc. 0026-0495/89/3805-0014$03.00/0

466

effect of sulfonylureas could increment the total pool of hexose 6-phosphates, and cooperate in this way to increase the cellular concentration of F-2,6-P*. The aim of this work was to test this hypothesis. Our results demonstrate that both tolbutamide and glipizide activate glycogen phosphorylase, possibly by a calciummediated mechanism. As a consequence of this effect, glycogenolysis is stimulated, the cellular concentration of hexose 6-phosphate and F-2,6-P* are incremented, and the flux through the glycolytic pathway is accelerated. Our findings also suggest that sulfonylureas direct the glycogenolytic flux mainly to lactate, and to a lesser extent to glucose. MATERIALS

AND METHODS

Collagenase and auxiliary enzymes were purchased from Boehringer Mannheim GmbH (Mannheim, West Germany). (IJ-‘4C)pyruvate, (U-‘4C)glucose, and “CaCl, were supplied by the Radiochemical Centre (Amersham, United Kingdom). F-2,6-P, used as standard and phenylephrine were obtained from Sigma Chemical Co (St Louis). Glipizide (lot MP605/7143) and tolbutamide (lot M86/ 0044) were kindly supplied by Farmitalia-Carlo Erba (Barcelona, Spain) and Boehringer Mannheim GmbH, respectively. A-23187 ionophore was purchased from Calbiochem-Behring (La Jolla, CA). Fed male Wistar rats from our inbred colony, weighing 200 to 300 g were used. The animals were maintained on a standard chow (A.04, Panlab S.L., Barcelona) and water ad libitum. Hepatocytes were isolated by perfusion of the liver with collagenase, following the procedure described by Hue et al.” The isolated cells were suspended in Krebs-Henseleit medium and their viability was evaluated by the Trypan blue test; usually 90% to 95% of the cells excluded the stain. Samples of cell suspension (I to 2 mL), containing 40 to 80 mg of cells, were shaken (120 strokes/min) in stoppered 20-mL vials, at 36OC. Glycogen phosphorylase a activity was assayed in 0.1 -mL aliquots of cell suspensions taken at the indicated times, as described elsewhere.‘* Hepatocyte F-2,6-P, was measured by the ability of this metabolite to activate potato tuber PPi:fructose (i-phosphate Iphosphotransferase” according to the method previously described3 For the determination of other glycolytic intermediates, aliquots of cell suspensions were taken at selected times and immediately mixed with one volume of ice-cooled 10% HClO,. After neutralization3 L-lactate, glucose 6-phosphate, and fructose 6-phosphate were measured in the extracts by enzymatic methods.‘4~‘s Hepatocyte glycogenolysis was evaluated by the rate of net glucose production. Glucose was assayed by the glucose oxidase method (Glucose GOD-Perid, Boehringer Mannheim). In addition, Metabolism, Vol 38, No 5 (May), 1989: pp 466-470

SULFONYLUREAS AND HEPATIC GLYCDGEN METABOLISM

467

glycogenolysis was measured isotopically by the rate of (“C)glucose formation by hepatocytes previously charged with (‘%)glycogen. The isotopic labeling of hepatocyte glycogen was performed by incubating the cells in the presence of 40 mmol/L (U-“C)glucose (0.5 ~Ci/~mol) for 40 minutes at 36OC; during all this period the incorporation of labeled glucose into glycogen was linear. Then, the cells were collected by centrifugation, washed three times with Krebs-Henseleit solution, and suspended in this medium. To reduce basal glycogenolysis, 20 mmol/L glucose was added to the cell suspension. The amount of (“C)glycogen remaining in the cells after glycogenolysis was also estimated.16 Calcium uptake studies were carried out with “CaCl,, following basically the method described by Keppens et al.” Hepatocytes were incubated for 30 minutes in Krebs-Henseleit medium in the presence of 20 mmol/L glucose; then, a trace amount of “CaCI, was added to cell suspensions (5 pCi/mL as final concentration), together with tolbutamide, glipizide, phenylephrine, ionophore, or saline. After 30 and 60 seconds of incubation, SO-rL aliquots of cell suspensions were pipetted into ice-cooled test tubes containing 10 mL of a solution composed of 10 mmol/L Hepes, 150 mmol/L NaCl, 5 mmol/L CaCl, and 0.1 mmol/L LaCI,, at pH 7.0. The diluted cell suspensions were immediately filtered under vacuum (25 cm Hg) through Whatman GF/A glass fiber filters (25 mm diameter); the cells retained by the filters were washed with 15 mL of the same ice-cooled solution. The filters were air-dried, and their radioactivity was measured in a liquid scintillation spectrometer. Protein was assayed by the method of Lowry et al,‘* using bovine serum albumin as standard; 1 g of packed hepatocytes corresponded to 220 f 5 mg of protein. Statistical significance of differences between values was calculated by the paired Student’s t test. The differences were considered statistically significant when the Pvalue was c.05.

RESULTS

As shown in Table 1, the presence of either tolbutamide (1 mmol/L) or glipizide (0.2 mmol/L) in the incubation medium slightly increased (respectively, 15% and 20% over the basal value) the rate of glucose production by hepatocytes isolated from fed rats and incubated in the absence of any energetic source. Under similar experimental conditions, phenylephrine (10 pmol/L) markedly stimulated the production of glucose by the cells (50% over the basal value). This glycogenolytic effect of sulfonylureas was accompanied by an accelerated formation of L-lactate (Table 1). Thus, the rate of L-lactate formation was increased by about 70% and 30%, respectively, in tolbutamide- and glipizide-treated cell incubations. By contrast, phenylephrine, which exhibited a

Table 2. Glycogenolytic Effect of Tolbutamide and Glipizide in Isolated Rat Hepatocytes Preloadad with (“CIGlycogen %Of Remaining Additions

glvcoasn

t’%KitRsbase (ccmd/gof cells x 10 min)

Saline

60 * 5

Tolbutamide (1 mmol/L)

47 f El*

6.1 + 0.23T

Glipizide (0.2 mmol/L)

49 f 7f

6.4 f 0.13T

Phenylephrine ( 10 pmol/L)

33 f 7*

7.2 + 0.27*

5.5 + 0.18

Cellular glycogen was isotopically labelled as indicated in “Materials and Methods.”

Then hepatocytes were incubated in Krebs-Henseleit

medium with 20 mmol/L glucose in the presence of different agents. Aliquots of cell suspensions were taken at 10 minutes of incubation to measure (“C)glucose

released to the medium. After 20 minutes of

incubation, hepatocytes were collected by centrifugation and their content of f”C)glycogen was measured. Values are the mean f SEM of four experiments. lP < .Ol “saline incubation. TP < .05 v saline incubation.

greater glycogenolytic action than sulfonylureas, caused only a 12% increment of the rate of L-lactate production. The stimulation of glycogenolysis by sulfonylureas was also demonstrated when the degradation of glycogen was estimated isotopically. As shown in Table 2, tolbutamide (1 mmol/L) and glipizide (0.2 mmol/L) caused a statistically significant increase (respectively, 10% and 16% over the basal value) of the rate of (“C)glucose formation by hepatocytes preloaded with ( r4C)glycogen. Simultaneously, the percentage of (‘4C)glycogen remaining in the cells at the end of the incubation was significantly reduced by the presence of sulfonylureas (Table 2). In accordance with its much higher glycogenolytic potency, phenylephrine provoked a more marked release of labeled glucose, as well as a greater reduction of the cellular content of ( “C)glycogen that than elicited by sulfonylureas. As a consequence of their glycogenolytic action, sulfonylureas significantly raised hepatocyte concentration of hexose B-phosphates (glucose 6-phosphate and fructose 6phosphate), as well as the cellular levels of F-2,6-P2 (Table 1). Similar effects were caused by phenylephrine. The ability of sulfonylureas to stimulate glycogenolysis in isolated rat hepatocytes prompted us to study the influence of these drugs on glycogen phosphorylase activity. Thus, the presence of tolbutamide (1 mmol/L) or glipizide (50 pmol/

Table 1. Effect of Tolbutamide and Glipiride on Glucose Release, L-Lactate Production, as well as on Fructose 2,6-bisphosphate, Hexose B-phosphate, and Fructose &phosphate Concentrations in Isolated Rat Hepatocyter GlucoseRelease Additions

L-LactateProduction

F-W-P,

bnnollg of cells x 30 min)

HeXls46-P

Fructose-6-P

fnmd/g of cells)

Saline

37.5

f 2

21.7

f 2.7

11.6 f 0.35

446 zt 22

Tolbutamide (1 mmol/L)

45.0

+ 1.5*

37.7

+ 2.6*

14.7 + 0.26*

576 f 30*

130 f 7 156 i 7’

Glipizida (0.2 mmol/L)

43.0

* 2f

28.8

f 2.7.

15.2 f 0.33*

556 f 27*

155 f 8+

Phenylephrine f 10 pmol/L)

57.0

+ 1.7.

25.2

+ 2.3t

13.6 f 0.43*

549 f 25.

150 i 8+

Hepatocytes were isolated from fed rats and incubated in Krebs-Henseleit medium in the absence of glucose for 30 minutes. Then aliquots of the cell suspensions were taken to measure glucose and L-lactate. F-2,6-P, Values are the mean f SEM of seven experiments. ‘P < .O 1 v saline incubation. fP < .05 “saline incubation.

and the hexose-6-P were measured in aliquots taken after 15 minutes of incubation.

468

MOJENA ET AL

L) in the incubation medium caused a statistically significant activation of glycogen phosphorylase in isolated rat hepatocytes (Fig 1). Maximal effects were observed two minutes after sulfonylurea addition; at this time, glycogen phosphorylase a activity was increased about 2- and 1S-fold, respectively, by tolbutamide and glipizide as compared with the values measured in saline incubations (5.0 + 1.l U/g of cells). Under similar conditions, both phenylephrine (10 rmol/L) and ionophore A-23187 (10 pmol/L) caused a marked stimulation of this enzyme activity (14.9 k 1.3 and 16.8 f 1.7 U/g of cells, respectively). After 20 minutes of incubation glycogen phosphorylase a activity of sulfonylurea-treated hepatocytes was still significantly higher than that measured in control liver cells. The activation of glycogen phosphorylase elicited by sulfonylureas was dose-dependent (Fig 2). Maximal effects were obtained when drugs were used at 0.5 mmol/L, the calcu-

‘*- I 10. 8.

i +*

‘_*

a5

,, v

8. 4. 2. 0

. 0

0.1

0.2 SULFONYLUREA

1

,(mM)

Fig 2. Effect of different concentrations of tolbutamide and glipizide on glycogen phosphorylare a acthrity in isolated rat hepatocytes. Experimental conditions were as indicated in Fig 1. The enzyme activity was measured in aliiuots of cell suspensions taken two minutes after sulfonylurea and phenylephrine add&ion. (0) tolbutamide, I) giipizide, and (A) 10 #mol/L phenyhphrine. Values are the maan + SEM of three experiments. lP < .OS and 9.P < .Ol Y basal activity.

lated concentrations of tolbutamide and glipizide responsible for the half-maximal effects being 60 and 30 rmol/L, respectively. Finally, the relative glycogenolytic potency of all these agents is in agreement with their specific ability to stimulate 45Ca uptake by the hepatocytes (Fig 3). DISCUSSION 1

/I

0

2

4 8 TIME(minutrs)

//

20

Fig 1. Effect of tolbutamide and glipizide on glycogen phosphorylase a activity in isolated rat hepatocytes. Liver ceils ware obtained from fed rats and preincubated for 30 minutes in KrebsHenseleit medium, in the presence of 10 mmol/L glucose. before the addition of the different agents. (0) saline, (0) 1 mmol/L tolbutamide, (WI 50 flmol/L glipizide, (Al 10 Nmol/L phenylaphrine, and (0 I 10 fimol/L A-23187 ionophore. Values are the mean + SEM of three experiments. lP c .05 and l*P < .Ol v saline incubation.

It has been demonstrated that, in addition to the stimulation of insulin release, sulfonylureas exert metabolic effects in extrapancreatic tissues, which in some cases cooperate with the hypoglycemic action of these drugsLS2’ In liver sulfonylureas may modulate carbohydrate metabolism. Thus, in vitro studies carried out in different liver preparations have shown that these compounds stimulate hepatic glycolytic flu~‘“~*~~and inhibit both basal and glucagonstimulated gluconeogenesis.3”g~*2~XMoreover, sulfonylureas have been reported to increase the concentration of F-2,6-P2 in perfused rat livers’S2and in isolated rat hepatocytes.‘c*9.‘0

469

SULFONYLUREAS AND HEPATIC GLYCOGEN METABOLISM

700 .c * zk

600

B F E‘

500

0 v 400

0

30 TIME

(seconds

60

)

Fig 3. Effect of tolbutamide and glipizide on %a uptake in isolated rat hepatocytes. Liver cells were incubated in the presence of 20 mmol/L glucose. The rest of the experimental conditions were as indicated in Fig 1. (0) ssline, (g) 1 mmol/L tolbutamide. (ml 0.2 mmol/L glipizide. (A) 10 pmol/L phenylephrine, and ( l ) 10 pmol/L A-23187 ionophore. Values are the mean f SEM of six experiments. lP < .06 and l*P < .Ol Y saline incubation.

However, the biochemical mechanism by which sulfonylureas increase hepatic F-2,6-P2 levels has not been completely clarified.4~9*‘0 These results demonstrate that the oral hypoglycemic agents tolbutamide and glipizide (in the micromolar range) exerted a paradoxical activation of glycogen phosphorylase in isolated rat hepatocytes. This sulfonylurea effect was much less marked than those elicited either by phenylephrine or by the A-23187 ionophore, both known glycogenolytic agents and activators of this enzyme.” As a consequence of the activation of glycogen phosphorylase, both tolbutamide and glipizide provoked a significant degradation of glycogen, which resulted in an increased concentration of hexose 6-phosphates (glucose 6-phosphate plus fructose 6-phosphate) and in an acceleration of glucose release by hepatocytes. Simultaneously, both the cellular concentration of F-2,6-P, and the rate of L-lactate production were markedly stimulated. Although these sulfonylurea effects were similar to those elicited by the alpha-adrenergic agonist phenylephrine, quantitative and qualitative differences existed concerning the stimulation of glycogenolysis. As mentioned above, sulfonylureas exert a lesser stimulation of glycogen phosphorylase a activity, as well as a smaller liberation of glucose, than that observed with phenylephrine. In contrast, the production of L-lactate was more markedly stimulated by sulfonylureas than by the alpha-adrenergic agonist.

Taken together, these results suggest that tolbutamide and glipizide canalize the glycogenolytic flux mainly to the formation of L-lactate and, to a lesser extent, to glucose. We have no clear explanation for these findings; however, we can speculate about reports that indicate a possible inhibitory effect of sulfonylureas on glucose-6-phosphatase activity.X*27 A partial blockade of this enzyme could direct most of the metabolic flux coming from glycogen to the glycolytic pathway. Furthermore, the slightly higher levels of hexose 6phosphate and F-2,6-P, present in sulfonylurea-treated hepatocytes, as compared to those measured in phenylephrine-treated cells, could be the consequence of this partial enzymatic blockade. However, the stimulation of 6-phosphofructo 2-kinase activity and the inhibition of fructose 2,6bisphosphatase elicited by sulfonylureasg9” could cooperate to increase hepatocyte F-2,6-P, levels and to promote glycolysis. At first glance, the small glycogenolytic effect caused by oral hypoglycemic agents tolbutamide and glipizide appears to be at least paradoxical. Previous reports have stressed the hepatic effect of different sulfonylureas antagonizing epinephrine- or glucagon-stimulated glycogenolysis2’ or potentiating either insulin-induced suppression of glycogenolysis? or insulin-dependent glycogenesis.29 However, our results are in agreement with those reported by Kramer et alM and Tan et al,” who showed that, in isolated perfused rat hearts, tolbutamide activates glycogen phosphorylase mobilizing the stores of glycogen and accelerates the glycolytic flux and the rate of L-lactate formation. It is well established that glycogen phosphorylase can be activated by phosphorylation mediated either by a cyclic AMP- or a Ca2+-dependent mechanism.‘7*32 The fact that neither tolbutamide or glipizide significantly affect the hepatic concentration of cyclic AMP,ls4 together with the evidence that these two sulfonylureas accelerate 45Ca-uptake by isolated rat hepatocytes, suggests that the activation of glycogen phosphorylase could be related to the modification of the intracellular Ca2+ concentration. In agreement with this suggestion, it has been demonstrated that tolbutamide (at concentrations similar to those able to activate hepatocyte glycogen phosphorylase) increases the cytosolic concentration of Ca2+ in cultured beta 41~“~~~ by cellular depolarization and subsequent entry of Ca2+ through voltagedependent channels. Our results offer an additional mechanism to explain the increase in F-2,6-P2 concentration caused by sulfonylureas in the liver. In the fed state the mobilization of the stored glycogen by sulfonylureas and its canalization to the glycolytic pathway could facilitate the formation of F-2,6-P,. This regulatory metabolite is the most potent activator of 6phosphofructo 1-kinase and an inhibitor of fructose 1,6bisphosphatase, and its accumulation in liver cells accelerates glycolysis and inhibits gluconeogenesis.7*8

ACKNOWLEDGMENT

The authors thank Dr Lisardo Bosd for critical reading of the manuscript and Martha Messman for her expert editorial assistance.

470

MOJENA ET AL

REFERENCES

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