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Modulation of Insulin Secretion and Glycemia by Selective Inhibition of Cyclic AMP Phosphodiesterase III
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 665–669 (1997)
RC977034
Modulation of Insulin Secretion and Glycemia by Selective Inhibition of Cyclic AMP Phosphodiesterase III Janice C. Parker,1 Maria A. VanVolkenburg, Nancy A. Nardone, Diane M. Hargrove, and Kim M. Andrews Pfizer Inc., Central Research Division, Groton, Connecticut 06340
Received June 19, 1997
The effects of selective inhibition of cyclic AMP phosphodiesterase type III on insulin and glucose levels during an oral glucose challenge were evaluated in obese, diabetic ob/ob mice and in lean, non-diabetic littermates using the selective inhibitor, milrinone. Oral administration of milrinone increased plasma insulin levels both in ob/ob and in lean mice. Glucose tolerance was improved in lean, but not in ob/ob mice, where glucose levels were increased by milrinone treatment. In isolated hepatocytes from normal rats incubation with 200mM milrinone caused a 30% increase in glucose release with a corresponding depletion of glycogen stores. Stimulation of isolated rat adipocytes with 200mM milrinone increased glycerol release 7-fold. We conclude that selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, but their therapeutic utility may be limited by their concurrent stimulation of lipolysis and hepatic glucose output. q 1997 Academic Press
Insulin secretion from pancreatic b-cells is stimulated by increases in the intracellular concentration of cyclic AMP (cAMP). The intracellular concentration of cAMP is regulated by the opposing activities of adenylate cyclase, which is responsible for the synthesis of cAMP, and of cAMP phosphodiesterase (PDE), which catalyzes its degradation. Agents such as forskolin and glucagon-like peptide-1 that increase intracellular levels of cAMP by activating adenylate cyclase have been shown to be effective insulin secretagogues (1, 2). It follows that agents that increase cAMP by inhibiting PDE should also increase insulin secretion, and there is evidence to suggest that this is the case (3-7). 1 Corresponding author. Fax: (860) 441-5719. E-mail: janice_c_ [email protected]. Abbreviations: cAMP, cyclic AMP, adenosine 3*:5*- cyclic monophosphate; DMSO, dimethylsulfoxide; OGTT, oral glucose tolerance test; PDE, adenosine 3*:5*- cyclic monophosphate phosphodiesterase.
PDE activity is ubiquitously expressed, however, multiple PDE isozymes and subtypes exist (8). PDE activity in any given tissue or cell type may be due to the activity of a small subset, or even a single member, of the many known isozymes of PDE. Hence therapeutic intervention based on specific modulation of the activity of an individual PDE may be feasible. A number of inhibitors have been identified that are selective with respect to PDE isozyme family and progress is being made in the design of inhibitors capable of distinguishing between isoforms within PDE families (9). The PDE isoform that modulates insulin secretion from the pancreatic b-cell appears to belong to the PDE III family insofar as insulin secretion is stimulated by PDE III-selective inhibitors such as milrinone and imazodan while inhibitors selective for other PDE isozymes are ineffective (5, 6). The purpose of the present study was to evaluate the potential therapeutic utility of isozyme-selective inhibition of PDE for the treatment of diabetic hyperglycemia. For this purpose, we tested the effect of milrinone, a widely-used PDE III-selective inhibitor, on circulating insulin levels and glycemia in ob/ob mice and in their lean littermates. We also assessed possible extrapancreatic effects of PDE III inhibition by testing milrinone in isolated rat hepatocytes and adipocytes. METHODS Oral glucose tolerance test. Ob/ob mice and their lean littermates were fasted overnight, then dosed by oral gavage with 1g/kg glucose and concurrently with either 10mg/kg milrinone in a 10% DMSO vehicle or with an equivalent volume of the vehicle. Blood samples were collected by orbital bleed at 30 min intervals for glucose measurement. At 90 min after dosing, animals were sacrificed by decapitation and blood collected for insulin measurement by radioimmunoassay. Isolated hepatocyte methods. Hepatocytes were isolated by collagenase digestion of livers from fed male
665
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characteristics of Experimental Mice Lean mice (//?) Body weight (g) Liver wet weight (g) Liver glycogen (mg/g wet weight) Plasma insulin (pM) Blood glucose (mM) Plasma glycerol (mM)
26.7 1.30 54 711 13.5 0.39
{ { { { { {
0.4 0.06 6 61 0.6 0.02
Obese mice (ob/ob)
(10) (10) (10) (10) (10) (10)
43.0 3.00 78 8363 28.1 0.56
{ { { { { {
0.9 0.06 10 876 1.8 0.02
(9) (9) (9) (9) (9) (9)
P 0.0001 0.0001 nsd 0.0001 0.0001 0.0001
Values obtained were for animals fed ad libitum and are means { S.E.M. for the numbers of animals indicated in parentheses. Significance (P) was assessed by Student’s t-test for unpaired data; nsd, no significant difference.
Sprague-Dawley rats as described elsewhere (10). Cells were suspended in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 1% (w/v) gelatin. Hepatocyte suspensions at a concentration of 1.0-3.0 1 106 cells/ml were incubated in 25ml flasks in a 377C shaking waterbath and continuously gassed with 95%O2/5%CO2 . Cells were incubated for one hour with milrinone in 0.1% DMSO or with 0.1% DMSO alone after which aliquots of cell suspension were removed. After centrifugation to sediment the cells, the glucose content of the buffer was measured. The cell pellet was retained for measurement of glycogen content (11). Isolated adipocyte methods. Adipocytes were prepared from the epididymal fat pads of normal male Sprague-Dawley rats. The fat pads were excised, minced finely, collagenase digested, filtered and centrifuged. Adipocytes were diluted to 2.5-5.0 1 106 cells/ ml and preincubated at 377C for 10 min. prior to drug treatment and for with drug. Lipolysis was terminated by placing the cells on ice. After centrifugation to sediment the cells, aliquots of buffer were removed for assay of glycerol. Free glycerol was measured enzymatically using a triglyceride reagent devoid of lipoprotein lipase. Animals. Normal male rats of the Sprague-Dawley line were obtained from Charles River. Male obese mice (C57BL/6J-ob/ob) and their lean littermates (either heterozygous ob// or the homozygous wild-type ///) were obtained from Jackson Labs (Bar Harbor, ME). Reagents. Cell culture media, antibiotics and sera were from BRL/Life Technologies (Grand Island, NY). Insulin radioimmunossay kits were from Binax (Portland, ME). Milrinone and other reagents were purchased from Sigma (St. Louis, MO). RESULTS The characteristics of the obese diabetic mice used in these experiments are presented in Table 1. These were male animals 6-10 weeks of age and the values represent samples from animals fed ad libitum. The
obese animals were hyperglycemic and hyperinsulinemic. While the liver glycogen content was similar in the two groups on a wet weight basis, the liver weight was significantly greater in the obese mice, so that the total liver glycogen content was approximately 3-fold greater than in the lean controls. When ob/ob mice and their lean littermates were subjected to an oral glucose tolerance test (OGTT) in conjunction with an oral dose of milrinone (10mg/kg) the plasma insulin levels were elevated in milrinonetreated animals relative to the corresponding untreated animals (Figure 1). This milrinone-stimulated increase in insulin levels was present both in ob/ob animals and in their lean littermates. However, when the blood glucose measurements taken during the course of the OGTT were compared (Figure 2), it was apparent that only in the lean animals did this increase in plasma insulin concentration translate into an improvement in glucose tolerance. In ob/ob mice treated with milrinone, blood glucose levels continued to climb for the duration of the experiment; 90 min after dosing, the blood glucose levels in the milrinone-treated ob/ob mice were 2-fold greater than in the untreated ob/ob controls. In order to assess the contribution of extra-pancreatic effects of milrinone to these in vivo observations, the effects of milrinone were examined in isolated hepatocytes and adipocytes. In isolated rat hepatocytes, milrinone stimulated glucose output in a concentrationdependent manner (Figure 3). In hepatocytes exposed to 200mM milrinone, glucose output was stimulated by 30% above basal levels. There was a corresponding decrease in the glycogen content of the milrinone-treated hepatocytes (Figure 4). In rat adipocytes, the effect of milrinone was greater than in hepatocytes on a percentage basis: 200mM milrinone increased glycerol release by 7-fold (Figure 5). However, to place this in context, this effect on lipolysis was approximately 50% of that induced by a maximally stimulating concentration (1mM) of the non-selective b-adrenergic agonist isoproterenol, while the effect of 200mM milrinone on glucose output from hepatocytes was approximately
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Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
55% of that of a maximally effective concentration (25nM) of glucagon (data not shown). DISCUSSION Selective inhibitors of PDE III have been shown to be effective insulin secretagogues both in vitro and in vivo (3-5), and here we show that they are effective in elevating insulin levels in an animal model of diabetes, the ob/ob mouse. However, in the ob/ob animals, unlike in their lean littermates, this increase in circulating insulin levels did not result in any improvement in glucose tolerance, nor in any lowering of blood glucose.
FIG. 1. Effect of PDE III inhibition on plasma insulin levels after a glucose challenge. Groups of 5 ob/ob mice or their lean littermates were fasted overnight, then dosed with 1g/kg glucose plus either 10mg/kg milrinone in 10% DMSO or an equivalent volume of 10% DMSO. Blood was collected for insulin measurement 90min after glucose challenge. Data are expressed as means { S.E.M.; *, significantly different from corresponding control value, P õ 0.001.
FIG. 2. Effect of PDE III inhibition on glucose tolerance. Groups of 5 ob/ob mice mice or their lean littermates were fasted overnight, then dosed with 10mg/kg milrinone in 10% DMSO (l) or an equivalent volume of vehicle (s) and immediately subjected to an oral glucose tolerance test as described in Methods. Blood was collected for glucose measurement prior to dosing and 30, 60 and 90 min after glucose challenge. Data are expressed as means { S.E.M.; *, significantly different from corresponding control value, P õ 0.005.
It is not unprecedented for an insulin secretagogue to be ineffective as a euglycemic agent in ob/ob mice; sulfonylureas which are effective for the therapy of human
667
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Effect of PDE III inhibition on glucose output from isolated rat hepatocytes. Isolated hepatocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of cell suspension were removed after 1hr for measurement of glucose content. Data are expressed as means { S.E.M. (nÅ3).
diabetes fail to lower blood glucose to any significant degree in these animals (12). This has been attributed to the fact that ob/ob mice are extremely insulin resis-
FIG. 4. Effect of PDE III inhibition on glycogen content of rat hepatocytes. Isolated hepatocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of cell suspension were removed after 1hr for measurement of glycogen content. Data are expressed as means { S.E.M. (nÅ3).
FIG. 5. Effect of PDE III inhibition on glycerol release from isolated rat adipocytes. Isolated adipocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of buffer were removed after 1hr for measurement of glycerol content. Data are expressed as means { S.E.M. (nÅ3).
tant and already markedly hyperinsulinemic. However in this particular study, milrinone treatment was actually associated with an increase in circulating blood glucose in ob/ob mice, leading us to suspect that inhibition of PDE III, which is expressed in liver, cardiac and adipose tissue as well as in pancreatic islets, was causing glucose mobilization by action at extra-pancreatic sites. We addressed this question by measuring the direct effects of milrinone in isolated hepatocytes and adipocytes from normal rats, and demonstrated that milrinone did increase glucose output from hepatocytes, and glycerol release from adipocytes, implying activation of both glycogenolysis and lipolysis, presumably via cAMP-mediated activation of glycogen phosphorylase and hormone-sensitive lipase respectively. The glycogen content of the milrinone-stimulated hepatocytes was reduced relative to controls, consistent with activation of glycogen phosphorylase. While stimulation of glycogenolysis liberates glucose directly into the bloodstream, increased lipolysis would tend to increase circulating glucose levels by liberating fatty acids that could serve as an alternate fuel source. Although in vitro, the effect of 200mM milrinone on lipolysis was greater than that on glycogenolysis in terms of stimulation above basal, the effects were similar relative to the effects of a maximally stimulatory physiological stimulus (1mM isoproterenol or 25nM glucagon). Thus the relative contributions of these two tissues to circulating glucose in vivo is likely to be a function of the total mass of each tissue. Assuming that milrinone stimulation of glycogenolysis and lipolysis occurs in in-
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tact animals of the ob/ob mouse strain as it does in the isolated cells of normal rats, each of these effects (i.e. stimulation of glycogenolysis and lipolysis) would be of greater magnitude in the obese animals than in their lean littermates, because of the greater adiposity and larger total glycogen content of the obese animals Table 1, (13, 14). This may account for the fact that milrinoneinduced stimulation of glycogenolysis and lipolysis, which presumably occurs in both lean and obese animals, exacerbates hyperglycemia in ob/ob mice, but not in their lean littermates. The extreme insulin resistance of the ob/ob mice (14) probably also contributes to the failure of milrinone-stimulated increases in insulin secretion to improve glycemia in these animals. It is possible that pre-existing insulin resistance may be exacerbated by milrinone, but this question was not addressed by the present study. These data suggest that while selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, they may have limited therapeutic utility for human diabetic subjects because of their concurrent stimulation of lipolysis and hepatic glucose output. However, it was only in the ob/ob mouse, an animal model of extreme hyperinsulinemia and insulin resistance (14), that these effects caused an increase in blood glucose; in their lean littermates glucose tolerance was improved. It may be that human diabetic subjects, who typically exhibit a far less extreme constellation of symptoms than that seen in the ob/ob mouse and whose disease can often be satisfactorily treated by therapeutic agents such as the sulfonylureas that are ineffective in that animal model, may benefit from treatment with appropriately selective inhibitors of PDE III. Theophylline, a non-specific PDE inhibitor, has been shown in normal human subjects to increase insulin secretion without deleterious effects on glucose levels (3). Furthermore, while the isoform of PDE that
modulates insulin secretion appears to be of the PDE III family (on the basis of its inhibition by type-selective inhibitors such as milrinone) this isozyme has yet to be cloned and may be of a subtype distinct from those found in other tissues. Identification of selective inhibitors of the b-cell specific form of PDE may permit the design of agents that stimulate insulin secretion without undesirable extra-pancreatic effects. REFERENCES 1. Hermansen, K. (1985) Endocrinology 116, 2251–2258. 2. Drucker, D. J., Phillippe, J., Mojsov, S., Chick, W. L., and Habener, J. F. (1987) Proc. Natl. Acad, Sci. USA 84, 3434–3438. 3. Arnman, K., Carlstrom, S., and Thorell, J. (1975) Acta Med. Scand. 197, 271–274. 4. Liebowitz, M., Biswas, C., Brady, E., Conti, M., Cullinan, C., Hayes, N., Manganiello, V., Saperstein, R., Wang, L., Zafian, P., and Berger, J. (1995) Diabetes 44, 67–74. 5. Parker, J. C., VanVolkenburg, M. A., Ketchum, R. J., Brayman, K. L., and Andrews, K. M. (1995) Biochem. Biophys. Res. Commun. 217, 916–923. 6. Shafiee-Nick, R., Pyne, N. J., and Furman, B. L. (1995) Brit. J. Pharmacol. 115, 1486–1492. 7. Sams, D. J., and Montague, W. (1972) Biochem. J. 129, 945– 952. 8. Beavo, J. (1995) Physiol Rev. 75, 725–748. 9. Beavo, J., and Reifsnyder, D. H. (1990) Trends Pharm. Sci. 11, 150–155. 10. Blackmore, P. F., and Exton, J. H. (1985) Methods Enzymol. 109, 550–558. 11. Hassid, W. Z., and Abraham, S. (1957) Methods Enzymol. 3, 34– 50. 12. Stevenson, R. W., Hutson, N. J., Krupp, M. N., Volkmann, R. A., Holland, G. F., Eggler, J. F., Clark, D. A., McPherson, R. K., Hall, K. L., Danbury, B. H., Gibbs, E. M., and Kreutter, D. K. (1990) Diabetes 39, 1218–1227. 13. van de Werve, G., Assimacopoulos-Jeannet, F., and Jeanrenaud, B. (1983) Biochem. J. 216, 273–280. 14. Herberg, L., and Coleman, D. L. (1977) Metabolism 26, 59–99.
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236, 665–669 (1997)
RC977034
Modulation of Insulin Secretion and Glycemia by Selective Inhibition of Cyclic AMP Phosphodiesterase III Janice C. Parker,1 Maria A. VanVolkenburg, Nancy A. Nardone, Diane M. Hargrove, and Kim M. Andrews Pfizer Inc., Central Research Division, Groton, Connecticut 06340
Received June 19, 1997
The effects of selective inhibition of cyclic AMP phosphodiesterase type III on insulin and glucose levels during an oral glucose challenge were evaluated in obese, diabetic ob/ob mice and in lean, non-diabetic littermates using the selective inhibitor, milrinone. Oral administration of milrinone increased plasma insulin levels both in ob/ob and in lean mice. Glucose tolerance was improved in lean, but not in ob/ob mice, where glucose levels were increased by milrinone treatment. In isolated hepatocytes from normal rats incubation with 200mM milrinone caused a 30% increase in glucose release with a corresponding depletion of glycogen stores. Stimulation of isolated rat adipocytes with 200mM milrinone increased glycerol release 7-fold. We conclude that selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, but their therapeutic utility may be limited by their concurrent stimulation of lipolysis and hepatic glucose output. q 1997 Academic Press
Insulin secretion from pancreatic b-cells is stimulated by increases in the intracellular concentration of cyclic AMP (cAMP). The intracellular concentration of cAMP is regulated by the opposing activities of adenylate cyclase, which is responsible for the synthesis of cAMP, and of cAMP phosphodiesterase (PDE), which catalyzes its degradation. Agents such as forskolin and glucagon-like peptide-1 that increase intracellular levels of cAMP by activating adenylate cyclase have been shown to be effective insulin secretagogues (1, 2). It follows that agents that increase cAMP by inhibiting PDE should also increase insulin secretion, and there is evidence to suggest that this is the case (3-7). 1 Corresponding author. Fax: (860) 441-5719. E-mail: janice_c_ [email protected]. Abbreviations: cAMP, cyclic AMP, adenosine 3*:5*- cyclic monophosphate; DMSO, dimethylsulfoxide; OGTT, oral glucose tolerance test; PDE, adenosine 3*:5*- cyclic monophosphate phosphodiesterase.
PDE activity is ubiquitously expressed, however, multiple PDE isozymes and subtypes exist (8). PDE activity in any given tissue or cell type may be due to the activity of a small subset, or even a single member, of the many known isozymes of PDE. Hence therapeutic intervention based on specific modulation of the activity of an individual PDE may be feasible. A number of inhibitors have been identified that are selective with respect to PDE isozyme family and progress is being made in the design of inhibitors capable of distinguishing between isoforms within PDE families (9). The PDE isoform that modulates insulin secretion from the pancreatic b-cell appears to belong to the PDE III family insofar as insulin secretion is stimulated by PDE III-selective inhibitors such as milrinone and imazodan while inhibitors selective for other PDE isozymes are ineffective (5, 6). The purpose of the present study was to evaluate the potential therapeutic utility of isozyme-selective inhibition of PDE for the treatment of diabetic hyperglycemia. For this purpose, we tested the effect of milrinone, a widely-used PDE III-selective inhibitor, on circulating insulin levels and glycemia in ob/ob mice and in their lean littermates. We also assessed possible extrapancreatic effects of PDE III inhibition by testing milrinone in isolated rat hepatocytes and adipocytes. METHODS Oral glucose tolerance test. Ob/ob mice and their lean littermates were fasted overnight, then dosed by oral gavage with 1g/kg glucose and concurrently with either 10mg/kg milrinone in a 10% DMSO vehicle or with an equivalent volume of the vehicle. Blood samples were collected by orbital bleed at 30 min intervals for glucose measurement. At 90 min after dosing, animals were sacrificed by decapitation and blood collected for insulin measurement by radioimmunoassay. Isolated hepatocyte methods. Hepatocytes were isolated by collagenase digestion of livers from fed male
665
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BBRC 7034
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6933$$$541
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AP: BBRC
Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characteristics of Experimental Mice Lean mice (//?) Body weight (g) Liver wet weight (g) Liver glycogen (mg/g wet weight) Plasma insulin (pM) Blood glucose (mM) Plasma glycerol (mM)
26.7 1.30 54 711 13.5 0.39
{ { { { { {
0.4 0.06 6 61 0.6 0.02
Obese mice (ob/ob)
(10) (10) (10) (10) (10) (10)
43.0 3.00 78 8363 28.1 0.56
{ { { { { {
0.9 0.06 10 876 1.8 0.02
(9) (9) (9) (9) (9) (9)
P 0.0001 0.0001 nsd 0.0001 0.0001 0.0001
Values obtained were for animals fed ad libitum and are means { S.E.M. for the numbers of animals indicated in parentheses. Significance (P) was assessed by Student’s t-test for unpaired data; nsd, no significant difference.
Sprague-Dawley rats as described elsewhere (10). Cells were suspended in Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 1% (w/v) gelatin. Hepatocyte suspensions at a concentration of 1.0-3.0 1 106 cells/ml were incubated in 25ml flasks in a 377C shaking waterbath and continuously gassed with 95%O2/5%CO2 . Cells were incubated for one hour with milrinone in 0.1% DMSO or with 0.1% DMSO alone after which aliquots of cell suspension were removed. After centrifugation to sediment the cells, the glucose content of the buffer was measured. The cell pellet was retained for measurement of glycogen content (11). Isolated adipocyte methods. Adipocytes were prepared from the epididymal fat pads of normal male Sprague-Dawley rats. The fat pads were excised, minced finely, collagenase digested, filtered and centrifuged. Adipocytes were diluted to 2.5-5.0 1 106 cells/ ml and preincubated at 377C for 10 min. prior to drug treatment and for with drug. Lipolysis was terminated by placing the cells on ice. After centrifugation to sediment the cells, aliquots of buffer were removed for assay of glycerol. Free glycerol was measured enzymatically using a triglyceride reagent devoid of lipoprotein lipase. Animals. Normal male rats of the Sprague-Dawley line were obtained from Charles River. Male obese mice (C57BL/6J-ob/ob) and their lean littermates (either heterozygous ob// or the homozygous wild-type ///) were obtained from Jackson Labs (Bar Harbor, ME). Reagents. Cell culture media, antibiotics and sera were from BRL/Life Technologies (Grand Island, NY). Insulin radioimmunossay kits were from Binax (Portland, ME). Milrinone and other reagents were purchased from Sigma (St. Louis, MO). RESULTS The characteristics of the obese diabetic mice used in these experiments are presented in Table 1. These were male animals 6-10 weeks of age and the values represent samples from animals fed ad libitum. The
obese animals were hyperglycemic and hyperinsulinemic. While the liver glycogen content was similar in the two groups on a wet weight basis, the liver weight was significantly greater in the obese mice, so that the total liver glycogen content was approximately 3-fold greater than in the lean controls. When ob/ob mice and their lean littermates were subjected to an oral glucose tolerance test (OGTT) in conjunction with an oral dose of milrinone (10mg/kg) the plasma insulin levels were elevated in milrinonetreated animals relative to the corresponding untreated animals (Figure 1). This milrinone-stimulated increase in insulin levels was present both in ob/ob animals and in their lean littermates. However, when the blood glucose measurements taken during the course of the OGTT were compared (Figure 2), it was apparent that only in the lean animals did this increase in plasma insulin concentration translate into an improvement in glucose tolerance. In ob/ob mice treated with milrinone, blood glucose levels continued to climb for the duration of the experiment; 90 min after dosing, the blood glucose levels in the milrinone-treated ob/ob mice were 2-fold greater than in the untreated ob/ob controls. In order to assess the contribution of extra-pancreatic effects of milrinone to these in vivo observations, the effects of milrinone were examined in isolated hepatocytes and adipocytes. In isolated rat hepatocytes, milrinone stimulated glucose output in a concentrationdependent manner (Figure 3). In hepatocytes exposed to 200mM milrinone, glucose output was stimulated by 30% above basal levels. There was a corresponding decrease in the glycogen content of the milrinone-treated hepatocytes (Figure 4). In rat adipocytes, the effect of milrinone was greater than in hepatocytes on a percentage basis: 200mM milrinone increased glycerol release by 7-fold (Figure 5). However, to place this in context, this effect on lipolysis was approximately 50% of that induced by a maximally stimulating concentration (1mM) of the non-selective b-adrenergic agonist isoproterenol, while the effect of 200mM milrinone on glucose output from hepatocytes was approximately
666
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Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
55% of that of a maximally effective concentration (25nM) of glucagon (data not shown). DISCUSSION Selective inhibitors of PDE III have been shown to be effective insulin secretagogues both in vitro and in vivo (3-5), and here we show that they are effective in elevating insulin levels in an animal model of diabetes, the ob/ob mouse. However, in the ob/ob animals, unlike in their lean littermates, this increase in circulating insulin levels did not result in any improvement in glucose tolerance, nor in any lowering of blood glucose.
FIG. 1. Effect of PDE III inhibition on plasma insulin levels after a glucose challenge. Groups of 5 ob/ob mice or their lean littermates were fasted overnight, then dosed with 1g/kg glucose plus either 10mg/kg milrinone in 10% DMSO or an equivalent volume of 10% DMSO. Blood was collected for insulin measurement 90min after glucose challenge. Data are expressed as means { S.E.M.; *, significantly different from corresponding control value, P õ 0.001.
FIG. 2. Effect of PDE III inhibition on glucose tolerance. Groups of 5 ob/ob mice mice or their lean littermates were fasted overnight, then dosed with 10mg/kg milrinone in 10% DMSO (l) or an equivalent volume of vehicle (s) and immediately subjected to an oral glucose tolerance test as described in Methods. Blood was collected for glucose measurement prior to dosing and 30, 60 and 90 min after glucose challenge. Data are expressed as means { S.E.M.; *, significantly different from corresponding control value, P õ 0.005.
It is not unprecedented for an insulin secretagogue to be ineffective as a euglycemic agent in ob/ob mice; sulfonylureas which are effective for the therapy of human
667
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07-11-97 18:57:19
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Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Effect of PDE III inhibition on glucose output from isolated rat hepatocytes. Isolated hepatocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of cell suspension were removed after 1hr for measurement of glucose content. Data are expressed as means { S.E.M. (nÅ3).
diabetes fail to lower blood glucose to any significant degree in these animals (12). This has been attributed to the fact that ob/ob mice are extremely insulin resis-
FIG. 4. Effect of PDE III inhibition on glycogen content of rat hepatocytes. Isolated hepatocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of cell suspension were removed after 1hr for measurement of glycogen content. Data are expressed as means { S.E.M. (nÅ3).
FIG. 5. Effect of PDE III inhibition on glycerol release from isolated rat adipocytes. Isolated adipocytes were prepared and incubated with the specified concentrations of the PDE III inhibitor milrinone as described in Methods. Aliquots of buffer were removed after 1hr for measurement of glycerol content. Data are expressed as means { S.E.M. (nÅ3).
tant and already markedly hyperinsulinemic. However in this particular study, milrinone treatment was actually associated with an increase in circulating blood glucose in ob/ob mice, leading us to suspect that inhibition of PDE III, which is expressed in liver, cardiac and adipose tissue as well as in pancreatic islets, was causing glucose mobilization by action at extra-pancreatic sites. We addressed this question by measuring the direct effects of milrinone in isolated hepatocytes and adipocytes from normal rats, and demonstrated that milrinone did increase glucose output from hepatocytes, and glycerol release from adipocytes, implying activation of both glycogenolysis and lipolysis, presumably via cAMP-mediated activation of glycogen phosphorylase and hormone-sensitive lipase respectively. The glycogen content of the milrinone-stimulated hepatocytes was reduced relative to controls, consistent with activation of glycogen phosphorylase. While stimulation of glycogenolysis liberates glucose directly into the bloodstream, increased lipolysis would tend to increase circulating glucose levels by liberating fatty acids that could serve as an alternate fuel source. Although in vitro, the effect of 200mM milrinone on lipolysis was greater than that on glycogenolysis in terms of stimulation above basal, the effects were similar relative to the effects of a maximally stimulatory physiological stimulus (1mM isoproterenol or 25nM glucagon). Thus the relative contributions of these two tissues to circulating glucose in vivo is likely to be a function of the total mass of each tissue. Assuming that milrinone stimulation of glycogenolysis and lipolysis occurs in in-
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Vol. 236, No. 3, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tact animals of the ob/ob mouse strain as it does in the isolated cells of normal rats, each of these effects (i.e. stimulation of glycogenolysis and lipolysis) would be of greater magnitude in the obese animals than in their lean littermates, because of the greater adiposity and larger total glycogen content of the obese animals Table 1, (13, 14). This may account for the fact that milrinoneinduced stimulation of glycogenolysis and lipolysis, which presumably occurs in both lean and obese animals, exacerbates hyperglycemia in ob/ob mice, but not in their lean littermates. The extreme insulin resistance of the ob/ob mice (14) probably also contributes to the failure of milrinone-stimulated increases in insulin secretion to improve glycemia in these animals. It is possible that pre-existing insulin resistance may be exacerbated by milrinone, but this question was not addressed by the present study. These data suggest that while selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, they may have limited therapeutic utility for human diabetic subjects because of their concurrent stimulation of lipolysis and hepatic glucose output. However, it was only in the ob/ob mouse, an animal model of extreme hyperinsulinemia and insulin resistance (14), that these effects caused an increase in blood glucose; in their lean littermates glucose tolerance was improved. It may be that human diabetic subjects, who typically exhibit a far less extreme constellation of symptoms than that seen in the ob/ob mouse and whose disease can often be satisfactorily treated by therapeutic agents such as the sulfonylureas that are ineffective in that animal model, may benefit from treatment with appropriately selective inhibitors of PDE III. Theophylline, a non-specific PDE inhibitor, has been shown in normal human subjects to increase insulin secretion without deleterious effects on glucose levels (3). Furthermore, while the isoform of PDE that
modulates insulin secretion appears to be of the PDE III family (on the basis of its inhibition by type-selective inhibitors such as milrinone) this isozyme has yet to be cloned and may be of a subtype distinct from those found in other tissues. Identification of selective inhibitors of the b-cell specific form of PDE may permit the design of agents that stimulate insulin secretion without undesirable extra-pancreatic effects. REFERENCES 1. Hermansen, K. (1985) Endocrinology 116, 2251–2258. 2. Drucker, D. J., Phillippe, J., Mojsov, S., Chick, W. L., and Habener, J. F. (1987) Proc. Natl. Acad, Sci. USA 84, 3434–3438. 3. Arnman, K., Carlstrom, S., and Thorell, J. (1975) Acta Med. Scand. 197, 271–274. 4. Liebowitz, M., Biswas, C., Brady, E., Conti, M., Cullinan, C., Hayes, N., Manganiello, V., Saperstein, R., Wang, L., Zafian, P., and Berger, J. (1995) Diabetes 44, 67–74. 5. Parker, J. C., VanVolkenburg, M. A., Ketchum, R. J., Brayman, K. L., and Andrews, K. M. (1995) Biochem. Biophys. Res. Commun. 217, 916–923. 6. Shafiee-Nick, R., Pyne, N. J., and Furman, B. L. (1995) Brit. J. Pharmacol. 115, 1486–1492. 7. Sams, D. J., and Montague, W. (1972) Biochem. J. 129, 945– 952. 8. Beavo, J. (1995) Physiol Rev. 75, 725–748. 9. Beavo, J., and Reifsnyder, D. H. (1990) Trends Pharm. Sci. 11, 150–155. 10. Blackmore, P. F., and Exton, J. H. (1985) Methods Enzymol. 109, 550–558. 11. Hassid, W. Z., and Abraham, S. (1957) Methods Enzymol. 3, 34– 50. 12. Stevenson, R. W., Hutson, N. J., Krupp, M. N., Volkmann, R. A., Holland, G. F., Eggler, J. F., Clark, D. A., McPherson, R. K., Hall, K. L., Danbury, B. H., Gibbs, E. M., and Kreutter, D. K. (1990) Diabetes 39, 1218–1227. 13. van de Werve, G., Assimacopoulos-Jeannet, F., and Jeanrenaud, B. (1983) Biochem. J. 216, 273–280. 14. Herberg, L., and Coleman, D. L. (1977) Metabolism 26, 59–99.
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