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Second messenger and its regulatory enzymes under protein nutrition
NUTRITION RESEARCH,Vol. 9, pp. 465-469, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
SECOND
MESSENGER
AND
ITS
REGULATORY NUTRITION
ENZYMES
Vinay Bhutani M.Sc., Vijay Kumar M.Sc., A.K. Tyagi Ph.D. Department of Biochemistry Vallabhbhai Patel Chest Institute University of Delhi Delhi-10007, INDIA
UNDER
PROTEIN
and U.K. Misra Ph.D.
ABSTRACT
In rat pancreatic tissue the levels of cAMP are elevated in protein deficient diet and lowered in the case of protein rich diet. The activity of the enzymes for the synthesis and degradation of this molecule namely adenylate cyclase (AC) and phosphodiesterase (PDE) is markedly lowered in the case of deficient diet feeding and elevated in the case of protein rich diet feeding. However, both the enzymes are influenced to a different magnitude under the different dietary conditions and the levels of cAMP seems to be governed by the net balance of these anabolic and catabolic activities. Thus dietary status of animals seems to exert significant control on the level of cAMP in pancreas. Key words: pancreas, adenylate cyclase, phesphodiesterase INTRODUCTION The precise role of cAMP, the second messenger, in hormone induced metabolic responses and several regulatory functions are well known. Indirect evidence has accumulated during recent years indicating that cAMP is also i m p o r t a n t for r e l e a s e of i n s u l i n from p a n c r e a t i c beta cells, although the precise manner in which this nucleotide participates in insulin secretion is obscure (i). Moreover, a modest increase in cAMP synthesis can be attributed to the activation of adenylate cyclase by C~-calmodulin. However, this seems to play only a limited role in the secretory response of pancreatic tissue (2). Cyclic AMP is generated fromSTP in a reaction catalyzed by the enzyme adenylate cyclase and requiring Mg as a cofactor(3). Degradation of cAMP proceeds through the action of cAMP phosphodiesterase. This enzyme exists in multiple forms associated with soluble and membrane bound fractions. Several agents can cause elevation of protein kinase activity either by raising the levels of cAMP through adenylate cyclase or by inhibiting phosphodiesterase. This is in turn may cause excessive secretion by insulin (2). Correspondence to: Prof. U.K. Misra, Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi-ll0007, INDIA
465
466
V. BHUTANI eL a l .
We have earlier reported that inadequate protein intake results in glucose intolerance, lowered serum insulin levels and elevated levels of pancreatic insulin (4). However, no study has appeared on the regulation of cAMP in pancreas under different dietary conditions. This report for the first time deals with the influence of dietary protein intake on the enzymes that regulate the concentration of this second messenger in pancreas.
MATERIALS AND METHODS Male rats (Wistar strain) weighing 40-50 grams raised in our Institute maintained colony were used in the study. The rats were divided into three groups fed the following isonitrogenous diets - Group A: Rice diet (protein content 8%) deficient in lysine and threonine (Deficient group); Group B: Rice diet (protein content 8%) supplemented with lysine and threonime (Supplemented group) and Group C: 20% casein diet (Casein group). The formulation of diets is described earlier (5). The respective diets were pair-fed with supplemented and casein diet against deficient diet ad libitum in the course of four weeks of feeding. The rats had free access to water all times. After feeding for four weeks rats were sacrificed, cut open, pancreas were removed, washed, wiped, weighed and processed for various measurements. Adenylate cyclase activity was determined in pancreatic homogenates by measuring the radioactivity in the cyclic AMP formed from (8-14C)-ATP by a modified [Homogenate was prepared in 0.32 M sucrose, 6.0 uM theophylline was used in the reaction mixture and instead of paper chromatography thin layer chromatography was performed] method of Wood et al (6) and phosphodiesterase activity was measured in pancreatic homogenates by the method of Butcher and Sutherland (7). One unit of adenylate cyclase is expressed as one umole of ATP converted to cyclic AMP in one hour and one unit of phosphodiesterase is expressed as one umole of inorganic phosphorus liberated in one hour. cAMP was measured in tissue extracts of pancreas using cAMP kit supplied by Radiochemical Centre, Amersham, U.K. (4). Values from four assays performed for each measurement from pancreas of eight animals in each dietary group were statistically analyzed by using student's 't' test.
RESULTS AND DISCUSSION Feeding of diets varying in quality and quantity of proteins to rats for four weeks resulted in approximately 42% and 70% gain in body weight in supplemented and casein groups respectively as compared to the deficient group. Similarly increases in pancreatic weight were approximately 26% and 48% in supplemented and casein groups respectively as compared to the deficient group (5). This shows that feeding diets of low protein quality and quantity affects growth of the animal. In an earlier communication we reported that under such circumstances pancreatic chemistry [Lipid profile, membrane transducing components, insulin and enzyme levels, microtubule assembly] remains consistent whether calculated on the basis of per organ or per gram weight (5,8). Higher levels of cAMP have been reported in rats fed low protein diets (4). But how the enzymes responsible for its regulation are affected under different dietary protein status of the subject is not known.
SECONDMESSENGER & ITS REGULATORYENZYMES
467
Figure l depicts that in pancreas the activity of adenylate cyclase was markedly lower in the deficient group as compared to the supplemented group. On the other hand, animals fed the protein rich diet, both in quality and quantity (casein group) exhibited a marginal elevation in the activity of adenylate cyclase as compared to the supplemented group. It would thus appear that the casein-fed animals would accumulate higher concentration of cAMP.
1200 -
200 -
12 a.b
~b
I
1.0
I000180 -
L 800
~
:r. > 0.8
-
140~ 600 -
~. 0 . 6
o
E 400
o.t
g
~o-
0
"60,a
s
S
-
O
~DE
s
0.2-
D
D
AC
-
C 200
RO-
-
D
O
cAMP
Figure 1. Effect of dietary protein on the activities of adenylate cyclase,phosphodiesterase and the levels of cyclic adenosine monophosphate in pancreas. Assays of the enzymes were performed by the standard procedures and their activity expressed in units as given in '~aterials and Methods". Values are mean • SE from four animals in each group. PDE = phosphodiesterase; phosphate. D - deficient
group;
AC = adenylate
S - supplemented
a - values are significantly b - values are significantly
cyclase;
group;
cAMP = cyclic adenosine mono-
C - casein group.
different at 5% level from supplemented group. different at 5% level from casein group.
468
V. BHUTANI et al.
The assay of this second messenger however, exhibited the results to be otherwise. The levels of pancreatic cAMP were lowest in the casein diet fed animals and highest in the animals fed deficient diet (5) - a trend exactly opposite to the levels of adenylate cyclase. Similar to the adenylate cyclase data phosphodiesterase activity was also significantly lower in the deficient group as compared to the supplemented and casein groups. Also phosphodiesterase level was significantly lower in supplemented group as compared to casein group (Fig. 1). Although, in pancreas, both adenylate cyclase and phosphodiesterase exhibited similar type of influence in all groups of animals the magnitude of this influence was at variance. ~ The ratio of adenylate cyclase: phosphodiesterase in pancreas was significantly higher in deficient group as compared to supplemented and casein groups. It was lower in casein group (Fig. I). Higher adenylate cyclase:phosphodiesterase ratio in pancreas of deficient group would explain relatively higher concentration of cAMP in deficient group followed by supplemented group and the lowest in the case of casein group (Fig. I). Thus it appears that the concentration of cAMP in a cell.is not directly related to enzyme responsible for its synthesis rather it is the dynamic interplay of synthetic and degradative activities which are more important and it is the net balance of these activities that determines the level of cAMP in the cell. Under conditions of poor protein quality the energy requirements are met by metabolizing carbohydrates and lipids - a situation that would demand higher levels of cAMP. The levels of cAMP as observed above were significantly higher in deficient diet fed group as compared to supplemented or casein diet fed groups. Here quality of protein can be related to increased cAMP levels. ~wever, cAMP levels were non-significantly higher in supplemented group as compared to casein group which may be inferred to the quantity of protein and simultaneously lowering of c A ~ levels. It shows that dietary status of the animal is an important factor in regulating the levels of cAMP. ACKNOWLEDGEMENTS Vinay Bhutani and Vijay Kumar are CSIR Fellows. REFERENCES I.
Grill V, Cerasi E. Stimulation by D-glucose of cyclic adenosine 3'-5'monophosphate accumulation and insulin release in isolated pancreatic islets of the rat. J Biol Chem 1974;249:4196-201.
2.
Malaisse WJ, Malaisse-Lagae F. The role of cyclic AMP in insulin release. Experientia 1984;40:1068-75.
3.
Greaves MF. Cellular recognition. In: Outlines studies in biology. New York; John Wiley & Sons. 1975; 26-32.
4.
Bhutani V, Kumar V, Misra UK. Effect of inadequate dietary proteins on pancreatic insulin and cAMP levels in rats. Nutr Rept Int 1985;32:1413-20.
5.
Bhutani V, Kumar V, Tyagi AK, Misra UK. Changes of fatty acid composition in pancreas of rats fed inadequate dietary protein. Nutr Res 1987;7':763-70.
SECONDMESSENGER & ITS REGULATORYENZYMES 6.
Wood HN, Lin MC, Braun AC. The inhibition of plant and animal adenosine 3'5'-cyclic monophosphate phosphodiesterase by a cell division promoting substance from tissues of higher plant species. Proc Natl Acad Sci USA 1972;69:403-6.
7.
Butcher RW, Sutherland EW. Adenosine 3'-5'-phosphate in biological materials. J Biol Chem 1962;237:1244-50.
8.
Bhutani V, Kumar V, Misra UK. Turnover of pancreas phospholipids in rats fed inadequate dietary protein. Int J Vit Nutr Res 1986;56:275-79.
Accepted for publication December 1, 1988.
469
SECOND
MESSENGER
AND
ITS
REGULATORY NUTRITION
ENZYMES
Vinay Bhutani M.Sc., Vijay Kumar M.Sc., A.K. Tyagi Ph.D. Department of Biochemistry Vallabhbhai Patel Chest Institute University of Delhi Delhi-10007, INDIA
UNDER
PROTEIN
and U.K. Misra Ph.D.
ABSTRACT
In rat pancreatic tissue the levels of cAMP are elevated in protein deficient diet and lowered in the case of protein rich diet. The activity of the enzymes for the synthesis and degradation of this molecule namely adenylate cyclase (AC) and phosphodiesterase (PDE) is markedly lowered in the case of deficient diet feeding and elevated in the case of protein rich diet feeding. However, both the enzymes are influenced to a different magnitude under the different dietary conditions and the levels of cAMP seems to be governed by the net balance of these anabolic and catabolic activities. Thus dietary status of animals seems to exert significant control on the level of cAMP in pancreas. Key words: pancreas, adenylate cyclase, phesphodiesterase INTRODUCTION The precise role of cAMP, the second messenger, in hormone induced metabolic responses and several regulatory functions are well known. Indirect evidence has accumulated during recent years indicating that cAMP is also i m p o r t a n t for r e l e a s e of i n s u l i n from p a n c r e a t i c beta cells, although the precise manner in which this nucleotide participates in insulin secretion is obscure (i). Moreover, a modest increase in cAMP synthesis can be attributed to the activation of adenylate cyclase by C~-calmodulin. However, this seems to play only a limited role in the secretory response of pancreatic tissue (2). Cyclic AMP is generated fromSTP in a reaction catalyzed by the enzyme adenylate cyclase and requiring Mg as a cofactor(3). Degradation of cAMP proceeds through the action of cAMP phosphodiesterase. This enzyme exists in multiple forms associated with soluble and membrane bound fractions. Several agents can cause elevation of protein kinase activity either by raising the levels of cAMP through adenylate cyclase or by inhibiting phosphodiesterase. This is in turn may cause excessive secretion by insulin (2). Correspondence to: Prof. U.K. Misra, Department of Biochemistry, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi-ll0007, INDIA
465
466
V. BHUTANI eL a l .
We have earlier reported that inadequate protein intake results in glucose intolerance, lowered serum insulin levels and elevated levels of pancreatic insulin (4). However, no study has appeared on the regulation of cAMP in pancreas under different dietary conditions. This report for the first time deals with the influence of dietary protein intake on the enzymes that regulate the concentration of this second messenger in pancreas.
MATERIALS AND METHODS Male rats (Wistar strain) weighing 40-50 grams raised in our Institute maintained colony were used in the study. The rats were divided into three groups fed the following isonitrogenous diets - Group A: Rice diet (protein content 8%) deficient in lysine and threonine (Deficient group); Group B: Rice diet (protein content 8%) supplemented with lysine and threonime (Supplemented group) and Group C: 20% casein diet (Casein group). The formulation of diets is described earlier (5). The respective diets were pair-fed with supplemented and casein diet against deficient diet ad libitum in the course of four weeks of feeding. The rats had free access to water all times. After feeding for four weeks rats were sacrificed, cut open, pancreas were removed, washed, wiped, weighed and processed for various measurements. Adenylate cyclase activity was determined in pancreatic homogenates by measuring the radioactivity in the cyclic AMP formed from (8-14C)-ATP by a modified [Homogenate was prepared in 0.32 M sucrose, 6.0 uM theophylline was used in the reaction mixture and instead of paper chromatography thin layer chromatography was performed] method of Wood et al (6) and phosphodiesterase activity was measured in pancreatic homogenates by the method of Butcher and Sutherland (7). One unit of adenylate cyclase is expressed as one umole of ATP converted to cyclic AMP in one hour and one unit of phosphodiesterase is expressed as one umole of inorganic phosphorus liberated in one hour. cAMP was measured in tissue extracts of pancreas using cAMP kit supplied by Radiochemical Centre, Amersham, U.K. (4). Values from four assays performed for each measurement from pancreas of eight animals in each dietary group were statistically analyzed by using student's 't' test.
RESULTS AND DISCUSSION Feeding of diets varying in quality and quantity of proteins to rats for four weeks resulted in approximately 42% and 70% gain in body weight in supplemented and casein groups respectively as compared to the deficient group. Similarly increases in pancreatic weight were approximately 26% and 48% in supplemented and casein groups respectively as compared to the deficient group (5). This shows that feeding diets of low protein quality and quantity affects growth of the animal. In an earlier communication we reported that under such circumstances pancreatic chemistry [Lipid profile, membrane transducing components, insulin and enzyme levels, microtubule assembly] remains consistent whether calculated on the basis of per organ or per gram weight (5,8). Higher levels of cAMP have been reported in rats fed low protein diets (4). But how the enzymes responsible for its regulation are affected under different dietary protein status of the subject is not known.
SECONDMESSENGER & ITS REGULATORYENZYMES
467
Figure l depicts that in pancreas the activity of adenylate cyclase was markedly lower in the deficient group as compared to the supplemented group. On the other hand, animals fed the protein rich diet, both in quality and quantity (casein group) exhibited a marginal elevation in the activity of adenylate cyclase as compared to the supplemented group. It would thus appear that the casein-fed animals would accumulate higher concentration of cAMP.
1200 -
200 -
12 a.b
~b
I
1.0
I000180 -
L 800
~
:r. > 0.8
-
140~ 600 -
~. 0 . 6
o
E 400
o.t
g
~o-
0
"60,a
s
S
-
O
~DE
s
0.2-
D
D
AC
-
C 200
RO-
-
D
O
cAMP
Figure 1. Effect of dietary protein on the activities of adenylate cyclase,phosphodiesterase and the levels of cyclic adenosine monophosphate in pancreas. Assays of the enzymes were performed by the standard procedures and their activity expressed in units as given in '~aterials and Methods". Values are mean • SE from four animals in each group. PDE = phosphodiesterase; phosphate. D - deficient
group;
AC = adenylate
S - supplemented
a - values are significantly b - values are significantly
cyclase;
group;
cAMP = cyclic adenosine mono-
C - casein group.
different at 5% level from supplemented group. different at 5% level from casein group.
468
V. BHUTANI et al.
The assay of this second messenger however, exhibited the results to be otherwise. The levels of pancreatic cAMP were lowest in the casein diet fed animals and highest in the animals fed deficient diet (5) - a trend exactly opposite to the levels of adenylate cyclase. Similar to the adenylate cyclase data phosphodiesterase activity was also significantly lower in the deficient group as compared to the supplemented and casein groups. Also phosphodiesterase level was significantly lower in supplemented group as compared to casein group (Fig. 1). Although, in pancreas, both adenylate cyclase and phosphodiesterase exhibited similar type of influence in all groups of animals the magnitude of this influence was at variance. ~ The ratio of adenylate cyclase: phosphodiesterase in pancreas was significantly higher in deficient group as compared to supplemented and casein groups. It was lower in casein group (Fig. I). Higher adenylate cyclase:phosphodiesterase ratio in pancreas of deficient group would explain relatively higher concentration of cAMP in deficient group followed by supplemented group and the lowest in the case of casein group (Fig. I). Thus it appears that the concentration of cAMP in a cell.is not directly related to enzyme responsible for its synthesis rather it is the dynamic interplay of synthetic and degradative activities which are more important and it is the net balance of these activities that determines the level of cAMP in the cell. Under conditions of poor protein quality the energy requirements are met by metabolizing carbohydrates and lipids - a situation that would demand higher levels of cAMP. The levels of cAMP as observed above were significantly higher in deficient diet fed group as compared to supplemented or casein diet fed groups. Here quality of protein can be related to increased cAMP levels. ~wever, cAMP levels were non-significantly higher in supplemented group as compared to casein group which may be inferred to the quantity of protein and simultaneously lowering of c A ~ levels. It shows that dietary status of the animal is an important factor in regulating the levels of cAMP. ACKNOWLEDGEMENTS Vinay Bhutani and Vijay Kumar are CSIR Fellows. REFERENCES I.
Grill V, Cerasi E. Stimulation by D-glucose of cyclic adenosine 3'-5'monophosphate accumulation and insulin release in isolated pancreatic islets of the rat. J Biol Chem 1974;249:4196-201.
2.
Malaisse WJ, Malaisse-Lagae F. The role of cyclic AMP in insulin release. Experientia 1984;40:1068-75.
3.
Greaves MF. Cellular recognition. In: Outlines studies in biology. New York; John Wiley & Sons. 1975; 26-32.
4.
Bhutani V, Kumar V, Misra UK. Effect of inadequate dietary proteins on pancreatic insulin and cAMP levels in rats. Nutr Rept Int 1985;32:1413-20.
5.
Bhutani V, Kumar V, Tyagi AK, Misra UK. Changes of fatty acid composition in pancreas of rats fed inadequate dietary protein. Nutr Res 1987;7':763-70.
SECONDMESSENGER & ITS REGULATORYENZYMES 6.
Wood HN, Lin MC, Braun AC. The inhibition of plant and animal adenosine 3'5'-cyclic monophosphate phosphodiesterase by a cell division promoting substance from tissues of higher plant species. Proc Natl Acad Sci USA 1972;69:403-6.
7.
Butcher RW, Sutherland EW. Adenosine 3'-5'-phosphate in biological materials. J Biol Chem 1962;237:1244-50.
8.
Bhutani V, Kumar V, Misra UK. Turnover of pancreas phospholipids in rats fed inadequate dietary protein. Int J Vit Nutr Res 1986;56:275-79.
Accepted for publication December 1, 1988.
469