- Email: [email protected]
Evidence of an age-related decline in mitochondrial glycerophosphate dehydrogenase activity of isolated rat islets
Metabolism Clinical and Experimental VOL. XXXII, NO. 11
NOVEMBER 1983
Evidence of an Age-Related Glycerophosphate Dehydrogenase S. Azhar,
Decline in Mitochondrial Activity of Isolated Rat Islets
H. Ho, E. P. Reaven,
and G. M. Reaven
The activities of three enzymes-two mitochondrial and one microsomal-were measured in isolated islets of Langerhans from 2-month-old and 12-month-old rats. Mitochondrial glycerophosphate dehydrogenase activity (expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein), decreased (P cl 0.01) from a mean (+SEM) of 73.2 t 11.2 (2-month-old) to 34.7 2 5.9 (12-month-old). In contrast, activities of neither mitochondrial monoamine oxidase nor microsomal NADH cytochrome-c reductase changed with age. These results demonstrate that the activity of the glycerophosphate shuttle decreases as rats grow older, and it raises the possibility that the consequent regenerating cytosolic NAD+ may play a role in the insulin secretory defect associated with aging.
P
REVIOUS studies from this laboratory have documented a progressive decline in glucose-stimulated insulin secretion per beta cell as rats age.’ This phenomenon could be demonstrated in both isolated islets of LangerhansIe3 and in the perfused pancreas,4 and it appeared to be an inevitable consequence of the aging process.*-.’ Preliminary evidence suggested that this reduction in beta cell function might be related to an age-related decrease in glycolysis,’ but the site of this possible defect has yet to be defined. Recently, MacDonald’ has pointed out that pancreatic islets contain 40 to 70 times the activity of mitochondrial glycerophosphate dehydrogenase than is present in other tissues. Since this enzyme plays a crucial role in transporting reducing equivalents from cytosol to mitochondria,7-.9 a decrease in its activity should lead to an accumulation of cytosolic NADH: As a consequence, an increase in the cytosolic NADH/NAD’ ratio, a decrease in glycolysis, and a reduction in glucose-stimulated insulin secretion could be anticipated. In support of this line of reasoning is the observation that diazoxide inhibition of insulin secretion is associated with a marked reduction in pancreatic islet mitochondrial glycerophosphate dehydrogenase activity.’ Given these considerations, the present study was initiated to assess the effect of age on the activity of mitochondrial glycerophosphate dehydrogenase present in collagenase-isolated rat islets. The results document an approximate 50% reduction
Merabolism,Vol.
32, No. 11 (November). 1983
difficulty
in
(P < 0.01) in the activity of this enzyme in islets of I2-month-old compared with 2-month-old rats. In contrast, there was no age-related change in activity of either monoamine oxidase (mitochondrial) or NADH cytochrome-c reductase (microsomal). These observations provide a possible mechanism to account for the fall in glucose-induced insulin secretory response associated with aging in rats.‘--4 MATERIALS Animals.
Two-month-old
male Sprague-Dawley Wilmington, standard
Mass)
AND
rats (Charles were
Isolation of pancreatic islets. Kostianovsky” digested
acinar
of collagenase as described tissue
River
used in present
rat food and maintained
a modification
METHODS
and 12-month-old
(retired
Breeding studies.
Rats
on a 12-hour light-dark Pancreatic incubation
previously.‘,”
by discontinuous
breeders)
Laboratories, were
fed
cycle.
islets were isolated procedure
of Lacy
Islets were separated Ficoll
density
by and
from
gradient
From the Department of Medicine, Stanford University School of Medirine. Geriatric Research, Education, and Clinical Center, Veterans Administrotion Medical Cetner. Palo Alto, California. Receivedfor publication June 18. 1983. This research was supported by grants from the Research Service of the Veterans Administration and the Cambridge Quest Foundation. Address reprint requests to Gerald M. Reovm. MD, Veterans Administration Medical Center (1828). 3801 Mirandu Ave. Palo Alto, CA 94304. 1~1983 by Grune & Stratton. Inc. 0026~0495/83/321 l~OOOlSOl.OO/O
1019
1020
AZHAR ET AL
Table 1. Mean (*SEMI Microsomal
Activities
of Mitochondrial
NADH Cytochrome-c
Glycerophosphate
Reductase
in Subcellular
Dshydrogenase,
1 P-Month-Old
Z-Month-Old
Glycerophosphate
73.2
Mitochondrial
Monoamine
Fractions of Islets from 2- and 12-Month-Old
+ 11.2 (6)
34.7
Oxidase, and Rats P
+ 5.9 (6)
< 0.01
dehydrogenase. Monoamine oxldaset
460.0
+ 40.0 18)
450.0
+ 48.0 (8)
NS
NADH cytochrome-c
185.0
+ 19.0 (6)
204.0
+ 10.0 (6)
NS
reductases The number of experiments are given in parentheses. *Expressed as nmol iodonitrotetrazolium reduced/min/mg t&pressed
as nmol benzaldehyde produced/min/mg
protein.
protein.
$Expressed as nmol 2.6-dichlorophenolindophenol reduced/min/ng
centrifugation,‘.”
DNA.
washed several times, selected under a dissecting
microscope, and used for the preparation of subcellular fractions. Preporation of mitochondrial fraction. Islet mitochondrial fractions were isolated by minor modifications of procedures described earlier.” Briefly, islets (approximately 800) were homogenized in 1 mL of ice-cold buffer (0.25 mol/L sucrose and 50 mmol/L mannitol, pH 7.4), the resulting homogenate centrifuged at 600 x g for 10 minutes to sediment nuclei and cell debris, and the pellet rehomogenized and centrifuged as above. Combined supernatant fractions from the first and second centrifugation were pooled and centrifuged at 1500 x g for 10 minutes to sediment mitochondria. The sedimented mitochondria were then washed two times in buffer, and the pellet resuspended and used immediately for biochemical assays. Mitochondrial glycerophosphate dehydrogenase activity was measured by following reduction of iodonitrophenytetrazolium violet.14 The enzyme activity was expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein. The radiochemical procedure of McCaman et al” was used to quantitate mitochondrial monoamine oxidase, with activity expressed as nanomoles of benzaldehyde produced per minute per milligram of protein. Microsomal NADH cytochrome-c reductase was measured by a combination of the procedures of Omura and Takesue16 and Strobe1 and Dignam” with activity expressed as nanomoles of 2,6-dichlorophenolindophenol reduced per minute per nanogram of DNA. Protein content of all fractions was measured by the modified procedure of Lowry et al as described by Markwell et al.” Islet DNA content was measured fluorometrically.19
RESULTS AND DISCUSSION
Activities of mitochondrial glycerophosphate dehydrogenase, mitochondrial monoamine oxidase, and microsomal NADH cytochrome-c reductase measured in islets from pancreases of 2-month-old and 12month-old rats are shown in Table 1. These results indicate that the activity of glycerophosphate dehydrogenase was decreased by approximately 50% in mitochondrial fractions of islets isolated from 12-monthold rats. In contrast, the activities of monoamine
oxidase, another mitochondrial enzyme, and microsoma1 NADH cytochrome-c reductase, were not altered as a result of age. Monoamine oxidase is located on the outer mitochondrial membrane and was chosen for study to serve as a control for the decline in enzyme activity associated with preparation of mitochondrial fractions. NADH cytochrome-c reductase is a conventional marker of microsomal activity and is a control for the possibility that age leads to a generalized decline in activity of islet enzymes. The fact that the activity of neither of these enzymes declined as the rats grew older provides support for the relative specificity of the age-related change in glycerophosphate dehydrogenase activity. Furthermore, since beta cell mass increases significantly as rats grow older,‘T2the decline in mitochondrial glycerophosphate dehydrogenase activity cannot be due to a relative decrease of islet tissue with age. In conclusion, the fall of activity of mitochondrial glycerophosphate dehydrogenase of islets isolated from 12-month-old rats seen in these studies provides an explanation for the age-related defect in beta cell function associated with aging.14 Thus, there is substantial evidence that glucose-induced insulin secretion depends upon the maintenance of glycolysis.20*2’which, in turn, requires the regeneration of NAD+ from NADH produced in the oxidation of glyceraldehyde 3-phosphate.6 This process depends upon the central role of mitochondrial glycerophosphate dehydrogenase in the glycerophosphate shuttle. It is possible at this time to at least speculate that the age-related decline in glucose-stimulated insulin secretion is related to a decrease in the activity of the glycerophosphate shuttle that occurs with aging.
REFERENCES 1. Reaven EP. Gold G. Reaven GM: Effect of age on glucosestimulated insulin release by the beta cell of the rat. J Clin Invest 64:591-599, 1979 2. Reaven EP. Reaven GM: Structure and function changes in the endocrine pancreas of aging rats with reference to the modulat-
ing effects of exercise and caloric restriction. J Clin Invest 68:75-84, 1981 3. Reaven E, Wright D, Mondon CE, et al: Effect of age and diet on insulin secretion and insulin action in the rat. Diabetes 32:175180.1983
ISLET GDH ACTIVITY
4. Reaven
1021
WITH AGE
E, Curry
D, Moore
J, et al: Effect
of age
and
environmental factors on insulin release from the perfused pancreas of the rat. J Clin Invest 71:345-350, 1983 5. Reaven GM, Reaven EP: Effect of age on glucose oxidation by isolated rat islets, Diabetologia 18:69-7 I, 1980 6. MacDonald MJ: High content of mitochondrial glycerol3-phosphate dehydrogenase in pancreatic islets and its inhibition by Diazoxide. J Biol Chem 256:8287-8290, 1981 7. Dawson AC: Oxidation of cytosolic NADH formed during aerobic metabolism in mammalian cells. Trends Biochem Sci 4:171116,1919 8. Werner HV, Berry MN: Stimulatory effects of thyroxine administration on reducing-equivalent transfer from substrate to oxygen during hepatic metabolism of sorbitol and glycerol. Em J Biochem 42:3 15-324, 1974 9. Wernette ME, Ochs RS, Lardy HA: Ca2+ stimulation of rat liver mitochondrial glycerophosphate dehydrogenase. J Biol Chem 256:12767-12771, 1981 10. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of Langerhans from rat pancreas. Diabetes 16:35-39, 1967 I I. Reaven EP, Solomon R, Azhar S, et al: Functional homogeneity of pancreatic islets of aging rats. Metabolism 31:859-860, 1982 12. Lacy PE, Finke EH, Conant S, et al: Longterm isolated rat islets in vitro. Diabetes 25:484493, 1976
perfusion
of
13. Johnson D, Lardy HA: Isolation of liver or kidney mitochondria. Methods Enzymol IO:94-96, 1967 14. Gardner RS: A sensitive calorimetric assay for mitochondrial oc-glycerophosphate dehydrogenase. Anal Biochem 59:272-276, 1974 15. McCaman RE, McCaman MW, Hunt JM, et al: Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarboxylase activities in nervous tissues. J Neurochem 12:15-23, 1965 16. Omura T, Takesue S: A new method for the simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J B&hem 67:249-257, 1970 17. Strobe1 HW, NADPH-cytochrome I IO, 1978
Dignam JC: Purification and properties of P-450 reductase. Methods Enzymol 52:89-
18. Markwell MAK, Haas SM. Tolbert NE, et al: Protein determination in membrane and lipoprotein samples: Manual and automated procedures. Methods Enzymol 72:296-303, 198 I 19. Vytasek R: A sensitive fluorometric tion of DNA. Anal Biochem 120:243-248,
assay for the determinaI98 I
20. Ashcroft SJH: Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18:5-l 5, 1980 21. Hedeskov CJ: Mechanism of secretion. Physiol Rev 60:442-509, 1980
glucose-induced
insulin
NOVEMBER 1983
Evidence of an Age-Related Glycerophosphate Dehydrogenase S. Azhar,
Decline in Mitochondrial Activity of Isolated Rat Islets
H. Ho, E. P. Reaven,
and G. M. Reaven
The activities of three enzymes-two mitochondrial and one microsomal-were measured in isolated islets of Langerhans from 2-month-old and 12-month-old rats. Mitochondrial glycerophosphate dehydrogenase activity (expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein), decreased (P cl 0.01) from a mean (+SEM) of 73.2 t 11.2 (2-month-old) to 34.7 2 5.9 (12-month-old). In contrast, activities of neither mitochondrial monoamine oxidase nor microsomal NADH cytochrome-c reductase changed with age. These results demonstrate that the activity of the glycerophosphate shuttle decreases as rats grow older, and it raises the possibility that the consequent regenerating cytosolic NAD+ may play a role in the insulin secretory defect associated with aging.
P
REVIOUS studies from this laboratory have documented a progressive decline in glucose-stimulated insulin secretion per beta cell as rats age.’ This phenomenon could be demonstrated in both isolated islets of LangerhansIe3 and in the perfused pancreas,4 and it appeared to be an inevitable consequence of the aging process.*-.’ Preliminary evidence suggested that this reduction in beta cell function might be related to an age-related decrease in glycolysis,’ but the site of this possible defect has yet to be defined. Recently, MacDonald’ has pointed out that pancreatic islets contain 40 to 70 times the activity of mitochondrial glycerophosphate dehydrogenase than is present in other tissues. Since this enzyme plays a crucial role in transporting reducing equivalents from cytosol to mitochondria,7-.9 a decrease in its activity should lead to an accumulation of cytosolic NADH: As a consequence, an increase in the cytosolic NADH/NAD’ ratio, a decrease in glycolysis, and a reduction in glucose-stimulated insulin secretion could be anticipated. In support of this line of reasoning is the observation that diazoxide inhibition of insulin secretion is associated with a marked reduction in pancreatic islet mitochondrial glycerophosphate dehydrogenase activity.’ Given these considerations, the present study was initiated to assess the effect of age on the activity of mitochondrial glycerophosphate dehydrogenase present in collagenase-isolated rat islets. The results document an approximate 50% reduction
Merabolism,Vol.
32, No. 11 (November). 1983
difficulty
in
(P < 0.01) in the activity of this enzyme in islets of I2-month-old compared with 2-month-old rats. In contrast, there was no age-related change in activity of either monoamine oxidase (mitochondrial) or NADH cytochrome-c reductase (microsomal). These observations provide a possible mechanism to account for the fall in glucose-induced insulin secretory response associated with aging in rats.‘--4 MATERIALS Animals.
Two-month-old
male Sprague-Dawley Wilmington, standard
Mass)
AND
rats (Charles were
Isolation of pancreatic islets. Kostianovsky” digested
acinar
of collagenase as described tissue
River
used in present
rat food and maintained
a modification
METHODS
and 12-month-old
(retired
Breeding studies.
Rats
on a 12-hour light-dark Pancreatic incubation
previously.‘,”
by discontinuous
breeders)
Laboratories, were
fed
cycle.
islets were isolated procedure
of Lacy
Islets were separated Ficoll
density
by and
from
gradient
From the Department of Medicine, Stanford University School of Medirine. Geriatric Research, Education, and Clinical Center, Veterans Administrotion Medical Cetner. Palo Alto, California. Receivedfor publication June 18. 1983. This research was supported by grants from the Research Service of the Veterans Administration and the Cambridge Quest Foundation. Address reprint requests to Gerald M. Reovm. MD, Veterans Administration Medical Center (1828). 3801 Mirandu Ave. Palo Alto, CA 94304. 1~1983 by Grune & Stratton. Inc. 0026~0495/83/321 l~OOOlSOl.OO/O
1019
1020
AZHAR ET AL
Table 1. Mean (*SEMI Microsomal
Activities
of Mitochondrial
NADH Cytochrome-c
Glycerophosphate
Reductase
in Subcellular
Dshydrogenase,
1 P-Month-Old
Z-Month-Old
Glycerophosphate
73.2
Mitochondrial
Monoamine
Fractions of Islets from 2- and 12-Month-Old
+ 11.2 (6)
34.7
Oxidase, and Rats P
+ 5.9 (6)
< 0.01
dehydrogenase. Monoamine oxldaset
460.0
+ 40.0 18)
450.0
+ 48.0 (8)
NS
NADH cytochrome-c
185.0
+ 19.0 (6)
204.0
+ 10.0 (6)
NS
reductases The number of experiments are given in parentheses. *Expressed as nmol iodonitrotetrazolium reduced/min/mg t&pressed
as nmol benzaldehyde produced/min/mg
protein.
protein.
$Expressed as nmol 2.6-dichlorophenolindophenol reduced/min/ng
centrifugation,‘.”
DNA.
washed several times, selected under a dissecting
microscope, and used for the preparation of subcellular fractions. Preporation of mitochondrial fraction. Islet mitochondrial fractions were isolated by minor modifications of procedures described earlier.” Briefly, islets (approximately 800) were homogenized in 1 mL of ice-cold buffer (0.25 mol/L sucrose and 50 mmol/L mannitol, pH 7.4), the resulting homogenate centrifuged at 600 x g for 10 minutes to sediment nuclei and cell debris, and the pellet rehomogenized and centrifuged as above. Combined supernatant fractions from the first and second centrifugation were pooled and centrifuged at 1500 x g for 10 minutes to sediment mitochondria. The sedimented mitochondria were then washed two times in buffer, and the pellet resuspended and used immediately for biochemical assays. Mitochondrial glycerophosphate dehydrogenase activity was measured by following reduction of iodonitrophenytetrazolium violet.14 The enzyme activity was expressed as nanomoles of iodonitrotetrazolium reduced per minute per milligram of protein. The radiochemical procedure of McCaman et al” was used to quantitate mitochondrial monoamine oxidase, with activity expressed as nanomoles of benzaldehyde produced per minute per milligram of protein. Microsomal NADH cytochrome-c reductase was measured by a combination of the procedures of Omura and Takesue16 and Strobe1 and Dignam” with activity expressed as nanomoles of 2,6-dichlorophenolindophenol reduced per minute per nanogram of DNA. Protein content of all fractions was measured by the modified procedure of Lowry et al as described by Markwell et al.” Islet DNA content was measured fluorometrically.19
RESULTS AND DISCUSSION
Activities of mitochondrial glycerophosphate dehydrogenase, mitochondrial monoamine oxidase, and microsomal NADH cytochrome-c reductase measured in islets from pancreases of 2-month-old and 12month-old rats are shown in Table 1. These results indicate that the activity of glycerophosphate dehydrogenase was decreased by approximately 50% in mitochondrial fractions of islets isolated from 12-monthold rats. In contrast, the activities of monoamine
oxidase, another mitochondrial enzyme, and microsoma1 NADH cytochrome-c reductase, were not altered as a result of age. Monoamine oxidase is located on the outer mitochondrial membrane and was chosen for study to serve as a control for the decline in enzyme activity associated with preparation of mitochondrial fractions. NADH cytochrome-c reductase is a conventional marker of microsomal activity and is a control for the possibility that age leads to a generalized decline in activity of islet enzymes. The fact that the activity of neither of these enzymes declined as the rats grew older provides support for the relative specificity of the age-related change in glycerophosphate dehydrogenase activity. Furthermore, since beta cell mass increases significantly as rats grow older,‘T2the decline in mitochondrial glycerophosphate dehydrogenase activity cannot be due to a relative decrease of islet tissue with age. In conclusion, the fall of activity of mitochondrial glycerophosphate dehydrogenase of islets isolated from 12-month-old rats seen in these studies provides an explanation for the age-related defect in beta cell function associated with aging.14 Thus, there is substantial evidence that glucose-induced insulin secretion depends upon the maintenance of glycolysis.20*2’which, in turn, requires the regeneration of NAD+ from NADH produced in the oxidation of glyceraldehyde 3-phosphate.6 This process depends upon the central role of mitochondrial glycerophosphate dehydrogenase in the glycerophosphate shuttle. It is possible at this time to at least speculate that the age-related decline in glucose-stimulated insulin secretion is related to a decrease in the activity of the glycerophosphate shuttle that occurs with aging.
REFERENCES 1. Reaven EP. Gold G. Reaven GM: Effect of age on glucosestimulated insulin release by the beta cell of the rat. J Clin Invest 64:591-599, 1979 2. Reaven EP. Reaven GM: Structure and function changes in the endocrine pancreas of aging rats with reference to the modulat-
ing effects of exercise and caloric restriction. J Clin Invest 68:75-84, 1981 3. Reaven E, Wright D, Mondon CE, et al: Effect of age and diet on insulin secretion and insulin action in the rat. Diabetes 32:175180.1983
ISLET GDH ACTIVITY
4. Reaven
1021
WITH AGE
E, Curry
D, Moore
J, et al: Effect
of age
and
environmental factors on insulin release from the perfused pancreas of the rat. J Clin Invest 71:345-350, 1983 5. Reaven GM, Reaven EP: Effect of age on glucose oxidation by isolated rat islets, Diabetologia 18:69-7 I, 1980 6. MacDonald MJ: High content of mitochondrial glycerol3-phosphate dehydrogenase in pancreatic islets and its inhibition by Diazoxide. J Biol Chem 256:8287-8290, 1981 7. Dawson AC: Oxidation of cytosolic NADH formed during aerobic metabolism in mammalian cells. Trends Biochem Sci 4:171116,1919 8. Werner HV, Berry MN: Stimulatory effects of thyroxine administration on reducing-equivalent transfer from substrate to oxygen during hepatic metabolism of sorbitol and glycerol. Em J Biochem 42:3 15-324, 1974 9. Wernette ME, Ochs RS, Lardy HA: Ca2+ stimulation of rat liver mitochondrial glycerophosphate dehydrogenase. J Biol Chem 256:12767-12771, 1981 10. Lacy PE, Kostianovsky M: Method for the isolation of intact islets of Langerhans from rat pancreas. Diabetes 16:35-39, 1967 I I. Reaven EP, Solomon R, Azhar S, et al: Functional homogeneity of pancreatic islets of aging rats. Metabolism 31:859-860, 1982 12. Lacy PE, Finke EH, Conant S, et al: Longterm isolated rat islets in vitro. Diabetes 25:484493, 1976
perfusion
of
13. Johnson D, Lardy HA: Isolation of liver or kidney mitochondria. Methods Enzymol IO:94-96, 1967 14. Gardner RS: A sensitive calorimetric assay for mitochondrial oc-glycerophosphate dehydrogenase. Anal Biochem 59:272-276, 1974 15. McCaman RE, McCaman MW, Hunt JM, et al: Microdetermination of monoamine oxidase and 5-hydroxytryptophan decarboxylase activities in nervous tissues. J Neurochem 12:15-23, 1965 16. Omura T, Takesue S: A new method for the simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J B&hem 67:249-257, 1970 17. Strobe1 HW, NADPH-cytochrome I IO, 1978
Dignam JC: Purification and properties of P-450 reductase. Methods Enzymol 52:89-
18. Markwell MAK, Haas SM. Tolbert NE, et al: Protein determination in membrane and lipoprotein samples: Manual and automated procedures. Methods Enzymol 72:296-303, 198 I 19. Vytasek R: A sensitive fluorometric tion of DNA. Anal Biochem 120:243-248,
assay for the determinaI98 I
20. Ashcroft SJH: Glucoreceptor mechanisms and the control of insulin release and biosynthesis. Diabetologia 18:5-l 5, 1980 21. Hedeskov CJ: Mechanism of secretion. Physiol Rev 60:442-509, 1980
glucose-induced
insulin