BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
344-349 (1991)
4,
Extracellular Phosphate Requirement for Insulin Action on Isolated Rat Hepatocytes KENNETH
S. ROGERS,* RIAZ A. MEMON,
CHANDRA MOHAN, AND SAMUEL P. BESSMAN
PAUL J. GEIGER,
*Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, and Department of Pharmacology and Nutrition, University of Southern California, Los Angeles, California 90033
Received November 19, 1990, and in revised form January 25, 1991 Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30” in the presence of tracer [2,3-“Clsuccinate and a 0.5 mr+rconcentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3“C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of “P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state. Q 1991 Academic Press, Inc.
Insulin stimulates the synthesis of lipid, protein, and glycogen in the hepatocyte and the phosphorylation of many metabolic intermediates. The uniformly required molecule in all these anabolic processes is ATP. Bessman (1) proposed that insulin, by increasing the supply of mitochondrially generated ATP, could accelerate nearly all of the reactions it is known to effect. He proposed that insulin increases hexokinase binding to mitochondria, thereby stimulating mitochondrial energy generation and glucose utilization. This process of energy (ATP) generation appears to be dependent upon the availability of phosphate and calcium in the medium. The data presented in this paper show that insulin responsiveness of isolated hepatocytes is significantly diminished when cells are washed and incubated in Krebs-Henseleit bicarbonate (KHB) buffer containing low levels of phosphate or calcium. The requirement of extracellular phosphate for insulin 344 08854505/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.
PHOSPHATE
REQUIREMENT
FOR INSULIN
ACTION
345
stimulation of mitochondrial Krebs cycle activity, as assessed by the oxidation of [2,3-‘4C]succinate, appears to be specific since lack of extracellular phosphate did not prevent the stimulation of succinate oxidation by either epinephrine or glucagon; whereas the lack of extracellular calcium in the presence of adequate phosphate prevented the stimulation of succinate oxidation by glucagon, epinephrine, and insulin. MATERIALS AND METHODS
Male Sprague-Dawley rats (145-175 g) were maintained on Purina Lab Chow and water ad libitum. Rats were anesthetized by an intraperitoneal injection of sodium barbital (45 mg/kg body wt). Sodium heparin (500 U/kg body wt) was injected into the femoral vein. Liver perfusion and isolation of hepatocytes were described earlier (3). Centrifugation of hepatocyte preparations did not exceed log as higher centrifugal forces have been shown to be damaging to the hepatocyte (4). To examine the effect of extracellular phosphate on stimulation by insulin and other hormones on Krebs cycle oxidation and protein incorporation of tracer carbons, the hepatocytes were washed and centrifuged twice in ice-cold KHB buffer, pH 7.4, that contained the appropriate reduced amount of sodium phosphate. Sodium chloride replaced sodium phosphate in the KHB buffer. Hepatocyte incubations were carried out in 25-ml Erlenmeyer flasks containing 1.6 ml of KHB buffer, pH 7.4, all 20 natural amino acids, each to a final concentration of 0.5 mM; 0.4 ml of hepatocyte suspension (S-10 mg protein); 0.1 ~Ci of [2,3-‘4C]succinate (ICN) or 50 PCi of 32P04 (ICN); + insulin (10 mu/ml) (Calbiochem, crystalline bovine); + glucagon (1 nM, Sigma); and + epinephrine (10 PM, Sigma). After the addition of the isotope, the flasks were either topped with a rubber stopper (32P experiments) or with rubber sleeve stoppers (14C experiments) that contained rolled filter paper in a polypropylene cup suspended from the center of the sleeve stopper. They were incubated for either 5 min at 30” (32P experiments) or 15 min at 30” (14C experiments) in a Dubnoff metabolic shaker (60 oscillations/min). The reaction was terminated by the addition of 0.6 ml of 3 N perchloric acid. CO2 analysis. One molar NaOH (0.2 ml) was injected into the cup to trap 14C02. Shaking of the flasks was continued for 1 hr to ensure transfer of the CO2 to the central cup. The filter paper and central cup contents were transferred to a scintillation vial. The cup was again washed with 0.4 ml of water and this was also added to the counting vial. Five milliliters of Safetysolve (Research Products International, Mount Prospect, IL) was added to the vial and the samples were counted after 12-18 hr. [2,3-‘4C]Succinate carbon incorporation into protein. The contents of the flasks were collected and centrifuged at 9OOg for 10 min. The precipitates were washed three times with 2.5 ml of 0.7 M perchloric acid. The protein precipitate was washed with 2 ml of ethanol: ether (3: 1) to remove lipids. After a wash with 1 ml of ether, the precipitate was centrifuged, dried in air, and dissolved in 1 ml of 1 N NaOH. An aliquot of 0.2 ml was used for scintillation counting. Protein was estimated by the method of Lowry et al. (5).
346
ROGERS
ET AL.
32P04 analysis. The perchloric acid filtrate was brought to about pH 7 with 10 KOH and 1 M KHC03 using bromothymol blue as an indicator. Samples were kept frozen in the dark to await chromatography. Separation of organic phosphates was carried out by anion-exchange chromatography and detected and quantified with the Bessman automated phosphate analyzer (6-8). Columns were 150 x 3 mm (Altex-Beckman) packed with AGMP-1 resin (Bio-Rad) carefully sized to the lo- to 20-pm particle range. A precolumn containing the same resin was used for loading the sample and for serving as a filter to protect the main column. A typical sample volume was 400 ~1. The elution was started with 10 mM Na2B407 at a flow rate of 0.3 ml per min. After 15 min, buffer A (0.05 M NH&l and 0.05 M Na2B407) was commenced. Thirty minutes later, a linear gradient of buffer A and buffer B (0.6 M NH&l and 0.05 M Na,B,O,) was passed through the column at about 300 lbs/sq. inch to separate inorganic and organic phosphates. Effluent fractions were collected at 1-min intervals from the phosphate analyzer and counted in minivials using Safetysolve. The nanomoles of compounds in individual peaks were estimated from integrator counts recorded with a Shimadzu C-R3A integrating recorder using inorganic phosphate (Pr) as a standard. The 32P counts per minute for the peaks were recorded with a Searle Delta-300 liquid scintillation counter. Calculations and statistical analyses of data were performed with an IBM-type PC, a spreadsheet program set up with templates to fit our needs, and the statistical program “Stata” (9). Data entered from the chromatograph and the scintillation counter conveniently and rapidly yielded the nanomoles, counts per minute corrected for decay, and specific activity in tabular form. Statistical analysis. A two-tailed paired Student t test (9) was used to ascertain levels of significance. Probability values of 0.05 or less were considered significant. M
RESULTS AND DISCUSSION The effect of extracellular phosphate on the insulin stimulation of hepatocyte [2,3-14C]succinate carbon incorporation into protein and oxidation to 14C02 is summarized in Table 1. The phosphate concentrations represent the levels used during the final two washings of the hepatocytes and the incubation medium. The intracellular perchloric acid soluble inorganic phosphate was about 22 nmole/mg hepatocyte protein. In the absence of extracellular phosphate, insulin failed to stimulate the oxidation of [‘4C]succinate carbons to CO* and their incorporation into hepatocyte protein. The addition of extracellular phosphate to the incubation medium resulted in a significant increase in the insulin stimulation of succinate oxidation and the incorporation of succinate carbons into protein. Phosphatedependent insulin stimulation of [2,3-14C] succinate oxidation plateaued at a 0.89 mM phosphate concentration in the medium. On the other hand, protein incorporation of 14C carbons reached a similar level at a 1.18 mM phosphate concentration. The requirement of extracellular phosphate for insulin stimulation of the Krebs cycle is very specific since lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon or epinephrine (Table 2), even though the basal oxidation and protein incorporation of the tracer were signifi-
PHOSPHATE
REQUIREMENT
FOR INSULIN
347
ACTION
TABLE 1 The Effect of Extracellular Phosphate on Insulin Stimulation of [2,3-“‘ClSuccinate Protein Incorporation by Isolated Rat Hepatocytes Extracelhdar phosphate concentration (W 0 0.05 0.10 0.20 0.50 0.89 1.18
‘TO,
14C protein
Control 820 776 874 978 1077 1234 1176
+k + 2 + k *
19 (7) 71 (5) 60 (6) 65 (6) 104 (6) 181 (6) 85 (7)
Oxidation and
Insulin 834 954 1061 1194 1236 1573 1498
+ 2 2 + * k *
Control
cpm/mg protein 22 (6) 237 107 (6)’ 239 73 (6)’ 249 60 (6) 232 115 (6)“ 240 238 (6) 260 198 (7) 331
k IT k + + + +
40 30 28 32 39 37 29
Insulin (5) (5) (5) (5) (5) (5) (5)
222 269 275 292 290 293 420
f k 2 2 r 2 2
35 35 33 41 43 48 28
(5) (5) (5)d (5) (5)’ (5) (5)
a SEM. Values in parentheses represent number of experiments. b P < 0.05. = P < 0.02. d P < 0.01. e P < 0.005. f P < 0.001.
cantly lower in phosphate-depleted preparations. Glucagon and epinephrine failed to stimulate protein synthesis in either the presence or absence of extracellular phosphate. An examination of the relative specific activity of 32P04 incorporation into organic phosphate (Table 3) indicates that insulin added to phosphate-depleted hepatocytes did not stimulate the turnover of the organic phosphates, phosphoglyceric acid, uridine-diphosphoglucose, UTP, P-ADP, or ATP; nor did insulin added to control hepatocytes (isolated and incubated with 1.18 mM phosphate)
The Effect of Extracellular Extracellular phosphate 1.18 mM 0.0
rnM
% Change
TABLE 2 Phosphate on Insulin, Glucagon, and Epinephrine of [2,3-Y]Succinate
Stimulated Oxidation
Control
Insulin
Glucagon
765 + 44”
964 f 14b
635 + 111
(26)
708 f 102 (10.3)
1820 f 262’ (137.9) 1161 f 164’ (82.9)
1428 f 83’ (86.7) 997 f 212b (57.0)
- 16.9
-36.1
-36
-30.2
Epinephrine
a Data are presented as ‘YZO, cpm/mg protein. Values are presented as means 2 SEM (n = 4). Numbers in parentheses indicate percentage stimulation over control. b Level of significance is P < 0.001.
348
ROGERS ET AL. TABLE 3 Relative Specific Activity” (“P) of Hepatocyte Organic Phosphates Control (%)
Inorganic phosphate Phosphoglyceric acid Uridinediphosphoglucose UTP P-ADP ATP
loo (3)b 1.67 f .18 0.92 f .18 1.82 f .27 3.91 k 1.41 3.69 k .28
(2) (3) (3) (3) (3)
Insulin (%) 100 (3) 1.08 f 0.66 f 1.51 ?z 1.61 2 2.59 f
.40 .43 56 .95 .94
(2) (2) (3) (3) (3)
’ Relative specific activity was calculated using the formula: (“P cpm/nmole organic phosphate t 3zP cpm/nmole inorganic phosphate) x 100. b Mean -C SEM. Values in parentheses represent number of individual observations.
increase the turnover of these organic phosphates (data not shown) [cf. muscle
w91*
We conclude that the demonstrable activity of insulin on Krebs cycle oxidation and protein incorporation of succinate carbons requires extracellular phosphate. It is possible that this phosphate is presented to the mitochondria in a manner separate from that of intracellular phosphate (we observed no change in perchloric acid-soluble intracellular phosphate fractions due to insulin treatment of the hepatocyte). Diminution of this separate phosphate path impairs insulin activity. Our results could not be explained by the inhibition of phosphofructokinase by low phosphate (11) because succinate oxidation and protein synthesis still occur at a relative high rate [70% of control (Table l)]. This work is in agreement with that of Mine et al. (12) who reported that insulin inhibition of cyclic-AMP induced glycogenolysis was abolished during a phosphate-free perfusion of rat liver, and we have shown that insulin “inhibition” of gluconeogenesis is a function of the mitochondrial Krebs cycle (13). These findings have important clinical implications. Low insulin sensitivity has been reported in hypophosphatemic patients (14). This insulin insensitivity in hypophosphatemic patients may be related to the mitochondrial requirement for extracellular phosphate. Mohan et al. (13) have shown the importance of a normally operating Krebs cycle for all anabolic effects of insulin. Thus insulin sensitivity in these patients may be restored when their plasma phosphate levels approach the physiological range. Calcium appeared to be essential for the oxidation of succinate carbons. Basal and hormone-stimulated (insulin, glucagon, and epinephrine) 14C02 formation from [2,3-‘4C]succinate decreased significantly in the absence of calcium in the medium. Although no insulin effect was evident in the absence of calcium, a partial stimulatory effect of glucagon (33.2%) and epinephrine (23.1%) was observed (Table 4). Without calcium, insulin did not stimulate [2,3-‘4C]succinate carbon incorporation into protein (data not shown). Removal of magnesium from the KHB buffer did not alter the stimulatory effects of insulin, epinephrine, or glucagon on succinate oxidation and protein synthesis (data not shown). We conclude that lowering the medium calcium lowered the intracellular and intramitochondrial levels of calcium. Foden and Randle (15) reported that the
PHOSPHATE
REQUIREMENT
FOR INSULIN
TABLE 4 The Effect of Calcium on Insulin, Glucagon, and Epinephrine of [2,3-‘4C]Succinate Calcium 1.9
rnM
Insulin
Glucagon
774 * 53
996 2 93’ (28.7) 291 L 32 (8.5) -70
1610 f 88
268 -c 22’
% Change
-65.4
a Data Numbers * Level ’ Level
Stimulated Oxidation
Control
rnM
0.0
349
ACTION
Epinephrine
357 f 26 (33.2)
1515 f 176’ (95.8) 330 + 41 (23.1)
-77.8
-78.2
U’W
are presented as 14COz cpm/mg protein. Values are presented as means ? SEM (n = 4). in parentheses indicate percentage stimulation over control. of significance is P < 0.05. of significance is P < 0.001.
calcium efflux of hepatocyte and mitochondria occurs within 2 min. Our incubation time was 15 min. Thomas and Reed (16) have reported that 1 to 5 hr of incubation of hepatocytes in a calcium-free medium induces oxidative stress and leakage of cellular constituents. Calcium appears to be essential for enhanced phosphate uptake in the presence of glucagon (17). In this work we showed that the hormone control of cellular oxidation and protein synthesis is lost within 15 min following incubation in a calcium-free medium. Insulin stimulation of mitochondrial oxidation is dependent on optimal levels of both calcium and phosphate whereas only calcium appears to be essential for epinephrine and glucagon effects on Krebs cycle oxidation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Bessman SP. Am J Med 40~740, 1966. Bessman SP, Mohan C, Zaidise I. Proc Nat1 Acad Sci USA 83:5067, 1986. Mohan C, Bessman SP. Arch Biochem Biophys 242:563, 1985. Memon RA, Mohan C, Geiger PJ, Bessman SP, Rogers KS. Biochem Med Metab Biol 42~216, 1989. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. J Biol Chem 193:265, 1951. Bessman SP, Geiger PJ, Lu T-C, McCabe ERB. Anal Biochem 59:533, 1974. Geiger PJ, Ahn S, Bessman SP. in “Methods in Carbohydrate Chemistry” (Whistler RL, BeMiller JN, Eds.). Academic Press, New York. Erickson-Viitanen S, Viitanen P, Geiger PJ, Yang WCT, Bessman SP. J Biol Chem 25214395, 1982. Edwards AL. “Statistical Analysis,” p. 127, Rinehart, New York, 1958; and Statistical Analysis Program, “Stata,” available from Computing Resources Center, Santa Monica, CA. Bessman SP, Borrebaek B, Geiger PJ, Ben-Or S. in “Microenvironments and Cellular Compartmentation” (Srere P, Estabrook RW, Eds.), p. 111. Academic Press, New York, 1978. Brautber N, Massry SG. Adv Exp Med Biol 178:363, 1984. Mine T, Kimura S, Koide Y, Ohsawa H, Ogata E. Horm Metabol Res 12438, 1985. Mohan C, Geiger PH, Bessman SP. Cur-r Top Cell ReguZ3Or105, 1989. DeFronzo RA, Lang R. N Engl J Med 303:1259, 1980. Foden S, Randle PJ. Biochem J 170:615, 1978. Thomas CE, Reed DJ. J Pharmacol Exp Therap 245:493, 1988. Bygrave FL, Lenton L, Altin JG, Setchell BA, Karjalsinen A. Biochem J 26269, 1990.
MEDICINE
AND
METABOLIC
BIOLOGY
344-349 (1991)
4,
Extracellular Phosphate Requirement for Insulin Action on Isolated Rat Hepatocytes KENNETH
S. ROGERS,* RIAZ A. MEMON,
CHANDRA MOHAN, AND SAMUEL P. BESSMAN
PAUL J. GEIGER,
*Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, and Department of Pharmacology and Nutrition, University of Southern California, Los Angeles, California 90033
Received November 19, 1990, and in revised form January 25, 1991 Isolated rat hepatocytes were prepared in KHB buffer, pH 7.4; were centrifuged and washed twice in KHB buffer containing various amounts of phosphate and calcium; and were incubated at 30” in the presence of tracer [2,3-“Clsuccinate and a 0.5 mr+rconcentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.1 mM phosphate failed to show any insulin stimulation of [2,3“C]succinate oxidation or protein incorporation of tracer carbons. The absence or presence of extracellular phosphate did not alter the specific activity of “P-adenine nucleotides; they remained the same in the presence or absence of insulin. The maximal insulin stimulatory effect on succinate oxidation and tracer incorporation into protein was observed in the presence of 1.18 mM phosphate and 1.9 mM calcium ion. The lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon on epinephrine, whereas removal of calcium from the medium abolished their hormonal effects. The lack of medium calcium also prevented the insulin stimulation of succinate oxidation and protein synthesis. Our data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state. Q 1991 Academic Press, Inc.
Insulin stimulates the synthesis of lipid, protein, and glycogen in the hepatocyte and the phosphorylation of many metabolic intermediates. The uniformly required molecule in all these anabolic processes is ATP. Bessman (1) proposed that insulin, by increasing the supply of mitochondrially generated ATP, could accelerate nearly all of the reactions it is known to effect. He proposed that insulin increases hexokinase binding to mitochondria, thereby stimulating mitochondrial energy generation and glucose utilization. This process of energy (ATP) generation appears to be dependent upon the availability of phosphate and calcium in the medium. The data presented in this paper show that insulin responsiveness of isolated hepatocytes is significantly diminished when cells are washed and incubated in Krebs-Henseleit bicarbonate (KHB) buffer containing low levels of phosphate or calcium. The requirement of extracellular phosphate for insulin 344 08854505/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.
PHOSPHATE
REQUIREMENT
FOR INSULIN
ACTION
345
stimulation of mitochondrial Krebs cycle activity, as assessed by the oxidation of [2,3-‘4C]succinate, appears to be specific since lack of extracellular phosphate did not prevent the stimulation of succinate oxidation by either epinephrine or glucagon; whereas the lack of extracellular calcium in the presence of adequate phosphate prevented the stimulation of succinate oxidation by glucagon, epinephrine, and insulin. MATERIALS AND METHODS
Male Sprague-Dawley rats (145-175 g) were maintained on Purina Lab Chow and water ad libitum. Rats were anesthetized by an intraperitoneal injection of sodium barbital (45 mg/kg body wt). Sodium heparin (500 U/kg body wt) was injected into the femoral vein. Liver perfusion and isolation of hepatocytes were described earlier (3). Centrifugation of hepatocyte preparations did not exceed log as higher centrifugal forces have been shown to be damaging to the hepatocyte (4). To examine the effect of extracellular phosphate on stimulation by insulin and other hormones on Krebs cycle oxidation and protein incorporation of tracer carbons, the hepatocytes were washed and centrifuged twice in ice-cold KHB buffer, pH 7.4, that contained the appropriate reduced amount of sodium phosphate. Sodium chloride replaced sodium phosphate in the KHB buffer. Hepatocyte incubations were carried out in 25-ml Erlenmeyer flasks containing 1.6 ml of KHB buffer, pH 7.4, all 20 natural amino acids, each to a final concentration of 0.5 mM; 0.4 ml of hepatocyte suspension (S-10 mg protein); 0.1 ~Ci of [2,3-‘4C]succinate (ICN) or 50 PCi of 32P04 (ICN); + insulin (10 mu/ml) (Calbiochem, crystalline bovine); + glucagon (1 nM, Sigma); and + epinephrine (10 PM, Sigma). After the addition of the isotope, the flasks were either topped with a rubber stopper (32P experiments) or with rubber sleeve stoppers (14C experiments) that contained rolled filter paper in a polypropylene cup suspended from the center of the sleeve stopper. They were incubated for either 5 min at 30” (32P experiments) or 15 min at 30” (14C experiments) in a Dubnoff metabolic shaker (60 oscillations/min). The reaction was terminated by the addition of 0.6 ml of 3 N perchloric acid. CO2 analysis. One molar NaOH (0.2 ml) was injected into the cup to trap 14C02. Shaking of the flasks was continued for 1 hr to ensure transfer of the CO2 to the central cup. The filter paper and central cup contents were transferred to a scintillation vial. The cup was again washed with 0.4 ml of water and this was also added to the counting vial. Five milliliters of Safetysolve (Research Products International, Mount Prospect, IL) was added to the vial and the samples were counted after 12-18 hr. [2,3-‘4C]Succinate carbon incorporation into protein. The contents of the flasks were collected and centrifuged at 9OOg for 10 min. The precipitates were washed three times with 2.5 ml of 0.7 M perchloric acid. The protein precipitate was washed with 2 ml of ethanol: ether (3: 1) to remove lipids. After a wash with 1 ml of ether, the precipitate was centrifuged, dried in air, and dissolved in 1 ml of 1 N NaOH. An aliquot of 0.2 ml was used for scintillation counting. Protein was estimated by the method of Lowry et al. (5).
346
ROGERS
ET AL.
32P04 analysis. The perchloric acid filtrate was brought to about pH 7 with 10 KOH and 1 M KHC03 using bromothymol blue as an indicator. Samples were kept frozen in the dark to await chromatography. Separation of organic phosphates was carried out by anion-exchange chromatography and detected and quantified with the Bessman automated phosphate analyzer (6-8). Columns were 150 x 3 mm (Altex-Beckman) packed with AGMP-1 resin (Bio-Rad) carefully sized to the lo- to 20-pm particle range. A precolumn containing the same resin was used for loading the sample and for serving as a filter to protect the main column. A typical sample volume was 400 ~1. The elution was started with 10 mM Na2B407 at a flow rate of 0.3 ml per min. After 15 min, buffer A (0.05 M NH&l and 0.05 M Na2B407) was commenced. Thirty minutes later, a linear gradient of buffer A and buffer B (0.6 M NH&l and 0.05 M Na,B,O,) was passed through the column at about 300 lbs/sq. inch to separate inorganic and organic phosphates. Effluent fractions were collected at 1-min intervals from the phosphate analyzer and counted in minivials using Safetysolve. The nanomoles of compounds in individual peaks were estimated from integrator counts recorded with a Shimadzu C-R3A integrating recorder using inorganic phosphate (Pr) as a standard. The 32P counts per minute for the peaks were recorded with a Searle Delta-300 liquid scintillation counter. Calculations and statistical analyses of data were performed with an IBM-type PC, a spreadsheet program set up with templates to fit our needs, and the statistical program “Stata” (9). Data entered from the chromatograph and the scintillation counter conveniently and rapidly yielded the nanomoles, counts per minute corrected for decay, and specific activity in tabular form. Statistical analysis. A two-tailed paired Student t test (9) was used to ascertain levels of significance. Probability values of 0.05 or less were considered significant. M
RESULTS AND DISCUSSION The effect of extracellular phosphate on the insulin stimulation of hepatocyte [2,3-14C]succinate carbon incorporation into protein and oxidation to 14C02 is summarized in Table 1. The phosphate concentrations represent the levels used during the final two washings of the hepatocytes and the incubation medium. The intracellular perchloric acid soluble inorganic phosphate was about 22 nmole/mg hepatocyte protein. In the absence of extracellular phosphate, insulin failed to stimulate the oxidation of [‘4C]succinate carbons to CO* and their incorporation into hepatocyte protein. The addition of extracellular phosphate to the incubation medium resulted in a significant increase in the insulin stimulation of succinate oxidation and the incorporation of succinate carbons into protein. Phosphatedependent insulin stimulation of [2,3-14C] succinate oxidation plateaued at a 0.89 mM phosphate concentration in the medium. On the other hand, protein incorporation of 14C carbons reached a similar level at a 1.18 mM phosphate concentration. The requirement of extracellular phosphate for insulin stimulation of the Krebs cycle is very specific since lack of external phosphate did not prevent the stimulation of succinate oxidation by either glucagon or epinephrine (Table 2), even though the basal oxidation and protein incorporation of the tracer were signifi-
PHOSPHATE
REQUIREMENT
FOR INSULIN
347
ACTION
TABLE 1 The Effect of Extracellular Phosphate on Insulin Stimulation of [2,3-“‘ClSuccinate Protein Incorporation by Isolated Rat Hepatocytes Extracelhdar phosphate concentration (W 0 0.05 0.10 0.20 0.50 0.89 1.18
‘TO,
14C protein
Control 820 776 874 978 1077 1234 1176
+k + 2 + k *
19 (7) 71 (5) 60 (6) 65 (6) 104 (6) 181 (6) 85 (7)
Oxidation and
Insulin 834 954 1061 1194 1236 1573 1498
+ 2 2 + * k *
Control
cpm/mg protein 22 (6) 237 107 (6)’ 239 73 (6)’ 249 60 (6) 232 115 (6)“ 240 238 (6) 260 198 (7) 331
k IT k + + + +
40 30 28 32 39 37 29
Insulin (5) (5) (5) (5) (5) (5) (5)
222 269 275 292 290 293 420
f k 2 2 r 2 2
35 35 33 41 43 48 28
(5) (5) (5)d (5) (5)’ (5) (5)
a SEM. Values in parentheses represent number of experiments. b P < 0.05. = P < 0.02. d P < 0.01. e P < 0.005. f P < 0.001.
cantly lower in phosphate-depleted preparations. Glucagon and epinephrine failed to stimulate protein synthesis in either the presence or absence of extracellular phosphate. An examination of the relative specific activity of 32P04 incorporation into organic phosphate (Table 3) indicates that insulin added to phosphate-depleted hepatocytes did not stimulate the turnover of the organic phosphates, phosphoglyceric acid, uridine-diphosphoglucose, UTP, P-ADP, or ATP; nor did insulin added to control hepatocytes (isolated and incubated with 1.18 mM phosphate)
The Effect of Extracellular Extracellular phosphate 1.18 mM 0.0
rnM
% Change
TABLE 2 Phosphate on Insulin, Glucagon, and Epinephrine of [2,3-Y]Succinate
Stimulated Oxidation
Control
Insulin
Glucagon
765 + 44”
964 f 14b
635 + 111
(26)
708 f 102 (10.3)
1820 f 262’ (137.9) 1161 f 164’ (82.9)
1428 f 83’ (86.7) 997 f 212b (57.0)
- 16.9
-36.1
-36
-30.2
Epinephrine
a Data are presented as ‘YZO, cpm/mg protein. Values are presented as means 2 SEM (n = 4). Numbers in parentheses indicate percentage stimulation over control. b Level of significance is P < 0.001.
348
ROGERS ET AL. TABLE 3 Relative Specific Activity” (“P) of Hepatocyte Organic Phosphates Control (%)
Inorganic phosphate Phosphoglyceric acid Uridinediphosphoglucose UTP P-ADP ATP
loo (3)b 1.67 f .18 0.92 f .18 1.82 f .27 3.91 k 1.41 3.69 k .28
(2) (3) (3) (3) (3)
Insulin (%) 100 (3) 1.08 f 0.66 f 1.51 ?z 1.61 2 2.59 f
.40 .43 56 .95 .94
(2) (2) (3) (3) (3)
’ Relative specific activity was calculated using the formula: (“P cpm/nmole organic phosphate t 3zP cpm/nmole inorganic phosphate) x 100. b Mean -C SEM. Values in parentheses represent number of individual observations.
increase the turnover of these organic phosphates (data not shown) [cf. muscle
w91*
We conclude that the demonstrable activity of insulin on Krebs cycle oxidation and protein incorporation of succinate carbons requires extracellular phosphate. It is possible that this phosphate is presented to the mitochondria in a manner separate from that of intracellular phosphate (we observed no change in perchloric acid-soluble intracellular phosphate fractions due to insulin treatment of the hepatocyte). Diminution of this separate phosphate path impairs insulin activity. Our results could not be explained by the inhibition of phosphofructokinase by low phosphate (11) because succinate oxidation and protein synthesis still occur at a relative high rate [70% of control (Table l)]. This work is in agreement with that of Mine et al. (12) who reported that insulin inhibition of cyclic-AMP induced glycogenolysis was abolished during a phosphate-free perfusion of rat liver, and we have shown that insulin “inhibition” of gluconeogenesis is a function of the mitochondrial Krebs cycle (13). These findings have important clinical implications. Low insulin sensitivity has been reported in hypophosphatemic patients (14). This insulin insensitivity in hypophosphatemic patients may be related to the mitochondrial requirement for extracellular phosphate. Mohan et al. (13) have shown the importance of a normally operating Krebs cycle for all anabolic effects of insulin. Thus insulin sensitivity in these patients may be restored when their plasma phosphate levels approach the physiological range. Calcium appeared to be essential for the oxidation of succinate carbons. Basal and hormone-stimulated (insulin, glucagon, and epinephrine) 14C02 formation from [2,3-‘4C]succinate decreased significantly in the absence of calcium in the medium. Although no insulin effect was evident in the absence of calcium, a partial stimulatory effect of glucagon (33.2%) and epinephrine (23.1%) was observed (Table 4). Without calcium, insulin did not stimulate [2,3-‘4C]succinate carbon incorporation into protein (data not shown). Removal of magnesium from the KHB buffer did not alter the stimulatory effects of insulin, epinephrine, or glucagon on succinate oxidation and protein synthesis (data not shown). We conclude that lowering the medium calcium lowered the intracellular and intramitochondrial levels of calcium. Foden and Randle (15) reported that the
PHOSPHATE
REQUIREMENT
FOR INSULIN
TABLE 4 The Effect of Calcium on Insulin, Glucagon, and Epinephrine of [2,3-‘4C]Succinate Calcium 1.9
rnM
Insulin
Glucagon
774 * 53
996 2 93’ (28.7) 291 L 32 (8.5) -70
1610 f 88
268 -c 22’
% Change
-65.4
a Data Numbers * Level ’ Level
Stimulated Oxidation
Control
rnM
0.0
349
ACTION
Epinephrine
357 f 26 (33.2)
1515 f 176’ (95.8) 330 + 41 (23.1)
-77.8
-78.2
U’W
are presented as 14COz cpm/mg protein. Values are presented as means ? SEM (n = 4). in parentheses indicate percentage stimulation over control. of significance is P < 0.05. of significance is P < 0.001.
calcium efflux of hepatocyte and mitochondria occurs within 2 min. Our incubation time was 15 min. Thomas and Reed (16) have reported that 1 to 5 hr of incubation of hepatocytes in a calcium-free medium induces oxidative stress and leakage of cellular constituents. Calcium appears to be essential for enhanced phosphate uptake in the presence of glucagon (17). In this work we showed that the hormone control of cellular oxidation and protein synthesis is lost within 15 min following incubation in a calcium-free medium. Insulin stimulation of mitochondrial oxidation is dependent on optimal levels of both calcium and phosphate whereas only calcium appears to be essential for epinephrine and glucagon effects on Krebs cycle oxidation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Bessman SP. Am J Med 40~740, 1966. Bessman SP, Mohan C, Zaidise I. Proc Nat1 Acad Sci USA 83:5067, 1986. Mohan C, Bessman SP. Arch Biochem Biophys 242:563, 1985. Memon RA, Mohan C, Geiger PJ, Bessman SP, Rogers KS. Biochem Med Metab Biol 42~216, 1989. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. J Biol Chem 193:265, 1951. Bessman SP, Geiger PJ, Lu T-C, McCabe ERB. Anal Biochem 59:533, 1974. Geiger PJ, Ahn S, Bessman SP. in “Methods in Carbohydrate Chemistry” (Whistler RL, BeMiller JN, Eds.). Academic Press, New York. Erickson-Viitanen S, Viitanen P, Geiger PJ, Yang WCT, Bessman SP. J Biol Chem 25214395, 1982. Edwards AL. “Statistical Analysis,” p. 127, Rinehart, New York, 1958; and Statistical Analysis Program, “Stata,” available from Computing Resources Center, Santa Monica, CA. Bessman SP, Borrebaek B, Geiger PJ, Ben-Or S. in “Microenvironments and Cellular Compartmentation” (Srere P, Estabrook RW, Eds.), p. 111. Academic Press, New York, 1978. Brautber N, Massry SG. Adv Exp Med Biol 178:363, 1984. Mine T, Kimura S, Koide Y, Ohsawa H, Ogata E. Horm Metabol Res 12438, 1985. Mohan C, Geiger PH, Bessman SP. Cur-r Top Cell ReguZ3Or105, 1989. DeFronzo RA, Lang R. N Engl J Med 303:1259, 1980. Foden S, Randle PJ. Biochem J 170:615, 1978. Thomas CE, Reed DJ. J Pharmacol Exp Therap 245:493, 1988. Bygrave FL, Lenton L, Altin JG, Setchell BA, Karjalsinen A. Biochem J 26269, 1990.
