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OF BIOCHEMISTRY
AND
EIOPHYSICS
Vol. 289, No. 1, August 15, pp. 83-89, 1991
Arn~hi~o~i~ Role of the Krebs Cycle in the Insulin-Stimulated Protein Synthesis Chandra deportment
Mohan,l
Riaz 14. Memon,
of P~r~co~~
Received February
and Samuel
and nutrition,
University
P. Bessman
of Southern C~~~forni~, School of Medicine, Los Angeles, Californ~
13, 1991, and in revised form May 1, 1991
It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14C02 formation and protein incoramino poration of several l- 14C-labeled or U- ‘*C-labeled acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced ‘“COz formation and protein incorporation primarily of those carbons of amino acids wlhich are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 MM), a potent inhibitor of the transamination reaction, it was shown that there exists an “insulin-sensitive” pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of ‘%-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitoc~ondri~ transamination of (Yketoglutarate to glutamate is essential for insulin stimulation of ‘*C incorporation into hepatocyte protein. 0 1991
Academic
Press,
90033
Inc.
Insulin in vitro enhances the inco~oration of a number of amino acids into muscle protein (1,!2). This is not due to any effect of insulin on amino acid transport (3, 4). Studies on the effects of insulin on liver protein synthesis have generated controversial data. Several studies using the incorporation of radio-labeled branched chain amino acids, which are poorly metabolized by the liver (5, 6) have shown that insulin has no significant effect on their incorporation into liver protein (7-9). It was, therefore, concluded that the major effect of insulin was on muscle protein synthesis and in liver it merely reduced proteolysis. Using several metabolizable amino acids and other ’ To whom correspondence
should be addressed.
0003.9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
intermediates we have shown that insulin stimulates their rate of incorporation by 20-30 percent (10-12). The interpretation of tracer studies depends upon information on the actual process by which tracer amino acid carbons are incorporated into protein and the ultimate location of these tracer carbons. Experimental data from various studies have suggested preferential utilization of amino acids from extracellular (13-l’?), intracellular (l&22), or specialized pools derived from both extraand intracellular amino acids (23). In this paper it is shown that insulin enhances the oxidation and incorporation into protein only of those carbons of amino acids which traverse the mitochondrial Krebs cycle. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats (150-200 g) used for this study were maintained on lab chow and water ad libitum. Rats were anesthetized by intraperitoneal injection of pentobarbital(45 mg/kg). The femoral vein was exposed through a smail incision in the skin and sodium heparin (500 U/kg) was injected intravenously. The abLiver perfusion and the preparation of isolated hepatocytes. dominal cavity was opened and the hepatic portal vein was cannulated. Liver perfusion and the isolation of hepatocytes were carried out by the method of Seglen (24) with a few modifications (10). The viability of the cells as assessed by the trypan blue exclusion method (24) was routinely 90% or better. Preparation of isolated dehorn pieces. The diaphragm was quickly removed as a whole by severing the central tendon and cutting around the sternal and rib boundaries. It was washed several times in ice-cold Krebs-Henseleit bicarbonate (KHB)’ buffer (118 mM NaC1; 4.7 mM KC1; 1.18 mM MgSOI; 1.18 mM KH,PO,; 2.52 mM CaCl,; and 25 mM NaHCO,), pH 7.4, containing 5 mM glucose equilibrated previously with a mixture of Oa-CO, (95:5) for 1 h. Each diaphragm was cut radially into eight pieces of approximately equal size (four from each half) and two pieces (one from each half) were pooled and used for each incubation. Incubation of hepatocytes. The incubation medium consisted of KHB buffer, pH 7.4, equilibrated with oxygen-carbon dioxide (95:5) containing 0.5 mM final concentration of each of the 20 natural amino acids and 0.4 ml of hepatocyte suspension (8-10 mgprotein). The final incubation
’ Abbreviations used: KHB, Krebs-Henseleit a-ketoglutarate dehydrogenase.
bicarbonate;
a-KGDH, 83
Inc. reserved.
84
MOHAN. MEMON. AND BESSMAN
volume was 2 ml. To the experimental flasks insulin (10 mu/ml; crystalline bovine, Calbiochem) was added at time 0, 15,30,45, and 60 min. At t = 60 min, tracer amino acid [0.25 PCi of L-[l-“Clglutamic acid (Amersham); 2.0 &i of DL-[5-‘*C]glutamic acid (Research Products International, IL); 2.0 j&i of L-[U-“Clalanine (ICN); 0.2 &i of DL-[l“Clmethionine (ICN); 0.25 &i of L-[l-‘4C]valine or L-[U-%]valine (ICN)] was added to the flasks and they were fitted with sleeve stoppers containing centrally suspended polypropylene cups. The incubations were terminated at t = 120 min by injecting 0.6 ml of 3 N perchloric acid into the flasks through the stopper. %O, was collected in the suspended cups and counted as described earlier (10). Inhibition of hepatocyte transaminase activity by aminoozyacetic acid. Preliminary dose-response studies with aminooxyacetic acid showed that maximum transaminase inhibition occurred at 0.5 mM concentration. Aminooxyacetic acid (0.5 mM; Mann Research Laboratories, New York) was added at time zero to the experimental flasks which contained the above medium and cells. Protein incorporation and oxidation of l-‘4C-labeled glutamic acid (0.1 &i/flask) was studied. Insulin was added to the designated flasks at times zero and 30 min and the reaction was terminated at t = 60 min by injecting 0.6 ml of 3 N perchloric acid into the flasks through the sleeve stopper and ‘%O, was collected as described (10). Incubation of diaphragm pieces was Incubation of diaphragmpieces. initiated by placing them in 25.ml Erlenmeyer flasks containing 2 ml of KHB buffer, pH 7.4, containing 5 mM glucose, 0.25 mM final concentration of each of the 20 natural amino acids, and 0.5 pCi of [l-‘4C]valine; 0.5 (Xi of [U-Wlvaline; or 2.0 &i of [2,3-3H]valine (ICN). Insulin (10 mu/ml) was added to the experimental flasks at time zero. Incubation was terminated at t = 60 min by the addition of 0.6 ml of 3 N perchloric acid to the flask. ‘%‘02 was collected (in l-i% and U-14C experiments) and counted as described (10). The diaphragm pieces with the contents of the flask were homogenized in a glass homogenizer connected to a motor-driven pestle. Analytical methods. Following the termination of incubation, the contents of the flasks (or homogenates in diaphragm experiments) were collected and centrifuged at 9OOg for 10 min. The precipitates were washed three times with 0.7 M perchloric acid followed by 2 ml of ethanol: ether (3:l) and 1 ml of ether. Ethanohether and ether washes were pooled and saved for the determination of lipid radioactivity. Protein precipitates were dissolved in 1 ml of 1 N sodium hydroxide and 0.2-ml aliquots were used for counting in a liquid scintillation counter. Protein was assayed by the method of Lowry et al. (25).
TABLE
I
Effect of Insulin on ‘%O, Formation from [ l-‘4C]Glutamate and Its Incorporation into Isolated Hepatocyte Protein and Lipids Specific activity COP(n=14) Control Insulin P
11143 + 595 11978 + 470 N.S.
(cpm/mg hepatocyte Protein
(n = 14)
571.3 + 36.2 660.5 + 34.9
OF BIOCHEMISTRY
AND
EIOPHYSICS
Vol. 289, No. 1, August 15, pp. 83-89, 1991
Arn~hi~o~i~ Role of the Krebs Cycle in the Insulin-Stimulated Protein Synthesis Chandra deportment
Mohan,l
Riaz 14. Memon,
of P~r~co~~
Received February
and Samuel
and nutrition,
University
P. Bessman
of Southern C~~~forni~, School of Medicine, Los Angeles, Californ~
13, 1991, and in revised form May 1, 1991
It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14C02 formation and protein incoramino poration of several l- 14C-labeled or U- ‘*C-labeled acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced ‘“COz formation and protein incorporation primarily of those carbons of amino acids wlhich are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 MM), a potent inhibitor of the transamination reaction, it was shown that there exists an “insulin-sensitive” pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of ‘%-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitoc~ondri~ transamination of (Yketoglutarate to glutamate is essential for insulin stimulation of ‘*C incorporation into hepatocyte protein. 0 1991
Academic
Press,
90033
Inc.
Insulin in vitro enhances the inco~oration of a number of amino acids into muscle protein (1,!2). This is not due to any effect of insulin on amino acid transport (3, 4). Studies on the effects of insulin on liver protein synthesis have generated controversial data. Several studies using the incorporation of radio-labeled branched chain amino acids, which are poorly metabolized by the liver (5, 6) have shown that insulin has no significant effect on their incorporation into liver protein (7-9). It was, therefore, concluded that the major effect of insulin was on muscle protein synthesis and in liver it merely reduced proteolysis. Using several metabolizable amino acids and other ’ To whom correspondence
should be addressed.
0003.9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
intermediates we have shown that insulin stimulates their rate of incorporation by 20-30 percent (10-12). The interpretation of tracer studies depends upon information on the actual process by which tracer amino acid carbons are incorporated into protein and the ultimate location of these tracer carbons. Experimental data from various studies have suggested preferential utilization of amino acids from extracellular (13-l’?), intracellular (l&22), or specialized pools derived from both extraand intracellular amino acids (23). In this paper it is shown that insulin enhances the oxidation and incorporation into protein only of those carbons of amino acids which traverse the mitochondrial Krebs cycle. MATERIALS
AND
METHODS
Animals. Male Sprague-Dawley rats (150-200 g) used for this study were maintained on lab chow and water ad libitum. Rats were anesthetized by intraperitoneal injection of pentobarbital(45 mg/kg). The femoral vein was exposed through a smail incision in the skin and sodium heparin (500 U/kg) was injected intravenously. The abLiver perfusion and the preparation of isolated hepatocytes. dominal cavity was opened and the hepatic portal vein was cannulated. Liver perfusion and the isolation of hepatocytes were carried out by the method of Seglen (24) with a few modifications (10). The viability of the cells as assessed by the trypan blue exclusion method (24) was routinely 90% or better. Preparation of isolated dehorn pieces. The diaphragm was quickly removed as a whole by severing the central tendon and cutting around the sternal and rib boundaries. It was washed several times in ice-cold Krebs-Henseleit bicarbonate (KHB)’ buffer (118 mM NaC1; 4.7 mM KC1; 1.18 mM MgSOI; 1.18 mM KH,PO,; 2.52 mM CaCl,; and 25 mM NaHCO,), pH 7.4, containing 5 mM glucose equilibrated previously with a mixture of Oa-CO, (95:5) for 1 h. Each diaphragm was cut radially into eight pieces of approximately equal size (four from each half) and two pieces (one from each half) were pooled and used for each incubation. Incubation of hepatocytes. The incubation medium consisted of KHB buffer, pH 7.4, equilibrated with oxygen-carbon dioxide (95:5) containing 0.5 mM final concentration of each of the 20 natural amino acids and 0.4 ml of hepatocyte suspension (8-10 mgprotein). The final incubation
’ Abbreviations used: KHB, Krebs-Henseleit a-ketoglutarate dehydrogenase.
bicarbonate;
a-KGDH, 83
Inc. reserved.
84
MOHAN. MEMON. AND BESSMAN
volume was 2 ml. To the experimental flasks insulin (10 mu/ml; crystalline bovine, Calbiochem) was added at time 0, 15,30,45, and 60 min. At t = 60 min, tracer amino acid [0.25 PCi of L-[l-“Clglutamic acid (Amersham); 2.0 &i of DL-[5-‘*C]glutamic acid (Research Products International, IL); 2.0 j&i of L-[U-“Clalanine (ICN); 0.2 &i of DL-[l“Clmethionine (ICN); 0.25 &i of L-[l-‘4C]valine or L-[U-%]valine (ICN)] was added to the flasks and they were fitted with sleeve stoppers containing centrally suspended polypropylene cups. The incubations were terminated at t = 120 min by injecting 0.6 ml of 3 N perchloric acid into the flasks through the stopper. %O, was collected in the suspended cups and counted as described earlier (10). Inhibition of hepatocyte transaminase activity by aminoozyacetic acid. Preliminary dose-response studies with aminooxyacetic acid showed that maximum transaminase inhibition occurred at 0.5 mM concentration. Aminooxyacetic acid (0.5 mM; Mann Research Laboratories, New York) was added at time zero to the experimental flasks which contained the above medium and cells. Protein incorporation and oxidation of l-‘4C-labeled glutamic acid (0.1 &i/flask) was studied. Insulin was added to the designated flasks at times zero and 30 min and the reaction was terminated at t = 60 min by injecting 0.6 ml of 3 N perchloric acid into the flasks through the sleeve stopper and ‘%O, was collected as described (10). Incubation of diaphragm pieces was Incubation of diaphragmpieces. initiated by placing them in 25.ml Erlenmeyer flasks containing 2 ml of KHB buffer, pH 7.4, containing 5 mM glucose, 0.25 mM final concentration of each of the 20 natural amino acids, and 0.5 pCi of [l-‘4C]valine; 0.5 (Xi of [U-Wlvaline; or 2.0 &i of [2,3-3H]valine (ICN). Insulin (10 mu/ml) was added to the experimental flasks at time zero. Incubation was terminated at t = 60 min by the addition of 0.6 ml of 3 N perchloric acid to the flask. ‘%‘02 was collected (in l-i% and U-14C experiments) and counted as described (10). The diaphragm pieces with the contents of the flask were homogenized in a glass homogenizer connected to a motor-driven pestle. Analytical methods. Following the termination of incubation, the contents of the flasks (or homogenates in diaphragm experiments) were collected and centrifuged at 9OOg for 10 min. The precipitates were washed three times with 0.7 M perchloric acid followed by 2 ml of ethanol: ether (3:l) and 1 ml of ether. Ethanohether and ether washes were pooled and saved for the determination of lipid radioactivity. Protein precipitates were dissolved in 1 ml of 1 N sodium hydroxide and 0.2-ml aliquots were used for counting in a liquid scintillation counter. Protein was assayed by the method of Lowry et al. (25).
TABLE
I
Effect of Insulin on ‘%O, Formation from [ l-‘4C]Glutamate and Its Incorporation into Isolated Hepatocyte Protein and Lipids Specific activity COP(n=14) Control Insulin P
11143 + 595 11978 + 470 N.S.
(cpm/mg hepatocyte Protein
(n = 14)
571.3 + 36.2 660.5 + 34.9
