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Insulin effect on amino acid transport in bone
L,
PRELIMINARY NOTES
675
BBA 21 262
Insulin effect on amino acid transport in bone Several polypeptide hormones alter bone synthesis or resorption when administered in vivo z. Direct effects in vitro, however, are limited to those hormones which control bone resorption (parathyroid hormone, calcitonin). Although insulin stimulates amino acid uptake and protein synthesis in several types of tissue 2, s, little is known of its effects on bone. We now report that insulin stimulates intracellular accumulation of amino acids in fetal membranous bone in vitro, and that this stimulation is dependent on protein synthesis. We isolated calvaria from Sprague-Dawley rat fetuses b y a method previously described 4. Tissues were placed in 2 ml of Krebs-Ringer bicarbonate buffer at p H 7.4 under O~-CO 2 (95:5, v/v), and preincubated with or without insulin in a metabolic shaker at 37 °. No glucose was added to the medium. Following the preincubation period, laC-labeled amino acids (o.I mM) were added and incubation was continued. Tissue and media radioactivities were determined 4 and used together with values for total tissue water and water spaces ~ to calculate distribution ratios (D.R.) (D.R. -~ [amino acid]lntraeellular nutd/~amino acid]extracellular fluid). Proline incorporation into acidic ethanol precipitable protein was determined b y previously described methods e. Glucose utilization was measured b y the production of x*CO~ from [14C61glucoseT. Recrystallized bovine pancreas insulin (Mann) was used at a concentration of o.2/~g/ml throughout. Values for total tissue water and extracellular fluid space measured with [z4C]inulin were 64 % and .39 % of wet tissue weight, respectively. Preincubation with insulin did not change these'values. Membrane transport was studied using ~-aminoisobutyric acid, a model amino acid which shares membrane transport mechanisms with neutral amino acids but is not further metabolized. Insulin did not significantly increase a-aminoisobutyric acid uptake when present only from the start of the experiment. However, when tissues were preincubated with insulin for at least 3o min prior to the addition of =-aminoisobutyric acid, a significant stimulation was observed. Furthermore, the amount of stimulation increased with longer periods of preincubation with insulin (Fig. I). After z2o min of insulin preincubation, the 3o-min D.R. for =-aminoisobutyric acid was increased by 37 % ( P < o . o I ) . Under similar conditions, insulin stimulated the trans-
m , ~ 1 1 / / 1 1 1 / / 1 1 / / 1 / / / / / ] " t0 ~ / / 1 ] / / / / / / / / / / / / / / / / "
Iflsulin
-
Neon ]; IIS E
Contro - Meon ± S.E m I
0
I
~0
i
I
I
I
I
60
90
120
150
180
MINUTES OF PREINCUBATION
Fig. z. Preincubation effect on insulin stimulation of 3o-min ~-aminoisobutyric acid (AIB) uptake Total incubation time was 2io min throughout. ~-Amino[laC]isobutyric acid was added after i8o min. Insulin was added at indicated intervals prior to the uptake period. Biochim. Biophys. Aaa, 184 (z969) 675--677
676
PRELIMINARY'NOTES
port of an imino acid, L-proline (D.R. insulin lO.5 -4- 0.6; control 7.8 zL 0.3, P < o . o I ) , but not of the model dibasic amino acid, diaminobutyric acid (D.R. insulin 9.7 zL 0.3; control 9.8 4- 0.2). a-Aminoisobutyric acid uptake was also stimulated when tissues were preincubated with insulin for 12o min, then washed and transferred to fresh buffer without insulin for a 3o-min uptake (D.R. insulin 13. 7 ± 0. 9 ; control lO.5 ± 0.7, P < o . o 5 ) . Hence, the presence of insulin in the medium was not essential for insulininduced stimulation after preliminary insulin exposure. These results suggest that insulin stimulates production of an intermediate which increases transport of certain amino acids into bone cells. To test this hypothesis, we studied the effect of puromycin, an inhibitor of protein synthesis, on insulin stimulation. Preincubation for 12o rain with puromycin (0.55 mM) alone reduced a-aminoisobutyric acid uptake from control values, a finding previously reported in rat diaphragm s and embryonic chick bone g. More importantly, addition of puromycin to the preincubation medium completely abolished insulin stimulation of a-aminoisobutyric acid transport (Table I). TABLE I INSULIN
EFFECT PUROMYCIN
ON
~-AMINOISOBUTYRIC
ACID
TRANSPORT
IN
TIlE
PRESENCE
AND
ABSENCE
OF
Preincubation time w a s 12o rain; uptake, 3o min. Values are expressed as the m e a n ± S.E. of six or m o r e observations. Student's t test w a s used to obtain P values.
Condition
Distribution ratio
P
Control
12.7
,( o.oi
Puromycin Puromycin + insulin
2~ i . o
9-3 • 0.8 9.2 i 0.8
.
Not significant 1 < o.oi < O,OOI
Insulin
17.1 ~ 0.9
.J
Protein synthesis in bone is dependent, at least in part, on intracellular amino acid concentrations e. We therefore, studied the effect of insulin on e-proline incorporation into acidic ethanol precipitable protein. Calvaria were preincubated with insulin and unlabeled i-proline (0.03 mM) for I2O min. Carrier-free L-[14C]proline was then added to the medium and the incubation continued for an additional x2o min. Under these conditions, insulin increased proline distribution ratios by 26 % ( P < o.ox) and L-proline incorporation into protein by 74 % (P
184 (1969) 675-677
PRELIMINARY NOTES
677
These findings demonstrate that insulin stimulates uptake of certain amino acids in fetal membranous bone in vitro. Our observations that the insulin effect (i) is not instantaneous, (ii) increases in magnitude with longer tissue exposure to insulin, (iii) does not require the presence of free insulin in the medium after suitable periods of exposure and (iv) is abolished by puromycin, suggest that insulin stimulation of amino acid transport in bone is mediated by protein synthesis. A similar mechanism has been proposed for insulin effects on amino acid transport in rat diaphragm 8. Whether the protein synthesized in response to insulin is directly involved in membrane transport cannot be determined from our data. Our results do, however, militate against the possibility that a rapid direct effect of insulin on bone cell membranes is responsible for the observed transport stimulation. Such a direct effect has been proposed as the mechanism for insulin stimulation of amino acid transport in muscle 1°. An association between amino acid transport and incorporation into protein in bone has been previously observed ~. It is, therefore, not surprising that insulin stimulation of proline transport is accompanied by increased proline incorporation into protein. Finally, it is of interest that insulin stimulates amino acid transport but not 3-0-methylglucose transport in fetal membranous bone. Indeed, we were not able to demonstrate active transport of 3-0-methylglucose in fetal calvaria, a finding also reported in embryonic chick bone rudiments n. This is in contrast to membrane transport in muscle where insulin-sensitive transport mechanisms for both 3-O-methylglucoseTMand amino acids 2 have been demonstrated. This work was supported in part by Public Health Service Special Fellowship Grant No. I-F3-CA-39, 622-Ol. THEODORE J. HAHN SYLVIA J. DOWNING JAMES M. PHANG
Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) I 2 3 4
W. A. PECK ANDT. R. DIRKSEN,Clin. Orthoped., 48 (I966) 243. I. G. WOOLANDIV[.E. KROHL,Am. J. Physiol., I96 (1959) 96I. R. LEVINE,New England J. Med., 277 (I967) I959. G. A. M. FINERMANAND L. E. ROSENBERG, J. Biol. Chem., 241 (1966) 1387.
5 L. E. ROSENBERG, S. J. D O W N I N G A N D S. SEGAL, A m . J. Physiol., 2oe (I962) 800. 6 G. A. IV[.FINERMAN, S. D O W N I N G A N D L. E. ROSENBERG, Biochim. Biophys. Acta, 135 (i967)
7 8 9 io
,008. L. E. ROSENBERG,A. N. WEINBERGAND S. SEGAL,Bioehim. Biophys. Aeta, 48 (I96I) 500. L. J. ELSAS,I. ALBRECHTAND L. E. ROSENEERG,J. Biol. Chem., 243 (I968) I846. L. F. ADAMSON,S. G. LANGELUTTIGANDC. S. ANAST,Bioehim. Biophys. Aeta, II5 (1966) 355. H. AKEDOAND H. N. CHRISTENSEN,J. Biol. Chem., 237 (1962) 118. L . F . ADAMSON, S. G . LANGELUTTIG AND C. S. ANAST, Bioehim. Biophys. Aeta, I I 5 ( I 9 6 6 ) 3 4 5 .
1I 12 L . S. W E I S AND H . T. NARAHARA,
Diabetes, 17 (1968) 3 0 4 .
Received June 2nd, 1969 Biochim. Biophys. Aeta, 184 (1969) 675-677
PRELIMINARY NOTES
675
BBA 21 262
Insulin effect on amino acid transport in bone Several polypeptide hormones alter bone synthesis or resorption when administered in vivo z. Direct effects in vitro, however, are limited to those hormones which control bone resorption (parathyroid hormone, calcitonin). Although insulin stimulates amino acid uptake and protein synthesis in several types of tissue 2, s, little is known of its effects on bone. We now report that insulin stimulates intracellular accumulation of amino acids in fetal membranous bone in vitro, and that this stimulation is dependent on protein synthesis. We isolated calvaria from Sprague-Dawley rat fetuses b y a method previously described 4. Tissues were placed in 2 ml of Krebs-Ringer bicarbonate buffer at p H 7.4 under O~-CO 2 (95:5, v/v), and preincubated with or without insulin in a metabolic shaker at 37 °. No glucose was added to the medium. Following the preincubation period, laC-labeled amino acids (o.I mM) were added and incubation was continued. Tissue and media radioactivities were determined 4 and used together with values for total tissue water and water spaces ~ to calculate distribution ratios (D.R.) (D.R. -~ [amino acid]lntraeellular nutd/~amino acid]extracellular fluid). Proline incorporation into acidic ethanol precipitable protein was determined b y previously described methods e. Glucose utilization was measured b y the production of x*CO~ from [14C61glucoseT. Recrystallized bovine pancreas insulin (Mann) was used at a concentration of o.2/~g/ml throughout. Values for total tissue water and extracellular fluid space measured with [z4C]inulin were 64 % and .39 % of wet tissue weight, respectively. Preincubation with insulin did not change these'values. Membrane transport was studied using ~-aminoisobutyric acid, a model amino acid which shares membrane transport mechanisms with neutral amino acids but is not further metabolized. Insulin did not significantly increase a-aminoisobutyric acid uptake when present only from the start of the experiment. However, when tissues were preincubated with insulin for at least 3o min prior to the addition of =-aminoisobutyric acid, a significant stimulation was observed. Furthermore, the amount of stimulation increased with longer periods of preincubation with insulin (Fig. I). After z2o min of insulin preincubation, the 3o-min D.R. for =-aminoisobutyric acid was increased by 37 % ( P < o . o I ) . Under similar conditions, insulin stimulated the trans-
m , ~ 1 1 / / 1 1 1 / / 1 1 / / 1 / / / / / ] " t0 ~ / / 1 ] / / / / / / / / / / / / / / / / "
Iflsulin
-
Neon ]; IIS E
Contro - Meon ± S.E m I
0
I
~0
i
I
I
I
I
60
90
120
150
180
MINUTES OF PREINCUBATION
Fig. z. Preincubation effect on insulin stimulation of 3o-min ~-aminoisobutyric acid (AIB) uptake Total incubation time was 2io min throughout. ~-Amino[laC]isobutyric acid was added after i8o min. Insulin was added at indicated intervals prior to the uptake period. Biochim. Biophys. Aaa, 184 (z969) 675--677
676
PRELIMINARY'NOTES
port of an imino acid, L-proline (D.R. insulin lO.5 -4- 0.6; control 7.8 zL 0.3, P < o . o I ) , but not of the model dibasic amino acid, diaminobutyric acid (D.R. insulin 9.7 zL 0.3; control 9.8 4- 0.2). a-Aminoisobutyric acid uptake was also stimulated when tissues were preincubated with insulin for 12o min, then washed and transferred to fresh buffer without insulin for a 3o-min uptake (D.R. insulin 13. 7 ± 0. 9 ; control lO.5 ± 0.7, P < o . o 5 ) . Hence, the presence of insulin in the medium was not essential for insulininduced stimulation after preliminary insulin exposure. These results suggest that insulin stimulates production of an intermediate which increases transport of certain amino acids into bone cells. To test this hypothesis, we studied the effect of puromycin, an inhibitor of protein synthesis, on insulin stimulation. Preincubation for 12o rain with puromycin (0.55 mM) alone reduced a-aminoisobutyric acid uptake from control values, a finding previously reported in rat diaphragm s and embryonic chick bone g. More importantly, addition of puromycin to the preincubation medium completely abolished insulin stimulation of a-aminoisobutyric acid transport (Table I). TABLE I INSULIN
EFFECT PUROMYCIN
ON
~-AMINOISOBUTYRIC
ACID
TRANSPORT
IN
TIlE
PRESENCE
AND
ABSENCE
OF
Preincubation time w a s 12o rain; uptake, 3o min. Values are expressed as the m e a n ± S.E. of six or m o r e observations. Student's t test w a s used to obtain P values.
Condition
Distribution ratio
P
Control
12.7
,( o.oi
Puromycin Puromycin + insulin
2~ i . o
9-3 • 0.8 9.2 i 0.8
.
Not significant 1 < o.oi < O,OOI
Insulin
17.1 ~ 0.9
.J
Protein synthesis in bone is dependent, at least in part, on intracellular amino acid concentrations e. We therefore, studied the effect of insulin on e-proline incorporation into acidic ethanol precipitable protein. Calvaria were preincubated with insulin and unlabeled i-proline (0.03 mM) for I2O min. Carrier-free L-[14C]proline was then added to the medium and the incubation continued for an additional x2o min. Under these conditions, insulin increased proline distribution ratios by 26 % ( P < o.ox) and L-proline incorporation into protein by 74 % (P
184 (1969) 675-677
PRELIMINARY NOTES
677
These findings demonstrate that insulin stimulates uptake of certain amino acids in fetal membranous bone in vitro. Our observations that the insulin effect (i) is not instantaneous, (ii) increases in magnitude with longer tissue exposure to insulin, (iii) does not require the presence of free insulin in the medium after suitable periods of exposure and (iv) is abolished by puromycin, suggest that insulin stimulation of amino acid transport in bone is mediated by protein synthesis. A similar mechanism has been proposed for insulin effects on amino acid transport in rat diaphragm 8. Whether the protein synthesized in response to insulin is directly involved in membrane transport cannot be determined from our data. Our results do, however, militate against the possibility that a rapid direct effect of insulin on bone cell membranes is responsible for the observed transport stimulation. Such a direct effect has been proposed as the mechanism for insulin stimulation of amino acid transport in muscle 1°. An association between amino acid transport and incorporation into protein in bone has been previously observed ~. It is, therefore, not surprising that insulin stimulation of proline transport is accompanied by increased proline incorporation into protein. Finally, it is of interest that insulin stimulates amino acid transport but not 3-0-methylglucose transport in fetal membranous bone. Indeed, we were not able to demonstrate active transport of 3-0-methylglucose in fetal calvaria, a finding also reported in embryonic chick bone rudiments n. This is in contrast to membrane transport in muscle where insulin-sensitive transport mechanisms for both 3-O-methylglucoseTMand amino acids 2 have been demonstrated. This work was supported in part by Public Health Service Special Fellowship Grant No. I-F3-CA-39, 622-Ol. THEODORE J. HAHN SYLVIA J. DOWNING JAMES M. PHANG
Metabolism Branch, National Cancer Institute, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) I 2 3 4
W. A. PECK ANDT. R. DIRKSEN,Clin. Orthoped., 48 (I966) 243. I. G. WOOLANDIV[.E. KROHL,Am. J. Physiol., I96 (1959) 96I. R. LEVINE,New England J. Med., 277 (I967) I959. G. A. M. FINERMANAND L. E. ROSENBERG, J. Biol. Chem., 241 (1966) 1387.
5 L. E. ROSENBERG, S. J. D O W N I N G A N D S. SEGAL, A m . J. Physiol., 2oe (I962) 800. 6 G. A. IV[.FINERMAN, S. D O W N I N G A N D L. E. ROSENBERG, Biochim. Biophys. Acta, 135 (i967)
7 8 9 io
,008. L. E. ROSENBERG,A. N. WEINBERGAND S. SEGAL,Bioehim. Biophys. Aeta, 48 (I96I) 500. L. J. ELSAS,I. ALBRECHTAND L. E. ROSENEERG,J. Biol. Chem., 243 (I968) I846. L. F. ADAMSON,S. G. LANGELUTTIGANDC. S. ANAST,Bioehim. Biophys. Aeta, II5 (1966) 355. H. AKEDOAND H. N. CHRISTENSEN,J. Biol. Chem., 237 (1962) 118. L . F . ADAMSON, S. G . LANGELUTTIG AND C. S. ANAST, Bioehim. Biophys. Aeta, I I 5 ( I 9 6 6 ) 3 4 5 .
1I 12 L . S. W E I S AND H . T. NARAHARA,
Diabetes, 17 (1968) 3 0 4 .
Received June 2nd, 1969 Biochim. Biophys. Aeta, 184 (1969) 675-677