Biochem. J. (1977) 164, 251-255 Printed in Great Britain
251
The Effect of Insulin on the Washout.of I45CalCalcium from Adipocytes and Soleus Muscle of the Rat By TORBEN CLAUSEN and BRUCE RICHARD MARTIN* Institute of Physiology, University of Arhus, 8000 Arhus C, Denmark (Received 26 November 1976)
Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure. In whole epididymal fat-pads of the rat, insulin was found to stimulate the washout of 45Ca (Clausen, 1969b). Preliminary studies indicated that isolated fat-cells showed a similar response (Martin et al., 1973), and in both liver plasma membranes (Marinetti et al., 1972) and 'ghosts' of fat-cells insulin was found to diminish the binding of Ca2+ (Kissebah et al., 1975). In a hyperosmolar milieu it could be demonstrated that insulin elicits a marked rise in the resting tension of rat soleus muscles (Clausen et al., 1974, 1975), and it was shown that, in cultured chick myoblasts, insulin stimulates the release of 45Ca (Schudt et al., 1976). All of these observations indicated that the hormone might exert a hitherto poorly characterized action on some cellular calcium pool, eventually leading to a mobilization of Ca2+ ions. The present paper describes the results of
experiments designed to detect and quantify possible effects of insulin on the exchange of 45Ca in intact fat-cells and muscles of the rat. Experimental Materials 45Ca (360 mCi/mmol) was obtained from the Isotope Laboratory, Danish Atomic Energy Commission, Ris0, DK 4000 Roskilde, Denmark. Monocomponent pig insulin (lot no. MC-S 823081, 25 units/mg) was a gift from the Novo Research Laboratories, Copenhagen, Denmark. Bovine serum albumin was purchased from Sigma Chemical Co., St. Louis, MO, U.S.A., and used after 24h of dialysis against water in the cold.
Methods
Experiments with adipocytes. The techniques for the preparation of isolated fat-cells, the incubation media and the procedures for sampling of cells, as * Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K. Vol. 164
well as counting of 45Ca radioactivity, were described previously (Martin et al., 1975). The washout of 45Ca from whole epididymal fatpads was measured as described elsewhere (Clausen, 1970), and the fraction of 45Ca released from the tissue per min was calculated as in analogous experiments with 3-O-[14C]methylglucose (Clausen, 1969a). Experiments with soleus muscles. Soleus muscles (wet wt. 25-35mg) were prepared from young fed rats (60-70g) as described by Kohn & Clausen (1971) and incubated in Krebs-Ringer bicarbonate buffer (Cohen, 1951). After loading with 45Ca for 60min, the washout of radioactivity into unlabelled medium was followed for 200min, and the fraction of 45Ca
released per min calculated (Clausen et al., 1975).
as
earlier reported
Results and Discussion We have shown (Martin et al., 1975) that the washout of 45Ca from isolated fat-cells displays a very rapid early phase lasting less than 1 min, followed by a much slower phase which probably represents release of 45Ca from intracellular pools. Table 1 shows the effect of adding insulin during the period 10-20min after the onset of washout. Insulin clearly accelerates the release of 45Ca from the cells, and the effect can be detected at concentrations down to 0.01 munit/ml, well within the physiological range (Blackshear & Alberti, 1974). When added during the later, slow phase of washout (60min after the onset of washing), insulin produced no significant change in the fractional loss of 45Ca. In whole epididymal fat-pads, the washout of 45Ca is much slower, and from around 60min after the onset of washout the fractional loss reaches an almost constant value (Clausen, 1970). Table 2 shows that during this slow phase of washout, insulin at concentrations ranging from 0.05 to lOmunits/ml increased the rate coefficient by 10-18 % in the period from 10 to 20min after its addition. At the higher
252
T. CLAUSEN AND B. R. MARTIN
Table 1. Effect of insulin on the washout of '5Ca from fat-cells Isolated fat-cells (1 ml packed volume) were incubated with shaking for 1 h at 37°C in 6ml of Krebs-Ringer bicarbonate buffer containing 1% bovine serum albumin and 1.27mM-45Ca (5 ,Ci/ml). At the end of this period the suspension was centrifuged (600g) and the medium was aspirated off. The cells were washed with 10ml of the same buffer without 45Ca and resuspended in 6ml of Krebs-Ringer bicarbonate buffer again with no 45Ca. The cells were incubated with shaking for 1Omin, divided into two equal portions, centrifuged, and the medium was aspirated off. Fresh medium was added to one portion of cells and fresh medium containing insulin to the other. Samples were taken immediately and after 10min incubation. Cells and medium were separated by centrifugation through dinonyl phthalate (Gliemann et al., 1972) and their 45Ca radioactivity was determined. Care was taken to ensure that the cells were evenly suspended before sampling, and that media were thoroughly gassed with 02/CO2 (19: 1). The fraction of cellular 45Ca released into the medium was calculated and the results expressed as the mean of three observations±s.E.M. Change in relation to Fraction of '"Ca controls Experimental conditions released/lOmin p (%/) Control 0.65 + 0.02 Insulin (0.01 munit/ml)
251
The Effect of Insulin on the Washout.of I45CalCalcium from Adipocytes and Soleus Muscle of the Rat By TORBEN CLAUSEN and BRUCE RICHARD MARTIN* Institute of Physiology, University of Arhus, 8000 Arhus C, Denmark (Received 26 November 1976)
Insulin stimulates the washout of 45Ca from preloaded isolated fat-cells, whole epididymal fat-pads and isolated soleus muscles of the rat. This effect occurs within 10min after the addition of the hormone, and it can be detected at concentrations down to those measured in rat plasma. When K+ is omitted from the washout medium, the effect on soleus muscles is more pronounced and increases with the time of exposure. In whole epididymal fat-pads of the rat, insulin was found to stimulate the washout of 45Ca (Clausen, 1969b). Preliminary studies indicated that isolated fat-cells showed a similar response (Martin et al., 1973), and in both liver plasma membranes (Marinetti et al., 1972) and 'ghosts' of fat-cells insulin was found to diminish the binding of Ca2+ (Kissebah et al., 1975). In a hyperosmolar milieu it could be demonstrated that insulin elicits a marked rise in the resting tension of rat soleus muscles (Clausen et al., 1974, 1975), and it was shown that, in cultured chick myoblasts, insulin stimulates the release of 45Ca (Schudt et al., 1976). All of these observations indicated that the hormone might exert a hitherto poorly characterized action on some cellular calcium pool, eventually leading to a mobilization of Ca2+ ions. The present paper describes the results of
experiments designed to detect and quantify possible effects of insulin on the exchange of 45Ca in intact fat-cells and muscles of the rat. Experimental Materials 45Ca (360 mCi/mmol) was obtained from the Isotope Laboratory, Danish Atomic Energy Commission, Ris0, DK 4000 Roskilde, Denmark. Monocomponent pig insulin (lot no. MC-S 823081, 25 units/mg) was a gift from the Novo Research Laboratories, Copenhagen, Denmark. Bovine serum albumin was purchased from Sigma Chemical Co., St. Louis, MO, U.S.A., and used after 24h of dialysis against water in the cold.
Methods
Experiments with adipocytes. The techniques for the preparation of isolated fat-cells, the incubation media and the procedures for sampling of cells, as * Present address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, U.K. Vol. 164
well as counting of 45Ca radioactivity, were described previously (Martin et al., 1975). The washout of 45Ca from whole epididymal fatpads was measured as described elsewhere (Clausen, 1970), and the fraction of 45Ca released from the tissue per min was calculated as in analogous experiments with 3-O-[14C]methylglucose (Clausen, 1969a). Experiments with soleus muscles. Soleus muscles (wet wt. 25-35mg) were prepared from young fed rats (60-70g) as described by Kohn & Clausen (1971) and incubated in Krebs-Ringer bicarbonate buffer (Cohen, 1951). After loading with 45Ca for 60min, the washout of radioactivity into unlabelled medium was followed for 200min, and the fraction of 45Ca
released per min calculated (Clausen et al., 1975).
as
earlier reported
Results and Discussion We have shown (Martin et al., 1975) that the washout of 45Ca from isolated fat-cells displays a very rapid early phase lasting less than 1 min, followed by a much slower phase which probably represents release of 45Ca from intracellular pools. Table 1 shows the effect of adding insulin during the period 10-20min after the onset of washout. Insulin clearly accelerates the release of 45Ca from the cells, and the effect can be detected at concentrations down to 0.01 munit/ml, well within the physiological range (Blackshear & Alberti, 1974). When added during the later, slow phase of washout (60min after the onset of washing), insulin produced no significant change in the fractional loss of 45Ca. In whole epididymal fat-pads, the washout of 45Ca is much slower, and from around 60min after the onset of washout the fractional loss reaches an almost constant value (Clausen, 1970). Table 2 shows that during this slow phase of washout, insulin at concentrations ranging from 0.05 to lOmunits/ml increased the rate coefficient by 10-18 % in the period from 10 to 20min after its addition. At the higher
252
T. CLAUSEN AND B. R. MARTIN
Table 1. Effect of insulin on the washout of '5Ca from fat-cells Isolated fat-cells (1 ml packed volume) were incubated with shaking for 1 h at 37°C in 6ml of Krebs-Ringer bicarbonate buffer containing 1% bovine serum albumin and 1.27mM-45Ca (5 ,Ci/ml). At the end of this period the suspension was centrifuged (600g) and the medium was aspirated off. The cells were washed with 10ml of the same buffer without 45Ca and resuspended in 6ml of Krebs-Ringer bicarbonate buffer again with no 45Ca. The cells were incubated with shaking for 1Omin, divided into two equal portions, centrifuged, and the medium was aspirated off. Fresh medium was added to one portion of cells and fresh medium containing insulin to the other. Samples were taken immediately and after 10min incubation. Cells and medium were separated by centrifugation through dinonyl phthalate (Gliemann et al., 1972) and their 45Ca radioactivity was determined. Care was taken to ensure that the cells were evenly suspended before sampling, and that media were thoroughly gassed with 02/CO2 (19: 1). The fraction of cellular 45Ca released into the medium was calculated and the results expressed as the mean of three observations±s.E.M. Change in relation to Fraction of '"Ca controls Experimental conditions released/lOmin p (%/) Control 0.65 + 0.02 Insulin (0.01 munit/ml)
