362
Increased Insulin Responsiveness in Isolated Rat Hepatocytes Incubated with Free Fatty Acid-Poor Albumin J. F. Caro, J. Hodges and M. K. Sinha Department of Medicine, Section of Endocrinology and Metabolism, East Carolina University School of Medicine, Greenville, North Carolina, U. S. A.
Materials and Methods
Freshly isolated rat hepatocytes incubated in a medium containing bovine serum albumin partially purified by charcoal treatment at pH 3 are three times more responsive to insulin with regard to a-aminoisobutyric acid uptake than those incubated in medium containing regular bovine serum albumin. This finding should facilitate future work in isolated hepatocytes, a cell system that for the most part has been considered relatively unresponsive to insulin.
Chemicals a-[l14C]-aminoisobutyric acid (51.6) mCi/mmol) was obtained from New England Nuclear, Boston, MA. Crude collagenase (4117) CLSII 41 K22) was purchased from Worthington Biochemical Corp., Freehold, NJ, and fraction V BSA from Armour Pharmaceutical Co., Kankakee, IL. Crystalline porcine insulin and [125ITyr] monoiodoinsulin (376 uCi/ug) were kindly provided by Eli Lilly & Co., Indianapolis, IN. Charcoal (Lot 12F 0314) was obtained from Sigma Chemical Co., St. Louis, MO, and Spectrapor membrane dialyzing tubing (#3487-F47) was obtained from Arthur Thomas Co., Philadelphia, PA. All other chemicals were reagent grade.
Key words Treatment Insulin — Hepatocytes — Insulin Action — Fatty Acid-free Albumin — Bovine Serum Albumin
Introduction Freshly isolated rat hepatocytes provide a powerful tool to study insulin action and metabolism at the cellular level. However, this cell system has not been extensively used because of its relative unresponsiveness to insulin in comparison to isolated rat adipocytes. In isolated adipocytes, maximal concentration of insulin stimulates basal metabolic rates several fold, whereas in isolated hepatocytes the response is, at its best, about 75% above basal. In the last several years, we have learned that in order to isolate hepatocytes even modestly responsive to insulin, special care should be given to the selection of both the "batches" of collagenase used during the cell isolation and of bovine serum albumin (BSA) used during the washing, preincubation, and incubation of the isolated hepatocytes. Each time new "batches" of either collagenase or BSA are purchased, several must be carefully screened to identify one that allows the isolation of insulin responsive hepatocytes. In this manuscript, we report that further purification of BSA by charcoal treatment at pH 3 eliminates one of these complicating factors. Isolated rat hepatocytes incubated with BSA treated in this manner are three times more responsive to insulin than those incubated with regular BSA.
Horm.metab.Res.23(1991)362-364 © Georg Thieme Verlag Stuttgart • New York
of BSA
The method of Chen (1967) was used to remove fatty acids from BSA. Activated charcoal 5% (w/v) was added to a 10% (w/v) solution of fraction V BSA in water. The pH was reduced to 3.0 by drop-wise addition of 1.0 N HC1 and mixed for 1 hour at 4 °C. The charcoal was removed by repeated centrifugation at 10,000 x g and filtration through Whatman 1 mm filter paper. The clear BSA solution was neutralized with 1.0 N NaOH and dialyzed against 50 volumes of 0.9% (w/v) NaCl and then against 50-100 volumes of water. Finally, the BSA was lyophilized and stored at 4 °C. Chen (1967) reported that Armour Fraction V BSA contains approximately 0.4 moles FFA/mole protein before charcoal treatment and < 0.01 moles FFA/mole protein after charcoal treatment. For additional experiments, fatty acids were added back to the purified BSA. Oleic acid (0.2 M in ethanol) was diluted to 2 mM Krebs-Ringer bicarbonate buffer pH 7.4, containing 30 g of purified BSA for 100 ml at 60 °C with gentle agitation for 60 min. Purified BSA was treated identically but without the addition of oleic acid. Then, Krebs-Ringer bicarbonate buffer without BSA was added to afinalBSA concentration of 1 % and 3 % for the execution of experiments described in the Results section.
Preparation of Hepatocytes Hepatocytes were isolated from adult male SpragueDawley rats (200—250 g) fed ad libitum. The rats were anesthetized with sodium pentobarbital (8 mg/100 g body weight). Liver perfusion was performed using the method of Exton (1975) in a liver perfusion cabinet designed and made at Vanderbilt University, Nashville, TN. The perfusion was Krebs-Ringer bicarbonate buffer-free of calcium and BSA and supplemented with 5 mM glutamate, 11 mM glucose, 5 mM sodium pyruvate, and 10% (v/v) washed outdated human erythrocytes at pH 7.4. The erythrocytes were washed three times with 0.9% (w/v) NaCl. After equilibration of the perfusion medium for 30 min at 37 °C with O2/CO2 (95:5, v/v), livers were perfused in a non-
Received: 17 May 1989
Accepted: 4 Jan. 1991 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Summary
Horm. metab. Res. 23 (1991)
Insulin Responsive Hepatocytes
363
Table 1 Insulin-stimulated AIB uptake in isolated hepatocytes. Hepatocytes from each liver perfusion were divided into two groups and incubated with either treated BSA or untreated BSA as described in the Methods section. The initial rate of AIB uptake (0.1 mM) was measured at 10 min after 120-min preincubation without insulin (basal) or with insulin (1 x 10- 9 M and 1 x 10- 7 M).The data from each separate parallel experiment are expressed as picomoles of AIB uptake per min per 106 cells. The mean ± SEM from the six separate experiments are shown at the end of each group. Cell Suspension Conditions and Experiment Number
AIB uptake pmol/min per 10 cells
Basal
Insulin 1 x 10- 9 M
Insulin 1 x 10-
1 2 3 4 5 6 Mean ± SEM
10 12 12 15 16 9 12±1
18 16 21 25 20 17 20+1
28 23 29 36 42 21 30±3
11 12 10 15 11 15 12±1
12 14 10 18 16 18 15+ 1
16 14 13 21 20 19 17±1
Untreated BSA
1 2 3 4 5 6 Mean ± SEM
Fig. 1 125l Insulin Binding. Incubations were performed with 125l insulin (1 x 10- 10 M) in the presence and absence of increasing concentrations of unlabeled insulin (log M indicated in the figure) at 4 °C for 16 hours as described in the Methods section. The data represent the mean + SEM of triplicates from a representative experiment.
Insulin
Binding
125
I insulin binding was determined as previously described by Kauffman and Caro (1983) in the presence of I insulin and increasing concentrations of unlabeled insulin for 16 hours at 4°C. circulating system for 15 min at 14 ml/min. The system was then set to recirculate and 20 mg of collagenase was added to the remaining 125150 ml of perfusion medium. After 35 min, the liver cells were isolated by the method of Feldhoff, Taylor and Jefferson (1977) with minor modifications as reported by Caro and Amatruda (1980). The liver was removed, minced gently with scissors, suspended in 50 ml of perfusion buffer (without erythrocytes), and filtered through nylon. The cells were centrifuged at 50 x g in 50 ml conical centrifuge tubes in a Dynac table-top centrifuge and washed three times with perfusion medium without erythrocytes, but containing 1 g of BSA/100 ml and 2.4 mM CaCl2. All buffers were maintained at 37 °C and equilibrated with O2/CO 2 (95 %, v/v). After three washes, cells were suspended in the same buffer containing 3 g of BSA/100 ml to yield ~ 3.0 x 106 cells/ml and were placed in 250 ml polycarbonate Erlenmeyer flasks with a maximum of 60 ml of suspended cells in one flask. After 30 min of incubation at 37 °C with a constant flow of O2/CO 2 mixture, the cells were centrifuged again and resuspended in the same buffer. Insulin
Action
The ability of insulin to stimulate the uptake of aaminoisobutyric acid (AIB), a nonmetabolizable analog of alanine, was used to assay insulin action as previously reported by Kauffman and Caro (1983). Briefly, freshly isolated hepatocytes were suspended (2-4 x 10 cells/ml) in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 3 % BSA. The cells were preincubated at 37 °C in the absence and presence of maximal (1 x 10- 7 M) and submaximal (1 x 10- M) concentrations of insulin for 2 hours. After preincubation, [14C] AIB (0.1 mM) was added to the incubation mixture. Since AIB uptake was linear for at least 20 minutes, the reaction was terminated at 10 min to obtain the initial rate of uptake by rapid centrifugation of hepatocytes through dibutylphthalate oil.
Results and Discussion Following the perfusion of liver with collagenase, the cell pellet was divided into two groups which were washed, preincubated, and incubated identically, except that in one group partially purified BSA was used and in the other group regular BSA was used. In both groups cell viability was over 90%, as measured by exclusion of trypan blue. Furthermore, hepatocytes from both groups incorporated C leucine (0.12 Ci/ml, 340 mCi/mmol) into trichloroacetic acid, precipitable material at an equal rate (cpm, 1 x 10 cells/hr regular BSA; 1100+140 cpm, 1 x 10 6 cells/hr purified BSA, mean + SEM, n = 6) for 4 hours. The data in Table 1 show the results of each of the six parallel experiments from six different animals, each with triplicate determinations. Only two concentrations of insulin, 1 x 1 0 - 9 M (ED50) and 1 x 1 0 - 7 M (Max), were used since an adequate supply of cells was necessary to perform parallel experiments with and without partially purified BSA. Basal AIB uptake was not afected by the different BSA preparations. The two hepatocyte suspensions responded to insulin 1 x 1 0 - 9 M (purified BSA p < 0.01 and regular BSA p < 0.02 paired t-test with basal) and 1 x 1 0 - 7 M (purified BSA p < 0.001 and regular BSA p < 0.01). However, the degree of responsiveness was markedly enhanced in the cells incubated with purified BSA (150% above basal) when compared with those incubated with regular BSA (41 % above basal, p < 0.001 unpaired t-test). Although we did not perform complete dose response curves, it appears that insulin sensitivity was not affected since about 50% of the maximal response
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Treated BSA
was achieved with 1 x 10 M insulin under the two experimental conditions. Also as demonstrated in Figure 1, the different BSA preparations did not alter I insulin binding at tracer concentrations of I insulin (1 x 10- M). The radioactivity bound to the receptors in the presence of purified and regular BSA was 24.3 % and 23.4% of total 125 I insulin, respectively. More than 90% of the I insulin bound at tracer concentration was displaced by a large excess of unlabeled insulin (1 x 1 0 - 6 M ) and 50% displacement was achieved with approximately 2 x 10-9 M insulin under both experimental conditions. Fatty acids have been demonstrated to interfere with many regulatory functions of the cells. Although never conclusively proven, it has been hypothesized by Randle, Garland, Hale and Newsholme (1963), and Reaven (1988) that elevated FFA in obesity or type II diabetes may be involved in the mechanism of insulin resistance in these diseases. Removal of FFA from BSA followed by improvement of insulin responsiveness is consistent with this hypothesis. Furthermore, the addition of oleic acid to the purified BSA significantly (p < 0.05) reduces insulin stimulated AIB uptake without altering basal uptake (purified BSA: basal 15 + 6; 1 x 1 0 ~ 7 M insulin 32 + 6; purified BSA plus oleic acid 0.2 mM: basal 13 + 4, 1 x 1 0 - 7 M insulin 20 + 3 AIB uptake pmol/min per 10 cells, mean ± SEM, n = 3). Juul and Jones (1982) and Sonne (1985) have demonstrated that the leakage of insulin-degrading enzymes from isolated rat hepatocytes is reduced when the cells are incubated in buffer containing FFA-poor BSA. However, this explanation is unlikely for the improved insulin responsiveness observed in hepatocytes incubated in FFA-poor BSA (Table 1) since the leakage of degrading enzymes in our cell preparation is minimal and the addition of bacitracin 1 mg/ml to the buffer to inhibit insulin degradation does not change insulin responsiveness in our cells {Caro and Amatruda 1981). It is also possible that treatment of BSA removed an insulin-like substance, however, this possibility is unlikely since basal AIB uptake was unchanged. Likewise, a nonspecific cytotoxic factor was probably not removed by the BSA treatment since cell viability was identical under the two experimental conditions. Following the conclusion of our study, Svedberg, Bjorntorp, Smith and Lonnroth (1990) also demonstrated that BSA-bound oleic acid 0.4 mM inhibited basal AIB uptake by 29% and insulin stimulated AIB uptake by 39% in isolated rat hepatocytes. In these studies, the control BSA, although reported to contain < 0.1 mM FFA, was not specifically purified to remove FFA, which may be one of the reasons why the insulin stimulation was only ~ 50% above basal. Furthermore, these investigators demonstrated the lack of a direct effect of FFA on the insulin receptor consistent with our experiments examining cell surface I insulin binding at
J. F. Caro, J. Hodges and M. K. Sinha 4 °C. However, they demonstrated that at 37 °C, FFA markedly decreased the rate of insulin receptor internalization and/or recycling and insulin degradation. In conclusion, our studies demonstrate that use of FFA-poor BSA prepared by charcoal treatment at acid pH, according to Chen (1967), improves insulin responsiveness of isolated rat hepatocytes. Although we do not understand the mechanism of this observation, we feel certain that this finding will facilitate future work on insulin action in isolated hepatocytes by other laboratories. Acknowledgements This work was supported by Grant #PO DK-36296 from the National Institutes of Health. References Caro, J. F., J. M. Amatruda: Insulin receptors in hepatocytes: postreceptor events mediate down regulation. Science 210: 1029—1031 (1980) Cam, J. F., J. M. Amatruda: Evidence for modulation of insulin action and degradation independently of insulin binding. Am. J. Physiol. 240:E325-E332(1981) Chen, R. F.: Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242:173 -181 (1967) Exton, J. H.: The perfused rat liver. Methods Enzymol. 39: 25—36 (1975) Feldhoff, R. C, J. M. Taylor, L. S. Jefferson: Synthesis and secretion of rat albumin in vitro in perfused liver and isolated hepatocytes. J. Biol. Chem. 252:3611-3616(1977) Juul, S. M., R. H. Jones: Evidence for a direct effect of bacitracin on cell-mediated insulin degradation in isolated hepatocytes. Biochem.J.295-299(1982) Kauffman, J. M., J. F. Caro: Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats. J. Clin. Invest. 71:698-708(1983) Randle, P. J., P. B. Garland, C. N. Hale, E. A. Newsholme: The glucose fatty-acid cycle: Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1:785 - 786 (1963) Reaven, G. M.: Role of insulin resistance in human. Diabetes 37: 1595-1607(1988) Sonne, O.: Increased inhibitory potency of free fatty acid-poor albumin on the release and activity of insulin-degrading enzymes from isolated rat adipocytes and hepatocytes. Anal. Biochem. 151: 109-117(1985) Svedberg, J., P. Bjorntorp, U. Smith, P. Lonnroth: Free-fatty acid inhibition of insulin binding, degradation, and action in isolated rat hepatocytes. Diabetes 39: 570 (1990) Requests for reprints should be addressed to: Jose F. Caro, M. D. Head, Section of Endocrinology Department of Medicine East Carolina University School of Medicine Greenville, North Carolina 27858-4354 (U. S. A.)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Horm. metab. Res. 23 (1991)
Increased Insulin Responsiveness in Isolated Rat Hepatocytes Incubated with Free Fatty Acid-Poor Albumin J. F. Caro, J. Hodges and M. K. Sinha Department of Medicine, Section of Endocrinology and Metabolism, East Carolina University School of Medicine, Greenville, North Carolina, U. S. A.
Materials and Methods
Freshly isolated rat hepatocytes incubated in a medium containing bovine serum albumin partially purified by charcoal treatment at pH 3 are three times more responsive to insulin with regard to a-aminoisobutyric acid uptake than those incubated in medium containing regular bovine serum albumin. This finding should facilitate future work in isolated hepatocytes, a cell system that for the most part has been considered relatively unresponsive to insulin.
Chemicals a-[l14C]-aminoisobutyric acid (51.6) mCi/mmol) was obtained from New England Nuclear, Boston, MA. Crude collagenase (4117) CLSII 41 K22) was purchased from Worthington Biochemical Corp., Freehold, NJ, and fraction V BSA from Armour Pharmaceutical Co., Kankakee, IL. Crystalline porcine insulin and [125ITyr] monoiodoinsulin (376 uCi/ug) were kindly provided by Eli Lilly & Co., Indianapolis, IN. Charcoal (Lot 12F 0314) was obtained from Sigma Chemical Co., St. Louis, MO, and Spectrapor membrane dialyzing tubing (#3487-F47) was obtained from Arthur Thomas Co., Philadelphia, PA. All other chemicals were reagent grade.
Key words Treatment Insulin — Hepatocytes — Insulin Action — Fatty Acid-free Albumin — Bovine Serum Albumin
Introduction Freshly isolated rat hepatocytes provide a powerful tool to study insulin action and metabolism at the cellular level. However, this cell system has not been extensively used because of its relative unresponsiveness to insulin in comparison to isolated rat adipocytes. In isolated adipocytes, maximal concentration of insulin stimulates basal metabolic rates several fold, whereas in isolated hepatocytes the response is, at its best, about 75% above basal. In the last several years, we have learned that in order to isolate hepatocytes even modestly responsive to insulin, special care should be given to the selection of both the "batches" of collagenase used during the cell isolation and of bovine serum albumin (BSA) used during the washing, preincubation, and incubation of the isolated hepatocytes. Each time new "batches" of either collagenase or BSA are purchased, several must be carefully screened to identify one that allows the isolation of insulin responsive hepatocytes. In this manuscript, we report that further purification of BSA by charcoal treatment at pH 3 eliminates one of these complicating factors. Isolated rat hepatocytes incubated with BSA treated in this manner are three times more responsive to insulin than those incubated with regular BSA.
Horm.metab.Res.23(1991)362-364 © Georg Thieme Verlag Stuttgart • New York
of BSA
The method of Chen (1967) was used to remove fatty acids from BSA. Activated charcoal 5% (w/v) was added to a 10% (w/v) solution of fraction V BSA in water. The pH was reduced to 3.0 by drop-wise addition of 1.0 N HC1 and mixed for 1 hour at 4 °C. The charcoal was removed by repeated centrifugation at 10,000 x g and filtration through Whatman 1 mm filter paper. The clear BSA solution was neutralized with 1.0 N NaOH and dialyzed against 50 volumes of 0.9% (w/v) NaCl and then against 50-100 volumes of water. Finally, the BSA was lyophilized and stored at 4 °C. Chen (1967) reported that Armour Fraction V BSA contains approximately 0.4 moles FFA/mole protein before charcoal treatment and < 0.01 moles FFA/mole protein after charcoal treatment. For additional experiments, fatty acids were added back to the purified BSA. Oleic acid (0.2 M in ethanol) was diluted to 2 mM Krebs-Ringer bicarbonate buffer pH 7.4, containing 30 g of purified BSA for 100 ml at 60 °C with gentle agitation for 60 min. Purified BSA was treated identically but without the addition of oleic acid. Then, Krebs-Ringer bicarbonate buffer without BSA was added to afinalBSA concentration of 1 % and 3 % for the execution of experiments described in the Results section.
Preparation of Hepatocytes Hepatocytes were isolated from adult male SpragueDawley rats (200—250 g) fed ad libitum. The rats were anesthetized with sodium pentobarbital (8 mg/100 g body weight). Liver perfusion was performed using the method of Exton (1975) in a liver perfusion cabinet designed and made at Vanderbilt University, Nashville, TN. The perfusion was Krebs-Ringer bicarbonate buffer-free of calcium and BSA and supplemented with 5 mM glutamate, 11 mM glucose, 5 mM sodium pyruvate, and 10% (v/v) washed outdated human erythrocytes at pH 7.4. The erythrocytes were washed three times with 0.9% (w/v) NaCl. After equilibration of the perfusion medium for 30 min at 37 °C with O2/CO2 (95:5, v/v), livers were perfused in a non-
Received: 17 May 1989
Accepted: 4 Jan. 1991 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Summary
Horm. metab. Res. 23 (1991)
Insulin Responsive Hepatocytes
363
Table 1 Insulin-stimulated AIB uptake in isolated hepatocytes. Hepatocytes from each liver perfusion were divided into two groups and incubated with either treated BSA or untreated BSA as described in the Methods section. The initial rate of AIB uptake (0.1 mM) was measured at 10 min after 120-min preincubation without insulin (basal) or with insulin (1 x 10- 9 M and 1 x 10- 7 M).The data from each separate parallel experiment are expressed as picomoles of AIB uptake per min per 106 cells. The mean ± SEM from the six separate experiments are shown at the end of each group. Cell Suspension Conditions and Experiment Number
AIB uptake pmol/min per 10 cells
Basal
Insulin 1 x 10- 9 M
Insulin 1 x 10-
1 2 3 4 5 6 Mean ± SEM
10 12 12 15 16 9 12±1
18 16 21 25 20 17 20+1
28 23 29 36 42 21 30±3
11 12 10 15 11 15 12±1
12 14 10 18 16 18 15+ 1
16 14 13 21 20 19 17±1
Untreated BSA
1 2 3 4 5 6 Mean ± SEM
Fig. 1 125l Insulin Binding. Incubations were performed with 125l insulin (1 x 10- 10 M) in the presence and absence of increasing concentrations of unlabeled insulin (log M indicated in the figure) at 4 °C for 16 hours as described in the Methods section. The data represent the mean + SEM of triplicates from a representative experiment.
Insulin
Binding
125
I insulin binding was determined as previously described by Kauffman and Caro (1983) in the presence of I insulin and increasing concentrations of unlabeled insulin for 16 hours at 4°C. circulating system for 15 min at 14 ml/min. The system was then set to recirculate and 20 mg of collagenase was added to the remaining 125150 ml of perfusion medium. After 35 min, the liver cells were isolated by the method of Feldhoff, Taylor and Jefferson (1977) with minor modifications as reported by Caro and Amatruda (1980). The liver was removed, minced gently with scissors, suspended in 50 ml of perfusion buffer (without erythrocytes), and filtered through nylon. The cells were centrifuged at 50 x g in 50 ml conical centrifuge tubes in a Dynac table-top centrifuge and washed three times with perfusion medium without erythrocytes, but containing 1 g of BSA/100 ml and 2.4 mM CaCl2. All buffers were maintained at 37 °C and equilibrated with O2/CO 2 (95 %, v/v). After three washes, cells were suspended in the same buffer containing 3 g of BSA/100 ml to yield ~ 3.0 x 106 cells/ml and were placed in 250 ml polycarbonate Erlenmeyer flasks with a maximum of 60 ml of suspended cells in one flask. After 30 min of incubation at 37 °C with a constant flow of O2/CO 2 mixture, the cells were centrifuged again and resuspended in the same buffer. Insulin
Action
The ability of insulin to stimulate the uptake of aaminoisobutyric acid (AIB), a nonmetabolizable analog of alanine, was used to assay insulin action as previously reported by Kauffman and Caro (1983). Briefly, freshly isolated hepatocytes were suspended (2-4 x 10 cells/ml) in Krebs-Ringer bicarbonate buffer, pH 7.4, supplemented with 3 % BSA. The cells were preincubated at 37 °C in the absence and presence of maximal (1 x 10- 7 M) and submaximal (1 x 10- M) concentrations of insulin for 2 hours. After preincubation, [14C] AIB (0.1 mM) was added to the incubation mixture. Since AIB uptake was linear for at least 20 minutes, the reaction was terminated at 10 min to obtain the initial rate of uptake by rapid centrifugation of hepatocytes through dibutylphthalate oil.
Results and Discussion Following the perfusion of liver with collagenase, the cell pellet was divided into two groups which were washed, preincubated, and incubated identically, except that in one group partially purified BSA was used and in the other group regular BSA was used. In both groups cell viability was over 90%, as measured by exclusion of trypan blue. Furthermore, hepatocytes from both groups incorporated C leucine (0.12 Ci/ml, 340 mCi/mmol) into trichloroacetic acid, precipitable material at an equal rate (cpm, 1 x 10 cells/hr regular BSA; 1100+140 cpm, 1 x 10 6 cells/hr purified BSA, mean + SEM, n = 6) for 4 hours. The data in Table 1 show the results of each of the six parallel experiments from six different animals, each with triplicate determinations. Only two concentrations of insulin, 1 x 1 0 - 9 M (ED50) and 1 x 1 0 - 7 M (Max), were used since an adequate supply of cells was necessary to perform parallel experiments with and without partially purified BSA. Basal AIB uptake was not afected by the different BSA preparations. The two hepatocyte suspensions responded to insulin 1 x 1 0 - 9 M (purified BSA p < 0.01 and regular BSA p < 0.02 paired t-test with basal) and 1 x 1 0 - 7 M (purified BSA p < 0.001 and regular BSA p < 0.01). However, the degree of responsiveness was markedly enhanced in the cells incubated with purified BSA (150% above basal) when compared with those incubated with regular BSA (41 % above basal, p < 0.001 unpaired t-test). Although we did not perform complete dose response curves, it appears that insulin sensitivity was not affected since about 50% of the maximal response
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Treated BSA
was achieved with 1 x 10 M insulin under the two experimental conditions. Also as demonstrated in Figure 1, the different BSA preparations did not alter I insulin binding at tracer concentrations of I insulin (1 x 10- M). The radioactivity bound to the receptors in the presence of purified and regular BSA was 24.3 % and 23.4% of total 125 I insulin, respectively. More than 90% of the I insulin bound at tracer concentration was displaced by a large excess of unlabeled insulin (1 x 1 0 - 6 M ) and 50% displacement was achieved with approximately 2 x 10-9 M insulin under both experimental conditions. Fatty acids have been demonstrated to interfere with many regulatory functions of the cells. Although never conclusively proven, it has been hypothesized by Randle, Garland, Hale and Newsholme (1963), and Reaven (1988) that elevated FFA in obesity or type II diabetes may be involved in the mechanism of insulin resistance in these diseases. Removal of FFA from BSA followed by improvement of insulin responsiveness is consistent with this hypothesis. Furthermore, the addition of oleic acid to the purified BSA significantly (p < 0.05) reduces insulin stimulated AIB uptake without altering basal uptake (purified BSA: basal 15 + 6; 1 x 1 0 ~ 7 M insulin 32 + 6; purified BSA plus oleic acid 0.2 mM: basal 13 + 4, 1 x 1 0 - 7 M insulin 20 + 3 AIB uptake pmol/min per 10 cells, mean ± SEM, n = 3). Juul and Jones (1982) and Sonne (1985) have demonstrated that the leakage of insulin-degrading enzymes from isolated rat hepatocytes is reduced when the cells are incubated in buffer containing FFA-poor BSA. However, this explanation is unlikely for the improved insulin responsiveness observed in hepatocytes incubated in FFA-poor BSA (Table 1) since the leakage of degrading enzymes in our cell preparation is minimal and the addition of bacitracin 1 mg/ml to the buffer to inhibit insulin degradation does not change insulin responsiveness in our cells {Caro and Amatruda 1981). It is also possible that treatment of BSA removed an insulin-like substance, however, this possibility is unlikely since basal AIB uptake was unchanged. Likewise, a nonspecific cytotoxic factor was probably not removed by the BSA treatment since cell viability was identical under the two experimental conditions. Following the conclusion of our study, Svedberg, Bjorntorp, Smith and Lonnroth (1990) also demonstrated that BSA-bound oleic acid 0.4 mM inhibited basal AIB uptake by 29% and insulin stimulated AIB uptake by 39% in isolated rat hepatocytes. In these studies, the control BSA, although reported to contain < 0.1 mM FFA, was not specifically purified to remove FFA, which may be one of the reasons why the insulin stimulation was only ~ 50% above basal. Furthermore, these investigators demonstrated the lack of a direct effect of FFA on the insulin receptor consistent with our experiments examining cell surface I insulin binding at
J. F. Caro, J. Hodges and M. K. Sinha 4 °C. However, they demonstrated that at 37 °C, FFA markedly decreased the rate of insulin receptor internalization and/or recycling and insulin degradation. In conclusion, our studies demonstrate that use of FFA-poor BSA prepared by charcoal treatment at acid pH, according to Chen (1967), improves insulin responsiveness of isolated rat hepatocytes. Although we do not understand the mechanism of this observation, we feel certain that this finding will facilitate future work on insulin action in isolated hepatocytes by other laboratories. Acknowledgements This work was supported by Grant #PO DK-36296 from the National Institutes of Health. References Caro, J. F., J. M. Amatruda: Insulin receptors in hepatocytes: postreceptor events mediate down regulation. Science 210: 1029—1031 (1980) Cam, J. F., J. M. Amatruda: Evidence for modulation of insulin action and degradation independently of insulin binding. Am. J. Physiol. 240:E325-E332(1981) Chen, R. F.: Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242:173 -181 (1967) Exton, J. H.: The perfused rat liver. Methods Enzymol. 39: 25—36 (1975) Feldhoff, R. C, J. M. Taylor, L. S. Jefferson: Synthesis and secretion of rat albumin in vitro in perfused liver and isolated hepatocytes. J. Biol. Chem. 252:3611-3616(1977) Juul, S. M., R. H. Jones: Evidence for a direct effect of bacitracin on cell-mediated insulin degradation in isolated hepatocytes. Biochem.J.295-299(1982) Kauffman, J. M., J. F. Caro: Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats. J. Clin. Invest. 71:698-708(1983) Randle, P. J., P. B. Garland, C. N. Hale, E. A. Newsholme: The glucose fatty-acid cycle: Its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1:785 - 786 (1963) Reaven, G. M.: Role of insulin resistance in human. Diabetes 37: 1595-1607(1988) Sonne, O.: Increased inhibitory potency of free fatty acid-poor albumin on the release and activity of insulin-degrading enzymes from isolated rat adipocytes and hepatocytes. Anal. Biochem. 151: 109-117(1985) Svedberg, J., P. Bjorntorp, U. Smith, P. Lonnroth: Free-fatty acid inhibition of insulin binding, degradation, and action in isolated rat hepatocytes. Diabetes 39: 570 (1990) Requests for reprints should be addressed to: Jose F. Caro, M. D. Head, Section of Endocrinology Department of Medicine East Carolina University School of Medicine Greenville, North Carolina 27858-4354 (U. S. A.)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Horm. metab. Res. 23 (1991)
