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Changes in insulin receptors on methylcholanthrene-induced sarcoma during growth
JOURNAL
OF SURGICAL
RESEARCH
48, 435-439 (19%))
Changes in Insulin Receptors on Methylcholanthrene-Induced Sarcoma during Growth’ LAWRENCE Division
D. WAGMAN, M.D., FACS, AND MARIANNE Z. METZ, B.A.
of Oncologic Surgery, City of Hope National
Presented at the Annual Meeting of the Association
Medical Center, 1500 East Dunrte Road, Duarte, California for Academic Surgery, Louisville,
Methylcholanthrene-induced sarcomas (MCA-S) have different growth patterns in diabetic (D) and nondiabetic (ND) rats. Diabetes delays the early phase of tumor growth and prolongs survival. This study evaluated MCA-S growth and its relation to insulin receptors (IR) and glucose uptake. Fisher 344 rats 150-200 g were assigned to two groups: Diabetic tumor bearers (DTB, n = 26) and nondiabetic tumor bearers (NDTB, n = 18). Diabetes was induced with iv streptozocin (40 mg/kg); MCA-S was inoculated (1 X 10’ cells) subcutaneously 10 days later. Animals were sacrificed during early growth (tumor volume 6 20 cc) or logarithmic growth (tumor volume > 20 cc). IR assay was performed (O-lo6 rig/ml cold insulin, 25 X lo3 cpm/tube Al4 “‘I-insulin, 90 min, 15”C, pH 7.8) on a single cell preparation. Serum glucose milligrams per deciliter and insulin nanograms per milliliter were assayed. Glucose uptake (dpm/g tissue/hr) was assayed 2 hr after an ip injection of 0.5 PCi 30[‘4C]methylglucose. Diabetic, tumor-bearing animals had a significantly increased number of insulin receptors at the small [<20 cc, 28.7 (D) vs 8.3 (ND)] and large [>20 cc, 82.8 (D) vs 27.8 (ND) ] tumor volumes. Glucose uptake was increased in the tumor at both volumes in the nondiabetic animals compared to the diabetic animals. The increase in insulin receptor number on the surface of the tumors of diabetic animals did not result in an increase in glucose uptake. Substrate (glucose) deprivation due to hypoinsulinemia remains the most likely explanation for the early phase growth delay in the tumors of diabetic animals. 0 1990 Academic Press. Inc.
The methylcholanthrene-induced sarcoma, grown in Fischer rats, has different growth patterns (changes in rate of linear and exponential phases) in streptozocininduced diabetic and normal animals [ 11. Previous reports have shown a relative increase in insulin receptor number with a relative decrease in receptor affinity when com1 Supported in part by an American Cancer Society Career Development Award (Lawrence D. Wagman, M.D.). 435
November
15-18, 1989
paring tumors from diabetic (D) and nondiabetic (ND) animals at the time of their death [2]. Analysis of glucose uptake with nonmetabolized glucose analogues (2-deoxyD-glUCOSE!) has demonstrated rapid uptake and retention in this tumor at levels exceeding liver, brain, and muscle [3]. In some tissues, insulin enhances the uptake of glucose by interaction with its receptor [4]. This series of experiments sought to answer the following questions: (1) Do changes in tumor growth rates correlate with changes in tumor insulin receptor number and/or affinity? (2) Do changes in tumor insulin receptor number and/or affinity correlate with the uptake of glucose by the tumor; and (3) Will tumor insulin receptor number and glucose uptake be altered if diabetic, tumor-bearing animals are treated with insulin? MATERIALS
AND METHODS
Animal Model Adult male 150- to 180-g Fischer 344 rats were obtained from Charles River Breeding Co. (Kingston, NY). This rat model has been previously shown to support reliable, consistent growth of a methylcholanthrene-induced sarcoma and tolerate diabetes. Animals were housed two per cage and fed rat chow (Wayne Pet Food Division, Continental Grain Co., Chicago, IL) and water ad Eibitum. Animals were weighed at the initiation of the study and every 3 days. Approval for animal use was obtained from the City of Hope Research Animal Care Committee. Experimental
INTRODUCTION
Kentucky,
91010
Groups
Seventy animals were assigned to one of four experimental groups: nondiabetic, nontumor bearer (NDNTB: n = 4); nondiabetic, tumor bearer (NDTB: ~20 cc tumor volume (TV), n = 7; ~20 cc TV n = 11); diabetic, nontumor bearer (DNTB: n = 4); and diabetic, tumor bearer (DTB: ~20 cc TV, n = 14; >20 cc, TV, n = 12). A separate group of diabetic, tumor-bearing animals was treated with insulin (~20 cc TV, n = 3; >20 cc TV, n = 2). Control (nontumor-bearing) animals were matched by experimental day (day of sacrifice based on tumor volume) to 0022-4804/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
436
JOURNAL
OF SURGICAL
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the tumor-bearing animals for comparisons of weight, glucose uptake, and serum glucose and insulin. The nontumor-bearing animals were subsequently referred to by tumor volume (<20 cc or >20 cc) for various comparisons. This method of pairing allowed chronologic-dependent comparisons to be made at equivalent times after initiation of the experiment. Tumor Model A methylcholanthrene-induced sarcoma (MCA-S) was inoculated on Day 0 of the experiment in the left flank at 1 X lo6 cells per milliliter dose. The tumor was grown in carrier animals and prepared as a single cell suspension. Experimental animals had tumor size measured in millimeters and recorded every 1 to 3 days. Tumor volume was calculated using the formula for a prolate spheroid: Volume = (4/3)A*B, where A = short axis and B = long axis. Tumor was weighed at the time of excision. Tumor index, a measure of tumor burden, was calculated. [Tumor volume/(body weight-tumor volume).] After the animals were euthanized with Halothane (Halocarbon Lab., Inc., Hackensack, NJ), the tumor was removed, rinsed with 0.5% collagenase (Worthington Biochemical Corp., Freehold, NJ), and incubated at 37°C for 30 min. The supernatant was removed and the remaining tumor passed through a stainless-steel wire mesh (l-mm mesh). Red blood cells were lysed, and the cells were washed and resuspended in cold assay buffer with 0.5% bovine serum albumin at pH 7.8. Tumor cell viability was assessed with the trypan blue exclusion technique. Diabetes Induction Under general anesthesia, 40 mg/kg of streptozocin (Upjohn Co., Kalamazoo, MI), was injected via the dorsal vein of the penis on Day -10 of the experimental protocol. Control animals were anesthetized and tagged. Hyperglycemia was documented prior to Day 0 by collection of 50 ~1 of tail vein blood. After centrifugation, 10 ~1 of plasma was analyzed using the glucose oxidase method on a Glucose Analyzer 2 (Beckman Instruments, Inc., Brea, CA). Insulin-Treated
Animals
On the day of tumor inoculation, 10 days following induction of diabetes, hyperglycemia was documented and treatment was started with insulin (Humulin, Eli Lilly 8z Co., Indianapolis, IN). Animals were treated at 12-hr intervals with 5 units of insulin delivered subcutaneously. Treatment continued through the morning dose of the day of glucose uptake and tumor analysis. Tumor Cell Insulin
Receptor Analysis
Animals were euthanized, tumor was completely excised, and following preparation of a single cell suspension of the tumor cells an assay of insulin receptors was per-
VOL. 48, NO. 5, MAY
1990
formed. Tumor cells (3 X lo6 cells per milliliter) were incubated for 90 min at 15“C, pH 7.8, with 2.5 X lo4 cpm of high-pressure liquid chromatography-purified Al4 1251insulin and increasing concentrations of unlabeled porcine insulin (O-lo5 rig/ml). At 90 min, duplicate aliquots of the cell suspension were washed and centrifuged twice in cold assay buffer. The remaining cellular pellet was counted in a gamma counter (Gamma 300, Beckman Instruments, Inc., Irvine, CA). Specific binding and insulin receptor number were calculated. Insulin degradation was less than 6%. Mean cell viability was 78 ? 1.32%. Glucose Uptake Studies After tumor growth to the designated volume (less than or equal to 20 cc and greater than 20 cc), animals were injected intraperitoneally with 0.5 &i of 3-0-[14C]methylglucose with a sp act of 10 mCi/mole (New England Nuclear, Boston, MA) in 0.5 cc of normal saline. Following a 2-hr incubation period, the animals were sacrificed and tumor, muscle, liver, and blood samples were collected. Blood was immediately centrifuged and a lOO-~1 aliquot of plasma was reserved for analysis. Tissue samples were rinsed in saline, weighed, and frozen in liquid nitrogen. After thawing, the tissues were homogenized in 50 mmol Tris buffer (pH 7.4) and precipitated with perchloric acid (final concentration 5%). After centrifugation the supernatant was neutralized (pH 7) and an aliquot (0.5 ml) was measured for the presence of 14C-labeled 3-O-methylglucose (LS 9800, Beckman Instruments, Irvine, CA). The rate of incorporation was calculated as disintegrations per minute per gram tissue. Quench curve evaluation showed efficiency of the counter at 68%. Insulin levels in plasma were determined by radioimmunoassay technique using rat insulin as a standard (INCSTAR Corp., Stillwater, MN). Labeled glucose levels in plasma were recorded as disintegrations per minute per milliliter plasma. Statistical
Evaluation
Comparison between and among treatment groups was performed using a Brown-Forsythe analysis of variance (variance not assumed equal). The Bonferroni correction for multiple comparisons was performed to reduce the risk of incorrectly identifying a significant difference due to the multiple comparisons. Differences were accepted as significant when corresponding P value was <0.05. RESULTS Tumor volume at the time of sacrifice was predetermined by group. Therefore, body weight and tumor index (tumor volume/[body weight - tumor volume]) were used to evaluate relative growth of host and tumor in the diabetic and normal environments. For tumors < 20 cc, the NDNTB and NDTB weighed an average of 268.2 and 276.9 g, respectively, whereas the DNTB and DTB
WAGMAN
TABLE Body
NDNTB NDTB DNTB DTB
Weight
AND
METZ:
INSULIN
RECEPTORS
Index
Tumor volume
n
Body weight
<20 >20 <20 >20 <20 >20 <20 220
2 2 7 11 2 2 14 12
268.3 293.1 276.9 305.3 197.1 189.8 193.2 228.1
Tumor index
+ 1.8 2~ 2.8 ?z 5.3 t 27.4 4 18.5 + 0.9 + 25.1 + 48.6
0.06 f 0.02 0.18 zk 0.09 0.08 f 0.02 0.03 f 0.11
Note. ND, nondiabetic; D, diabetic; NTB, nontumor bearer; TB, tumor bearer. Tumor volume reported in cubic centimeters. n, number of animals in each group. Body weight reported in grams. Data presented as the means + the standard error of the mean.
weighed 197.1 and 193.2 g, respectively. The presence of tumor did not significantly change body weight (P = 0.96). However, the diabetic state resulted in a significant reduction in body weight (P = 0.007). For tumors > 20 cc, the NDNTB and NDTB weighted an average of 293.1 and 306.2 g, respectively. The DNTB and DTB weighed 189.8 and 228.1 g, respectively. In this category, both the diabetic state and the tumor-bearing state were associated independently with significant changes in body weight (P < 0.0001 and P = 0.006, respectively) (Table 1). Tumor index functions as a measure of tumor burdenthe fraction of the total body weight made up by tumor. Because tumor volume correlates closely with tumor weight, these measurements can be used interchangeably [l, 51. The calculated tumor index for NDTB and DTB was 0.055 and 0.083 respectively for tumors < 20 cc. In the larger tumor group (>20 cc), the tumor index was 0.177 and 0.308 for NDTB and DTB, respectively. The differences were significant when analyzed for tumor volume or diabetic status. At both tumor volumes, the tumor burden was greater for the diabetic animals.
TABLE Experimental
NDTB DTB DTBRx
Tumor volume <20 >20 <20 220 <20 >20
TUMOR
437
GROWTH
The serum glucose was significantly increased (P < 0.001) comparing diabetic to nondiabetic animals regardless of whether or not they were tumor bearing. Diabetic, tumor-bearing animals had persistent hyperglycemia and hypoinsulinemia throughout the experiment. Diabetic tumor-bearing animals treated with twice daily insulin injections had normal to low glucose levels and a lower body weight (~20 cc 194.6 g; >20 cc 208.5 mg/dl) compared to nondiabetic tumor bearers. These findings were consistent at both tumor volumes (Table 2). Tumor surface insulin receptor binding and receptor number were analyzed in the diabetic and nondiabetic groups at ~20 cc and >20 cc TV. Specific binding was significantly greater comparing DTB < 20 cc vs NDTB < 20 cc and DTB < 20 cc vs >20 cc. There was no difference at the two tumor sizes for ND animals or comparing larger tumors in D vs ND groups. The significant increase in specific binding in small tumors from diabetic animals was not present in the larger diabetic or nondiabetic animals. Insulin-treated diabetic tumor-bearing animals had similar receptor number and affinity to nondiabetic animals (Table 2). Insulin receptor number (calculated from Scatchard analysis) was 8.3 and 27.8 (X105) sites per cell in the ~20 cc and >20 cc tumors from nondiabetic animals. Diabetic, tumor bearers had receptor numbers of 28.7 X lo5 sites per cell at TV < 20 cc and 82.8 X lo5 sites per cell at TV > 20 cc. Significant increase in sites per cell were recorded for both tumor sizes of the D vs ND animals. Within the groups (D and ND) the increase in receptors was significant comparing small and large tumors (D 28.7 vs 82.8, ND 8.3 vs 27.8) (Figs. 1 and 2). Diabetic animals treated with insulin had receptor numbers in the range of nondiabetic animals. Glucose uptake was measured in diabetic and normal animals at the two tumor volumes. There was a significant decrease in glucose uptake in diabetic vs nondiabetic animals at <20 cc TV (258.1 (D) vs 623.8 (ND), P < 0.05). No significant difference was identified at the larger tumor
1
and Tumor
AND
2
Characteristics
n
Serum glucose
Serum insulin
7 11 14 12 3 2
157.4 153.9 519.4 519.1 147.3 79.5
7.37 4.27 0.72 0.29 WA N/A
Percentage specific binding 0.278 0.270 0.377 0.310 0.278 0.291
f f f + + f
0.070 0.060 0.060 0.060 0.040 0.040
Tumor glucose uptake 623.8 562.0 258.1 350.3 466.3 912.2
f 72.0 + 112.0 + 118.0 2 72.0 + 7.4 f 22.0
Insulin receptor number 8.3 27.8 28.7 82.8 7.7 16.9
Note. NDTB, nondiabetic, tumor bearer; DTB, diabetic, tumor bearer; DTBRx, insulin-treated DTB. Tumor volumes reported in cubic centimeters; n, number of animals per group; serum glucose in milligrams per deciliter; serum insulin in nanograms per milliliter, tumor glucose uptake in milligrams glucose per gram tumor. Insulin receptor number reported as number of receptors per cell times 10’. N/A, not assayed. Data presented as the means * the standard error of the mean.
438
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SOUND (nM1
FIG. 1. Scatchard analysis of insulin receptor binding data for nondiabetic, tumor-bearing animals. x axis, bound insulin in nanomoles; y axis, bound to free insulin ratio.
volume. Diabetic animals treated with insulin had significantly increased glucose uptake in the tumor compared to the tumor of diabetic animals at ~20 cc and >20 cc tumor volume. Of interest, diabetic treated animals in the larger tumor group had increased glucose uptake compared to the diabetic and nondiabetic animals. In muscle, the uptake was similar for diabetic and nondiabetic animals. Treatment with twice daily doses of subcutaneous injections of regular insulin resulted in an increase of glucose uptake at the larger tumor size compared to the diabetic animals. DISCUSSION
Growth of normal and neoplastic tissue requires adequate amount of available substrates. The MCA sarcoma has been shown to require glucose for growth [3]. Because diabetes has been associated with decrease in glucose uptake, presumably due to the absence of insulin, a model of hypoinsulinemia was used to study the interaction of substrate availability and tumor growth. Previous studies showed a biphasic growth pattern of the MCA sarcoma with a delay in the early phase (O-20 cc tumor volume) of tumor growth. The latter phase of tumor growth (2060 cc tumor volume) was parallel in normal and diabetic animals [ 11. Previous study of the insulin receptor from the MCA sarcoma had shown an increase number just prior to the animal’s death from tumor; but, no correlation of receptor number and growth had been performed [2]. It was hypothesized that in response to the hypoinsulinemia of streptozocin-induced diabetes, the number of receptors on the tumor cell surface would be “up-regulated” allowing for equivalent glucose uptake despite the low insulin levels. This receptor up-regulation would lead to increase substrate uptake correlating with the similarity of the latter growth pattern overcoming the delayed early growth due to substrate deprivation. In this set of experiments, glucose was chosen as the “substrate.” Although insulin is known to enhance amino acid as well as glucose uptake, the previous work of Sloan [2], Kern [6], and Moley [7] provided documentation of the specific interaction of the MCA sarcoma and glucose.
VOL. 48, NO. 5, MAY
1990
Tumor growth patterns, animal body weight changes, and glucose and insulin levels were consistent in this experiment based on published reports [l, 21. Sacrifice of animals was based on tumor volume, not the time period from day of inoculation. Variation may have been introduced by unequal distribution in tumor size within the two groups for analysis of IR and specific binding. Analysis of mean TV for the D versus ND for 2 vol of tumor showed no statistically significant difference. Insulin receptor analysis on smaller and larger tumors from diabetic animals documented changes anticipated with hypoinsulinemia, i.e., up-regulation of IR number. Up-regulation is the increase in receptor number in the absence of the specific receptor ligand. The up-regulation was consistent with the insulin levels at the two tumor volumes. Of interest, was the relative increase in receptor number with growth of the tumor. This has not been described for other tumor models. Because most analyses have been performed on completely differentiated cells (red blood cells, lymphocytes, skeletal muscle, mature hepatocytes) this phenomena of increased receptor number with cell (tissue) growth in tumors may have been overlooked. Changes similar to those seen in this experiment may be expected in regenerating hepatocytes following resection of a portion of liver or after tumor debulking. If the number of insulin receptors was directly proportional to the uptake of glucose, then the diabetic animals should have had an initial increase in glucose uptake compared to nondiabetics. This was not demonstrated. In fact, the glucose uptake was significantly lower in the diabetic animals at both tumor volumes. When diabetic animals were treated with insulin (as outlined under Materials and Methods), glucose uptake increased compared to the diabetic group at both tumor volumes. This would suggest that the increase in number of insulin receptors did not augment glucose uptake and that the presence of insulin was required for glucose transport across the tumor cell membrane. A report by Goto et al. [8] showed improvement in the uptake of glucose in soleus muscles of streptozocin diabetic mice that were treated with insulin. A similar finding was noted for the diabetic animals at the lower tumor volume. In as much as the MCA sarcoma has its purported derivation from fibroblasts or muscle
Ai2O.x. r>2Qcc.
287 I 10’ wks/csll 828x ti sites/cell
BOUND
hM)
FIG. 2. Scatchard analysis of insulin receptor binding data for diabetic tumor-bearing animals. z axis, bound insulin in nanomoles; y axis, bound to free insulin ratio.
WAGMAN
AND
METZ:
INSULIN
RECEPTORS
cells, this pattern of glucose uptake enhancement with insulin was physiologically consistent. Previous reported changes in growth cannot be explained on the basis of receptor number. In the two groups (diabetic and nondiabetic), the ratio of insulin receptors at ~20 cc to >20 cc was 0.35 and 0.30, respectively. Thus, no relative increase in receptor number was identified to be causing the latter growth effects. In addition, the specific binding in the larger volume tumors in diabetic animals was less than the early phase. An enhancement of specific binding would be necessary to explain the change in growth pattern. This is consistent with current theory describing large amounts of excess insulin receptors on the surface of cells. It has been suggested that only approximately 3% of the insulin receptor occupancy is required for the maximal biologic action of insulin. Considering the reversal of the glucose uptake deficit when the diabetic animals were treated with twice daily doses of insulin, the hypoinsulinemia of streptozocin-induced diabetes appears to be the significant metabolic aberration in this tumor system. In summary, these experiments show parallel increases in insulin receptor numbers during the growth of MCA sarcomas regardless of diabetic status. Although there was a decrease in specific insulin binding (decreased affinity) to the receptors on >20 cc tumors of diabetic rats, no significant change in glucose uptake was noted. The explanation for the early phase growth delay in diabetic
AND
TUMOR
GROWTH
439
animals cannot be explained on the basis of insulin receptor number. Substrate deprivation due to hypoinsulinemia remains the most likely explanation for the early phase growth delay in the tumors of diabetic animals. REFERENCES 1.
Wagman, L. D., and Brennan, M. F. The effects of streptozocininduced diabetes and weight-matched pain feeding on tumor growth and survival in Fisher rats. J. Surg. Res. 36: 354, 1984. 2. Sloan, G. M., Harrison, L. C., Underhill, L. H., and Brennan, M. F. Inhibition of tumor size by streptozocin-induced diabetes mellitus. J. Surg. Res. 301: 463, 1981. 3. Kern, K. A., and Norton, J. A. Inhibition of established rat fibrosarcoma growth by the glucose antagonist 2-deoxy-D-glucose. Surgery 102:(2) 380,1987. 4. Cherrington, A. D., and Steiner, K. E. The effects of insulin on carbohydrate metabolism in-vivo. Clin. Endocrinol. Metub. 1 l:(2) 307,1982. 5. Reilly, J. J., Goodgame, J. T., Jones, D. C., and Brennan, M. F. DNA synthesis in rat sarcoma and liver: The effect of starvation. J. Surg. Res. 22: 281, 1977. imaging of rat sar6. Kern, K., and Norton, J. A. Autoradiographic coma in different anatomical sites using 2-[“Cl-deoxyglucose. Cancer Res. 47: 4706, 1987. 7. Moley, J. F., Morrison, S. D., and Norton, J. A. Insulin reversal of cancer cachexia in rats. Cancer Res. 45: 4925, 1985. 8. Goto, Y., Kida, K., Kaino, I., Inove, T., Ikeuchi, M., Miyagawa, T., and Matsuda, H. Insulin action on glucose uptake by soleus muscles of nonobese diabetic mice and streptozocin diabetic mice. Metabolism 37:(l) 74, 1988.
OF SURGICAL
RESEARCH
48, 435-439 (19%))
Changes in Insulin Receptors on Methylcholanthrene-Induced Sarcoma during Growth’ LAWRENCE Division
D. WAGMAN, M.D., FACS, AND MARIANNE Z. METZ, B.A.
of Oncologic Surgery, City of Hope National
Presented at the Annual Meeting of the Association
Medical Center, 1500 East Dunrte Road, Duarte, California for Academic Surgery, Louisville,
Methylcholanthrene-induced sarcomas (MCA-S) have different growth patterns in diabetic (D) and nondiabetic (ND) rats. Diabetes delays the early phase of tumor growth and prolongs survival. This study evaluated MCA-S growth and its relation to insulin receptors (IR) and glucose uptake. Fisher 344 rats 150-200 g were assigned to two groups: Diabetic tumor bearers (DTB, n = 26) and nondiabetic tumor bearers (NDTB, n = 18). Diabetes was induced with iv streptozocin (40 mg/kg); MCA-S was inoculated (1 X 10’ cells) subcutaneously 10 days later. Animals were sacrificed during early growth (tumor volume 6 20 cc) or logarithmic growth (tumor volume > 20 cc). IR assay was performed (O-lo6 rig/ml cold insulin, 25 X lo3 cpm/tube Al4 “‘I-insulin, 90 min, 15”C, pH 7.8) on a single cell preparation. Serum glucose milligrams per deciliter and insulin nanograms per milliliter were assayed. Glucose uptake (dpm/g tissue/hr) was assayed 2 hr after an ip injection of 0.5 PCi 30[‘4C]methylglucose. Diabetic, tumor-bearing animals had a significantly increased number of insulin receptors at the small [<20 cc, 28.7 (D) vs 8.3 (ND)] and large [>20 cc, 82.8 (D) vs 27.8 (ND) ] tumor volumes. Glucose uptake was increased in the tumor at both volumes in the nondiabetic animals compared to the diabetic animals. The increase in insulin receptor number on the surface of the tumors of diabetic animals did not result in an increase in glucose uptake. Substrate (glucose) deprivation due to hypoinsulinemia remains the most likely explanation for the early phase growth delay in the tumors of diabetic animals. 0 1990 Academic Press. Inc.
The methylcholanthrene-induced sarcoma, grown in Fischer rats, has different growth patterns (changes in rate of linear and exponential phases) in streptozocininduced diabetic and normal animals [ 11. Previous reports have shown a relative increase in insulin receptor number with a relative decrease in receptor affinity when com1 Supported in part by an American Cancer Society Career Development Award (Lawrence D. Wagman, M.D.). 435
November
15-18, 1989
paring tumors from diabetic (D) and nondiabetic (ND) animals at the time of their death [2]. Analysis of glucose uptake with nonmetabolized glucose analogues (2-deoxyD-glUCOSE!) has demonstrated rapid uptake and retention in this tumor at levels exceeding liver, brain, and muscle [3]. In some tissues, insulin enhances the uptake of glucose by interaction with its receptor [4]. This series of experiments sought to answer the following questions: (1) Do changes in tumor growth rates correlate with changes in tumor insulin receptor number and/or affinity? (2) Do changes in tumor insulin receptor number and/or affinity correlate with the uptake of glucose by the tumor; and (3) Will tumor insulin receptor number and glucose uptake be altered if diabetic, tumor-bearing animals are treated with insulin? MATERIALS
AND METHODS
Animal Model Adult male 150- to 180-g Fischer 344 rats were obtained from Charles River Breeding Co. (Kingston, NY). This rat model has been previously shown to support reliable, consistent growth of a methylcholanthrene-induced sarcoma and tolerate diabetes. Animals were housed two per cage and fed rat chow (Wayne Pet Food Division, Continental Grain Co., Chicago, IL) and water ad Eibitum. Animals were weighed at the initiation of the study and every 3 days. Approval for animal use was obtained from the City of Hope Research Animal Care Committee. Experimental
INTRODUCTION
Kentucky,
91010
Groups
Seventy animals were assigned to one of four experimental groups: nondiabetic, nontumor bearer (NDNTB: n = 4); nondiabetic, tumor bearer (NDTB: ~20 cc tumor volume (TV), n = 7; ~20 cc TV n = 11); diabetic, nontumor bearer (DNTB: n = 4); and diabetic, tumor bearer (DTB: ~20 cc TV, n = 14; >20 cc, TV, n = 12). A separate group of diabetic, tumor-bearing animals was treated with insulin (~20 cc TV, n = 3; >20 cc TV, n = 2). Control (nontumor-bearing) animals were matched by experimental day (day of sacrifice based on tumor volume) to 0022-4804/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
436
JOURNAL
OF SURGICAL
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the tumor-bearing animals for comparisons of weight, glucose uptake, and serum glucose and insulin. The nontumor-bearing animals were subsequently referred to by tumor volume (<20 cc or >20 cc) for various comparisons. This method of pairing allowed chronologic-dependent comparisons to be made at equivalent times after initiation of the experiment. Tumor Model A methylcholanthrene-induced sarcoma (MCA-S) was inoculated on Day 0 of the experiment in the left flank at 1 X lo6 cells per milliliter dose. The tumor was grown in carrier animals and prepared as a single cell suspension. Experimental animals had tumor size measured in millimeters and recorded every 1 to 3 days. Tumor volume was calculated using the formula for a prolate spheroid: Volume = (4/3)A*B, where A = short axis and B = long axis. Tumor was weighed at the time of excision. Tumor index, a measure of tumor burden, was calculated. [Tumor volume/(body weight-tumor volume).] After the animals were euthanized with Halothane (Halocarbon Lab., Inc., Hackensack, NJ), the tumor was removed, rinsed with 0.5% collagenase (Worthington Biochemical Corp., Freehold, NJ), and incubated at 37°C for 30 min. The supernatant was removed and the remaining tumor passed through a stainless-steel wire mesh (l-mm mesh). Red blood cells were lysed, and the cells were washed and resuspended in cold assay buffer with 0.5% bovine serum albumin at pH 7.8. Tumor cell viability was assessed with the trypan blue exclusion technique. Diabetes Induction Under general anesthesia, 40 mg/kg of streptozocin (Upjohn Co., Kalamazoo, MI), was injected via the dorsal vein of the penis on Day -10 of the experimental protocol. Control animals were anesthetized and tagged. Hyperglycemia was documented prior to Day 0 by collection of 50 ~1 of tail vein blood. After centrifugation, 10 ~1 of plasma was analyzed using the glucose oxidase method on a Glucose Analyzer 2 (Beckman Instruments, Inc., Brea, CA). Insulin-Treated
Animals
On the day of tumor inoculation, 10 days following induction of diabetes, hyperglycemia was documented and treatment was started with insulin (Humulin, Eli Lilly 8z Co., Indianapolis, IN). Animals were treated at 12-hr intervals with 5 units of insulin delivered subcutaneously. Treatment continued through the morning dose of the day of glucose uptake and tumor analysis. Tumor Cell Insulin
Receptor Analysis
Animals were euthanized, tumor was completely excised, and following preparation of a single cell suspension of the tumor cells an assay of insulin receptors was per-
VOL. 48, NO. 5, MAY
1990
formed. Tumor cells (3 X lo6 cells per milliliter) were incubated for 90 min at 15“C, pH 7.8, with 2.5 X lo4 cpm of high-pressure liquid chromatography-purified Al4 1251insulin and increasing concentrations of unlabeled porcine insulin (O-lo5 rig/ml). At 90 min, duplicate aliquots of the cell suspension were washed and centrifuged twice in cold assay buffer. The remaining cellular pellet was counted in a gamma counter (Gamma 300, Beckman Instruments, Inc., Irvine, CA). Specific binding and insulin receptor number were calculated. Insulin degradation was less than 6%. Mean cell viability was 78 ? 1.32%. Glucose Uptake Studies After tumor growth to the designated volume (less than or equal to 20 cc and greater than 20 cc), animals were injected intraperitoneally with 0.5 &i of 3-0-[14C]methylglucose with a sp act of 10 mCi/mole (New England Nuclear, Boston, MA) in 0.5 cc of normal saline. Following a 2-hr incubation period, the animals were sacrificed and tumor, muscle, liver, and blood samples were collected. Blood was immediately centrifuged and a lOO-~1 aliquot of plasma was reserved for analysis. Tissue samples were rinsed in saline, weighed, and frozen in liquid nitrogen. After thawing, the tissues were homogenized in 50 mmol Tris buffer (pH 7.4) and precipitated with perchloric acid (final concentration 5%). After centrifugation the supernatant was neutralized (pH 7) and an aliquot (0.5 ml) was measured for the presence of 14C-labeled 3-O-methylglucose (LS 9800, Beckman Instruments, Irvine, CA). The rate of incorporation was calculated as disintegrations per minute per gram tissue. Quench curve evaluation showed efficiency of the counter at 68%. Insulin levels in plasma were determined by radioimmunoassay technique using rat insulin as a standard (INCSTAR Corp., Stillwater, MN). Labeled glucose levels in plasma were recorded as disintegrations per minute per milliliter plasma. Statistical
Evaluation
Comparison between and among treatment groups was performed using a Brown-Forsythe analysis of variance (variance not assumed equal). The Bonferroni correction for multiple comparisons was performed to reduce the risk of incorrectly identifying a significant difference due to the multiple comparisons. Differences were accepted as significant when corresponding P value was <0.05. RESULTS Tumor volume at the time of sacrifice was predetermined by group. Therefore, body weight and tumor index (tumor volume/[body weight - tumor volume]) were used to evaluate relative growth of host and tumor in the diabetic and normal environments. For tumors < 20 cc, the NDNTB and NDTB weighed an average of 268.2 and 276.9 g, respectively, whereas the DNTB and DTB
WAGMAN
TABLE Body
NDNTB NDTB DNTB DTB
Weight
AND
METZ:
INSULIN
RECEPTORS
Index
Tumor volume
n
Body weight
<20 >20 <20 >20 <20 >20 <20 220
2 2 7 11 2 2 14 12
268.3 293.1 276.9 305.3 197.1 189.8 193.2 228.1
Tumor index
+ 1.8 2~ 2.8 ?z 5.3 t 27.4 4 18.5 + 0.9 + 25.1 + 48.6
0.06 f 0.02 0.18 zk 0.09 0.08 f 0.02 0.03 f 0.11
Note. ND, nondiabetic; D, diabetic; NTB, nontumor bearer; TB, tumor bearer. Tumor volume reported in cubic centimeters. n, number of animals in each group. Body weight reported in grams. Data presented as the means + the standard error of the mean.
weighed 197.1 and 193.2 g, respectively. The presence of tumor did not significantly change body weight (P = 0.96). However, the diabetic state resulted in a significant reduction in body weight (P = 0.007). For tumors > 20 cc, the NDNTB and NDTB weighted an average of 293.1 and 306.2 g, respectively. The DNTB and DTB weighed 189.8 and 228.1 g, respectively. In this category, both the diabetic state and the tumor-bearing state were associated independently with significant changes in body weight (P < 0.0001 and P = 0.006, respectively) (Table 1). Tumor index functions as a measure of tumor burdenthe fraction of the total body weight made up by tumor. Because tumor volume correlates closely with tumor weight, these measurements can be used interchangeably [l, 51. The calculated tumor index for NDTB and DTB was 0.055 and 0.083 respectively for tumors < 20 cc. In the larger tumor group (>20 cc), the tumor index was 0.177 and 0.308 for NDTB and DTB, respectively. The differences were significant when analyzed for tumor volume or diabetic status. At both tumor volumes, the tumor burden was greater for the diabetic animals.
TABLE Experimental
NDTB DTB DTBRx
Tumor volume <20 >20 <20 220 <20 >20
TUMOR
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GROWTH
The serum glucose was significantly increased (P < 0.001) comparing diabetic to nondiabetic animals regardless of whether or not they were tumor bearing. Diabetic, tumor-bearing animals had persistent hyperglycemia and hypoinsulinemia throughout the experiment. Diabetic tumor-bearing animals treated with twice daily insulin injections had normal to low glucose levels and a lower body weight (~20 cc 194.6 g; >20 cc 208.5 mg/dl) compared to nondiabetic tumor bearers. These findings were consistent at both tumor volumes (Table 2). Tumor surface insulin receptor binding and receptor number were analyzed in the diabetic and nondiabetic groups at ~20 cc and >20 cc TV. Specific binding was significantly greater comparing DTB < 20 cc vs NDTB < 20 cc and DTB < 20 cc vs >20 cc. There was no difference at the two tumor sizes for ND animals or comparing larger tumors in D vs ND groups. The significant increase in specific binding in small tumors from diabetic animals was not present in the larger diabetic or nondiabetic animals. Insulin-treated diabetic tumor-bearing animals had similar receptor number and affinity to nondiabetic animals (Table 2). Insulin receptor number (calculated from Scatchard analysis) was 8.3 and 27.8 (X105) sites per cell in the ~20 cc and >20 cc tumors from nondiabetic animals. Diabetic, tumor bearers had receptor numbers of 28.7 X lo5 sites per cell at TV < 20 cc and 82.8 X lo5 sites per cell at TV > 20 cc. Significant increase in sites per cell were recorded for both tumor sizes of the D vs ND animals. Within the groups (D and ND) the increase in receptors was significant comparing small and large tumors (D 28.7 vs 82.8, ND 8.3 vs 27.8) (Figs. 1 and 2). Diabetic animals treated with insulin had receptor numbers in the range of nondiabetic animals. Glucose uptake was measured in diabetic and normal animals at the two tumor volumes. There was a significant decrease in glucose uptake in diabetic vs nondiabetic animals at <20 cc TV (258.1 (D) vs 623.8 (ND), P < 0.05). No significant difference was identified at the larger tumor
1
and Tumor
AND
2
Characteristics
n
Serum glucose
Serum insulin
7 11 14 12 3 2
157.4 153.9 519.4 519.1 147.3 79.5
7.37 4.27 0.72 0.29 WA N/A
Percentage specific binding 0.278 0.270 0.377 0.310 0.278 0.291
f f f + + f
0.070 0.060 0.060 0.060 0.040 0.040
Tumor glucose uptake 623.8 562.0 258.1 350.3 466.3 912.2
f 72.0 + 112.0 + 118.0 2 72.0 + 7.4 f 22.0
Insulin receptor number 8.3 27.8 28.7 82.8 7.7 16.9
Note. NDTB, nondiabetic, tumor bearer; DTB, diabetic, tumor bearer; DTBRx, insulin-treated DTB. Tumor volumes reported in cubic centimeters; n, number of animals per group; serum glucose in milligrams per deciliter; serum insulin in nanograms per milliliter, tumor glucose uptake in milligrams glucose per gram tumor. Insulin receptor number reported as number of receptors per cell times 10’. N/A, not assayed. Data presented as the means * the standard error of the mean.
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OF SURGICAL
RESEARCH:
SOUND (nM1
FIG. 1. Scatchard analysis of insulin receptor binding data for nondiabetic, tumor-bearing animals. x axis, bound insulin in nanomoles; y axis, bound to free insulin ratio.
volume. Diabetic animals treated with insulin had significantly increased glucose uptake in the tumor compared to the tumor of diabetic animals at ~20 cc and >20 cc tumor volume. Of interest, diabetic treated animals in the larger tumor group had increased glucose uptake compared to the diabetic and nondiabetic animals. In muscle, the uptake was similar for diabetic and nondiabetic animals. Treatment with twice daily doses of subcutaneous injections of regular insulin resulted in an increase of glucose uptake at the larger tumor size compared to the diabetic animals. DISCUSSION
Growth of normal and neoplastic tissue requires adequate amount of available substrates. The MCA sarcoma has been shown to require glucose for growth [3]. Because diabetes has been associated with decrease in glucose uptake, presumably due to the absence of insulin, a model of hypoinsulinemia was used to study the interaction of substrate availability and tumor growth. Previous studies showed a biphasic growth pattern of the MCA sarcoma with a delay in the early phase (O-20 cc tumor volume) of tumor growth. The latter phase of tumor growth (2060 cc tumor volume) was parallel in normal and diabetic animals [ 11. Previous study of the insulin receptor from the MCA sarcoma had shown an increase number just prior to the animal’s death from tumor; but, no correlation of receptor number and growth had been performed [2]. It was hypothesized that in response to the hypoinsulinemia of streptozocin-induced diabetes, the number of receptors on the tumor cell surface would be “up-regulated” allowing for equivalent glucose uptake despite the low insulin levels. This receptor up-regulation would lead to increase substrate uptake correlating with the similarity of the latter growth pattern overcoming the delayed early growth due to substrate deprivation. In this set of experiments, glucose was chosen as the “substrate.” Although insulin is known to enhance amino acid as well as glucose uptake, the previous work of Sloan [2], Kern [6], and Moley [7] provided documentation of the specific interaction of the MCA sarcoma and glucose.
VOL. 48, NO. 5, MAY
1990
Tumor growth patterns, animal body weight changes, and glucose and insulin levels were consistent in this experiment based on published reports [l, 21. Sacrifice of animals was based on tumor volume, not the time period from day of inoculation. Variation may have been introduced by unequal distribution in tumor size within the two groups for analysis of IR and specific binding. Analysis of mean TV for the D versus ND for 2 vol of tumor showed no statistically significant difference. Insulin receptor analysis on smaller and larger tumors from diabetic animals documented changes anticipated with hypoinsulinemia, i.e., up-regulation of IR number. Up-regulation is the increase in receptor number in the absence of the specific receptor ligand. The up-regulation was consistent with the insulin levels at the two tumor volumes. Of interest, was the relative increase in receptor number with growth of the tumor. This has not been described for other tumor models. Because most analyses have been performed on completely differentiated cells (red blood cells, lymphocytes, skeletal muscle, mature hepatocytes) this phenomena of increased receptor number with cell (tissue) growth in tumors may have been overlooked. Changes similar to those seen in this experiment may be expected in regenerating hepatocytes following resection of a portion of liver or after tumor debulking. If the number of insulin receptors was directly proportional to the uptake of glucose, then the diabetic animals should have had an initial increase in glucose uptake compared to nondiabetics. This was not demonstrated. In fact, the glucose uptake was significantly lower in the diabetic animals at both tumor volumes. When diabetic animals were treated with insulin (as outlined under Materials and Methods), glucose uptake increased compared to the diabetic group at both tumor volumes. This would suggest that the increase in number of insulin receptors did not augment glucose uptake and that the presence of insulin was required for glucose transport across the tumor cell membrane. A report by Goto et al. [8] showed improvement in the uptake of glucose in soleus muscles of streptozocin diabetic mice that were treated with insulin. A similar finding was noted for the diabetic animals at the lower tumor volume. In as much as the MCA sarcoma has its purported derivation from fibroblasts or muscle
Ai2O.x. r>2Qcc.
287 I 10’ wks/csll 828x ti sites/cell
BOUND
hM)
FIG. 2. Scatchard analysis of insulin receptor binding data for diabetic tumor-bearing animals. z axis, bound insulin in nanomoles; y axis, bound to free insulin ratio.
WAGMAN
AND
METZ:
INSULIN
RECEPTORS
cells, this pattern of glucose uptake enhancement with insulin was physiologically consistent. Previous reported changes in growth cannot be explained on the basis of receptor number. In the two groups (diabetic and nondiabetic), the ratio of insulin receptors at ~20 cc to >20 cc was 0.35 and 0.30, respectively. Thus, no relative increase in receptor number was identified to be causing the latter growth effects. In addition, the specific binding in the larger volume tumors in diabetic animals was less than the early phase. An enhancement of specific binding would be necessary to explain the change in growth pattern. This is consistent with current theory describing large amounts of excess insulin receptors on the surface of cells. It has been suggested that only approximately 3% of the insulin receptor occupancy is required for the maximal biologic action of insulin. Considering the reversal of the glucose uptake deficit when the diabetic animals were treated with twice daily doses of insulin, the hypoinsulinemia of streptozocin-induced diabetes appears to be the significant metabolic aberration in this tumor system. In summary, these experiments show parallel increases in insulin receptor numbers during the growth of MCA sarcomas regardless of diabetic status. Although there was a decrease in specific insulin binding (decreased affinity) to the receptors on >20 cc tumors of diabetic rats, no significant change in glucose uptake was noted. The explanation for the early phase growth delay in diabetic
AND
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GROWTH
439
animals cannot be explained on the basis of insulin receptor number. Substrate deprivation due to hypoinsulinemia remains the most likely explanation for the early phase growth delay in the tumors of diabetic animals. REFERENCES 1.
Wagman, L. D., and Brennan, M. F. The effects of streptozocininduced diabetes and weight-matched pain feeding on tumor growth and survival in Fisher rats. J. Surg. Res. 36: 354, 1984. 2. Sloan, G. M., Harrison, L. C., Underhill, L. H., and Brennan, M. F. Inhibition of tumor size by streptozocin-induced diabetes mellitus. J. Surg. Res. 301: 463, 1981. 3. Kern, K. A., and Norton, J. A. Inhibition of established rat fibrosarcoma growth by the glucose antagonist 2-deoxy-D-glucose. Surgery 102:(2) 380,1987. 4. Cherrington, A. D., and Steiner, K. E. The effects of insulin on carbohydrate metabolism in-vivo. Clin. Endocrinol. Metub. 1 l:(2) 307,1982. 5. Reilly, J. J., Goodgame, J. T., Jones, D. C., and Brennan, M. F. DNA synthesis in rat sarcoma and liver: The effect of starvation. J. Surg. Res. 22: 281, 1977. imaging of rat sar6. Kern, K., and Norton, J. A. Autoradiographic coma in different anatomical sites using 2-[“Cl-deoxyglucose. Cancer Res. 47: 4706, 1987. 7. Moley, J. F., Morrison, S. D., and Norton, J. A. Insulin reversal of cancer cachexia in rats. Cancer Res. 45: 4925, 1985. 8. Goto, Y., Kida, K., Kaino, I., Inove, T., Ikeuchi, M., Miyagawa, T., and Matsuda, H. Insulin action on glucose uptake by soleus muscles of nonobese diabetic mice and streptozocin diabetic mice. Metabolism 37:(l) 74, 1988.