- Email: [email protected]
Interorgan glutamine metabolism in the tumor-bearing rat
JOURNAL
OF SURGICAL
Interorgan
RESEARCH
4,
720-726 (1988)
Glutamine Metabolism
in the Tumor-Bearing
Rat
WILEY W. SOUBA, M.D., Sc.D., FREDERICK R. STREBEL, B.S., JOAN M. BULL, M.D., EDWARD M. COPELAND, M.D., HEINRICH TEAGTMEYER, M.D., PH.D., AND KAREN CLEARY, M.D. Department of Surgery and Biochemistry, University of Florida School of Medicine, Gainesville, Florida 32610, Departments of Surgery and Pathology, M. D. Anderson Hospital and Tumor Institute, Houston, Texas; and Department of Surgery and Medicine, University of Texas Medical School, Houston, Texas
Presentedat the Annual Meeting of the Association of Academic Surgery, Orlando, Florida, November l-4, 1987 The effectsof progressive malignant diseaseon gut/liver glutamine metabolism were studied in order to gain further insight into the altered glutamine metabolism that characterizes the host with cancer and to further elucidate the causes and consequences of glutamine depletion in tumor-bearing rats. Rats were inoculated on Day 0 with 2 X lo6 viable fibrosarcoma cells and blood glutamine was measured every 6 days. On Day 24 the animals underwent laparotomy and sampling of arterial, portal venous, and hepatic venous blood. Arterial glutamine fell by more than one-third in tumor-bearing rats and the arterial-portal venous concentration difference for glutamine across the intestinal tract was diminished by 50% (P i 0.01). Simultaneously the fractional extraction of glutamine by the gut was reduced from 2 1 to 15%(P < 0.05). The liver switched from an organ of near glutamine balance in control rats to one of marked glutamine output in tumor-beating rats (P < 0.01). The wet weight ofthe small intestine was diminished by 15% in tumor-bearing rats and villous height was uniformly decreasedin tumor-bearing rats with an average reduction in villous height of 26% (P < 0.05). The causes of glutamine depletion in this tumor-bearing rat model remain unclear. The growing tumor may behave as a glutamine trap but also appears to alter glutamine metabolism in vital metabolic processing centers such as the gut and liver. Malignant cells may compete with gut mucosal cells for glutamine resulting in a diminished gut glutamine utilization and detrimental changes in mucosal architecture. The altered glutamine metabolism that characterizes these tumor-bearing rats may be one component of cancer cachexia. 0 1988 Academic Press,Inc.
Cancer cachexia is commonly observed in patients with advanced malignant disease.The etiology of this syndrome of weight loss, anorexia, and negative nitrogen balance remains obscure [l-3]. While loss of appetite and diminished food intake are likely to contribute to the development of cancer cachexia, it is also likely that certain growing tumors alter normal host metabolism by behaving as a nitrogen trap or by regulating nitrogen flux via some, as yet undefined, distant metabolic effect.The accelerated muscle catabolism and loss of lean body tissue characteristic of many cancer patients may be a reflection of the malignant tumor’s ability to alter amino acid metabolism in vital metabolic processing centers such as the intestine and liver and change the balance of flow of amino acids from skeletal muscle to the splanchnic viscera. 0022-4804/88 $1.50 Copyright 0 1988 by Academic Press,Inc. All rights of reproduction in any form reserved.
Tumors and other rapidly replicating tissues such as the intestinal epithelium are major glutamine consumers [4-81. Glutamine is the most abundant amino acid in blood and tissues and comprises nearly twothirds of the free amino acid pool in skeletal muscle [9-l I]. Glutamine is the principal amino acid consumed by tumor cells and often servesas their principal respiratory fuel [5]. With progressive tumor growth, altered glutamine metabolism becomes a characteristic feature of the tumor-bearing host. In rats bearing large fast-growing malignancies, plasma and hepatocyte intracellular glutamine concentrations fall at a time when glutamine utilization by the tumor may be excessive [ 121. Studies on skeletal muscle glutamine metabolism in tumor-bearing rats demonstrate accelerated glutamine release
720
SOUBA
ET AL.: INTERORGAN
[ 131 and probable depletion of intracellular glutamine levels. The resultant depletion of tissue glutamine stores and persistent catabolism in skeletal muscle that characterize progressive malignant disease may be etiologic factors in the development of cancer cachexia. Previous studies have demonstrated that the altered skeletal muscle glutamine metabolism that characterizes catabolic states is intimately related to alterations in splanchnic glutamine metabolism [9, 11, 141.This study was designed to examine glutamine metabolism by the intestine and liver in tumorbearing rats to seewhether there are splanchnit alterations in glutamine metabolism that parallel and reflect the presumed changes observed in skeletal muscle. We hypothesized that as the tumor grows it alters normal interorgan glutamine relationships. We suggest that with advanced malignant disease the principal tissues of glutamine utilization “outconsume” the glutamine producers leading to a depletion of glutamine stores in blood and tissues and further tissue catabolism. The purpose of this study was to (a) compare arterial glutamine levels in pair-fed tumor-bearing and control rats, (b) compare the flux of glutamine across the intestine and liver in control and tumor-bearing rats, and (c) examine the consequences of glutamine depletion on small intestinal morphology. MATERIALS
AND METHODS
Selectionand Maintenance of Rats Female inbred Fischer 344 rats weighing 160- 170 g were utilized for the studies. The rats were obtained from a nearby breeder and maintained in standard cages (5 rats maximum/cage) in the animal laboratories. The rats were fed a standard laboratory rat chow and water ad libitum and subjected to alternate 12-hr periods of dark and light. Following tumor cell inoculation, control and study rats were pair-fed since previous studies have demonstrated that rats bearing relatively fast-growing tumors may have a depressedfood intake relative to ad libitum fed
GLUTAMINE
METABOLISM
721
nontumor-bearing rats [ 151.The daily ration of the pair-fed nontumor-bearing animals was the same as that amount of food consumed by the tumor-bearing animals on the preceding day.
Transplantation and Choice of the Fibrosarcoma Cell Line Eighteen rats were randomly selected to undergo either subcutaneous injection on the back with a single cell suspension of 2 X 1O6viable fibrosarcoma cells or to undergo sham injection with an identical volume of sterile saline. This number of cells resulted in the growth of a palpable 3- to 4-cm-diameter tumor in 3-4 weeks. This tumor model has been used by other investigators to study tumor-host metabolism alterations [ 161. This tumor cell line is locally aggressive,metastasizes rarely, and never regressesspontaneously. This cell line is a relatively fastgrowing tumor; faster growing tumors alter host amino acid metabolism significantly and may behave as a nitrogen trap [ 1, 17, 181.Glutamine utilization rates are high for such tumors and we have found the concentration of glutamine to be decreased in the blood of our tumor-bearing rats. Glutamine utilization by a number of tumors correlates with mitochondrial glutaminase activity [ 18-201 and glutamine supply to the tumor [15, 211. These observations suggest that early changes in intestinal and hepatic glutamine metabolism may be altered to a greater degree in fast-growing tumors.
Study Procedure Control and tumor rats were weighed on the day of tumor cell inoculation or saline injection (Day 0), placed in individual metabolic cages for paired-feeding, and weighed every 6 days thereafter. Tumor diameters were measured regularly. Arterial blood (0.5 cc) (by heart puncture under light ether anesthesia) was drawn on Days 6, 12 and 18 post tumor cell or saline injection and analyzed for whole blood glutamine.
722
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988
On Day 24 (post inoculation) control and tumor-bearing rats were anesthetized with ether and underwent laparotomy for blood sampling, tissue sampling and processing, and measurement of tumor weight. Blood (0.5 cc) was removed from the aorta, portal vein, and hepatic vein as described by Welboume et al. [22]. Briefly, the abdomen was opened, the sternum was gently elevated with a hemostat, and the central lobe of the liver was depressed, exposing the hepatic venous drainage of the middle hepatic lobe. This vein was carefully cannulated by directing a 26-gauge needle fitted to heparized Silastic tubing into the vein about 6-8 mm, which allows sampling of hepatic venous blood. The intestines were then gently displaced exposing the portal vein which was surrounded with a 5-O silk ligature. Flow to the liver was momentarily occluded and blood was sampled by cannulation of the portal vein with a 26-gauge needle fitted to Silastic tubing attached to a heparinized 1-cc syringe. Arterial blood was withdrawn directly from the abdominal aorta. All studies were performed in the postabsorptive state. Arteriovenous concentration differences were calculated from paired data, that is, the arterial and venous samples came from the same rat. Following blood sampling, the small intestine was removed, gently rinsed free of debris, and placed in 10% buffered formalin so multiple jejunal sections could later be prepared for morphometric (histologic) analysis. The tumor was removed from the flank of each tumor-bearing rat, measured, and weighed.
2. Morphometric analysis. Villous height was examined using the light microscope. 3. Tumor measurements. Maximum tumor diameters were measured on Days 6, 12, 18, and 24. Tumor weight was obtained on Day 24 when the animals were sacrificed. RESULTS
Relationship between Tumor Growth, Body Weight, Food Intake, and Jejunal Morphology
Changes in tumor size and weight, body weight, and wet weight of the small intestine are shown in Table 1. Food intake remained fairly constant (approximately 11 g/24 hr/ rat) until Day 14 post tumor implantation at which time there was a gradual decrease in food consumption to approximately 7 g/24 hr/rat on Day 24. This decrease in food intake in control rats explains the stabilization of body weight and failure to gain weight between Days 14 and 24. Body weight also remained stable in tumor-bearing rats, though by Day 24 the animals were beginning to show signs of cachexia (decreased hindquarter skeletal muscle mass on physical exam, thinning of abdominal wall musculature). The latter observation can be explained, at least in part, by the growing malignancy which by Day 24 demonstrated an averageweight of 15 g and measured approximately 3.5 X 3.5 cm. Thus, carcass weight (total body weight minus tumor weight) in the tumor-bearing rats was 158 f 3 g on Day 24, representing a 5% decrease in body weight. Control rats, on the other hand, demonstrated a 4% increase in body weight despite having to control food intake (P < 0.05). Analytical Procedures Wet weight of the small intestine (Table 1) 1. Whole blood glutamine. Heparinized and jejunal villous height were measured on whole blood was mixed with an equal vol- Day 24. Tumor-bearing rats had small intesume of 10% ice-cold perchloric acid, vor- tines that weighed significantly less than texed, and centrifuged. The supematant was those of the control animals (3.10 f 0.10 vs neutralized to pH 4.75 and stored at -70°C. 3.58 f 0.09 g, P < 0.05). Jejunal epithelial Glutamine was measured employing micro- morphology was examined by light microsfluorometric enzymatic assaysadapted from copy in six control and six tumor-bearing the method described by Bergmeyer [23]. rats. Villous height was reduced by an aver-
SOUBA ET AL.: INTERORGAN
GLUTAMINE
723
METABOLISM
TABLE 1 Days post tumor implantation
Control (n = 9) Gut weight (g) (small bowel) Body weight (g) Tumor (n = 9) Gut weight (g) (small bowel) Body weight (g) Approximate tumor size (mm X mm)
0
6
12
165 + 3
168 253
171 k 3
173k3
3.58 f 0.09 172+3
166 * 3
169k 3
172+3
173+2
3.10 * 0.10* 172 + 3
10 x 10
20 x 20
35 x 35 (15 g)
-
1x1
Body weight minus tumor weight
18
163 f 4
24
158 + 3*
Note. Data are expressed as means + SEM. * P K 0.05 compared to controls.
glutamine by the intestinal tract (A-PV concentration difference/arterial concentration) was also diminished in tumor-bearing rats (2 1% in controls vs 15% in tumor-bearing rats, P < 0.05). Arterial Glutamine Concentration Significant alterations in liver glutamine metabolism also occurred in the tumor rats. Arterial glutamine (GLN) concentration The arterial-hepatic venous (A-HV) concenshowed a slight decreasing but not statistitration difference was positive in controls but cally significant reduction in the control rats switched to slightly negative with tumor imover time (Table 2). Tumor-bearing rats, on plantation (78 f 17 vs -9 f 4 pmoles/liter, P the other hand, demonstrated a fall in whole < 0.01). The PV-HV concentration differblood glutamine over time which was signifience was significantly more negative in the cantly lower than control levels on Day 24 tumor group (-39 + 9 vs -67 f 11 pmoles/ (4 16 f 36 vs 58 1 + 32 pmoles/liter, P < 0.05, liter, P < 0.05). Table 2). age of 26% (P < 0.05) in tumor-bearing rats and was consistently diminished in all six TBR rats compared to controls. In addition, the villi were blunted and thickened.
Arteriovenous Concentration D@erences and Glutamine Flux on Day 24 There was a significant decreasein the arterial-portal venous (A-PV) concentration difference for glutamine in tumor-bearing rats (63 f 9 vs 120 f 10 pmoles/liter in controls, P < 0.01) (Table 3). This reduction in uptake by the small intestine occurred at a time when jejunal villous height and mucosal cellularity were reduced. Extraction of
DISCUSSION
Advanced malignant diseaseand other catabolic disease states are characterized by a reduction in tissue stores and plasma concentrations of glutamine [9, 10, 12, 131. In this research project we hypothesized that with progressive tumor growth circulating glutamine stores become depleted and alterations in gut/liver glutamine metabolism develop. Such was the case.
724
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988 TABLE 2 ARTERIAL GLUTAMINE CONCENTRATIONDAYS POSTTUMOR IMPLANTATION
Control Arterial GLN (rmol/l) Tumor Arterial GLN (pmole/liter)
6
12
18
24
647 2 17
636 + 32
578 f 38
581 f 32
652+31
605 + 26
523 + 21
416 + 36*
Note. Data are expressed as means + SEM; n = 6 in each group for Days 6, 12, 18; n = 9 in each group on Day 24. * P < 0.0 1 compared to controls.
The tumor cell line utilized in these studies, the methylcholanthrene-induced fibrosarcoma, has been used by others [ 161. Although it is a fairly rapidly growing tumor, it is representative of the rates of growth of some tumors observed in the clinical setting. This cell line was chosen for initial studies since previous studies have demonstrated that fast-growing tumors are avid glutamine consumers and alterations in glutamine metabolism develop in rats bearing these tumors. It was felt that these rapidly growing tumors would alter gut-liver glutamine relationships fairly quickly and, hence, be appropriate for initial investigations in the area of interorgan glutamine metabolism. Food intake was controlled with the daily ration of the pair-fed nontumor-bearing rats being equivalent to that amount of food consumed by the tumor-bearing animals on the preceding day. The modest decrease in arterial glutamine (10%) in control rats over
the 24-day period is most likely due to the diminished food intake. Partial starvation has been shown to result in decreased levels of circulating glutamine [lo, 241. The 36% fall in arterial glutamine in tumor-bearing rats by Day 24 supports the concept that the growing tumor contributes to a reduction in plasma glutamine. Whether this occurs from excessiveglutamine utilization by the tumor, alterations in glutamine synthetic pathways in other tissues, or some other mechanism is not clear from our study. Other studies demonstrate that as tumors grow they become significant glutamine consumers; the rate of glutamine utilization parallels the rate of tumor growth [6, 181. Eventually the arteriovenous concentration differences across gut and liver become altered (Table 3). The net result may be that the glutamine utilization is in excessof glutamine production resulting in glutamine depletion. Skeletal muscle glutamine release
TABLE 3 ARTERIOVENOUSCONCENTRATIONDIFFERENCES FORGLN (rmole/liter) ON DAY 24
Control (n = 9) Tumor (n = 9)
A
PV
HV
A-PV
A-HV
PV-HV
581 + 32 416 rf: 26**
458 + 23 348 + 18*
502 * 16 420 f 16*
121* 10 63 2 9**
7s* 17 -9 f 4**
-39 _+9 -67 f 1l**
Note. Data are expressed as means + SEM. A, arterial; PV, portal vein; HV, hepatic vein; A-PV, arterial-portal venous concentration difference; A-HV, arterial-hepatic venous concentration difference; PV-HV, portal venous-hepatic venous concentration difference; - = release; + = uptake. * P < 0.05 compared to controls. ** P < 0.01 compared to controls.
SOUBA ET AL.: INTERORGAN
is accelerated in the tumor-bearing rat [ 131as it is in other catabolic disease states [9, 1l] and intracellular stores and circulating levels of this key amino acid become depleted. In the current study, when blood glutamine levels fell, the liver switched to an organ of net glutamine release, a phenomenon we have observed in other catabolic states [9, 141. This may occur in part in an effort to normalize circulating levels of glutamine. This may be of significance since it has been demonstrated that the intracellular concentration of glutamine is a regulator of protein catabolism in skeletal muscle [ 111 and liver [25]. Low intracellular glutamine levels appear to mediate net protein catabolism in these tissues. In the nontumor-bearing animal, the mucosa of the small intestine is the principal site of glutamine utilization [8, 261. In the rat, approximately 20-30% of circulating glutamine is removed by the enterocytes with each pass through the intestinal vasculature [8]. Metabolism of glutamine by the intestinal tract is an integral aspect of the intestine’s role as a regulator of nitrogen metabolism in normal and catabolic states [9, 10, 14, 271. The growing tumor may “compete” with the small intestine for glutamine. Both tumor cells and gut mucosal cells demonstrate a striking similarity in substrate utilization and glutamine metabolism [ 15, 2 11. Abnormal intestinal glutamine metabolism (perhaps resulting from distant effects of the tumor) may result in a diminished utilization by glutamine by the enterocytes which may be linked to the alterations in intestinal morphology noted in the jejunum of the tumorbearing rats. Our findings in this study may be consistent with other studies which suggest that glutamine is essential for maintenance of normal intestinal structure and function. For example, in one study, the reduction of plasma glutamine concentrations to undetectable levels in monkeys and rabbits resulted in vomiting, diarrhea, mild villous atrophy, mucosal alterations, and intestinal necrosis [28]. In a study in rats, Okabe and colleagues [29] demonstrated that gluta-
GLUTAMINE
METABOLISM
725
mine protects against aspirin-induced gastric ulcerations. Others [3 I] have demonstrated that the provision of glutamine-enriched parenteral solutions to rats resulted in an increasein jejunal mucosal weight, DNA content, and villous height when compared to animals fed isonitrogenous glutamine-depleted intravenous diets. These studies suggest that the delivery of adequate amounts of glutamine from exogenous or endogenous sources may be a requirement for maintaining the integrity of the mucosa of the small intestine during periods of glutamine depletion. REFERENCES 1. Kawamura, I., Moldawer, L. L., Keenan, R. A., et al. Altered amino acid kinetics in rats with progressive tumor growth. Cancer Res. 4t: 824, 1982. 2. Bennegard, K., Lindmark, L., Eden, E., et al. Flux of amino acids acrossthe leg in weight-losing cancer patients. Cancer Rex 44: 386, 1984. 3. Popp, M. B., Morrison, S. D., and Brennan, M. F. Total parenteral nutrition in a methylcholanthreneinduced rat sarcoma model. Cancer Treat. Rep. 65: 137, 1981. 4. Coles, N. W., and Johnstone, R. M. Glutamine metabolism in ehrlich ascites-carcinoma cells. Biothem. J. 83: 284, 1962. 5. Kovacevic, Z. The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells. Biochem. J. 125: 757, 1971. 6. Kovacevic, Z., and Morris, H. P. The role of glutamine in the oxidative metabolism of malignant cells. Cancer Res. 32: 326, 1972. 7. Windmueller, H. G., and Spaeth, A. E. Uptake and metabolism of plasma glutamine by the small intestine. J. Biol. Chem. 249: 5070, 1974. 8. Windmueller, H. G. Glutamine utilization by the small intestine. Adv. Enzymol. 53: 202, 1982. 9. Souba, W. W., Smith, R. J., and Wilmore, D. W. Glutamine metabolism by the intestinal tract. JPEN 9(5): 608, 1985. 10. Souba, W. W. Glutamine Metabolism in Catabolic States: Role of the Intestinal Tract. Thesis, Department of Nutritional Biochemistry, Harvard School of Public Health, Cambridge, MA, 1984. 11. Muhlbacher, F., Kapadia, C. R., Colpoys, M. F., et al. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Amer. J. Physiol. 247: E75, 1984. 12. Wu, C., and Gauer, J. M. A study of free amino acids and of glutamine synthesis in tumor-bearing rats. Cancer Res. 20: 848, 1960. 13. Yamamoto, H., Aikawa, T., Matsutka, H., and
726
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988
Ishikawa, E. Relative uptake of plasma amino acids by fetal and tumor tissues. Metabolism 23: 1017, 1974. 14. Souba, W. W., Smith, R. J., and Wilmore, D. W. Effect of glucocotticoids on glutamine metabolism in visceral organs. Metabolism 34(5): 450, 1985. 15. Sauer, L. A., Stayman, J. W., III, and Dauchy, R. T. Amino acid, glucose and lactic acid utilization in vivo by rat tumors. Cancer Rex 42: 4090, 1982. 16. Popp, M. B., Waganer, S. C., and Brito, 0. J. Host and tumor responsesto increasing levels of intravenous nutritional support. Surgery 94: 300, 1983. 17. Carrascosa, J. M., Martinez, P., and decastro, I. N. Nitrogen movement between host and tumor in mice inoculated with ehrlich ascitic tumor cells. Cancer Res. 44: 3831, 1984. 18. Knox, W. E., Linder, M., and Friedell, G. H. A series of transplantable rat mammary tumors with graded differentiation, growth rate, and glutaminase content. Cancer Res. 30: 283, 1970. 19. Knox, W. E., Horowitc, M. L., and Friedell, G. H. The proportionality of glutaminase content to growth rate and morphology of rat neoplasma. Cancer Res. 29: 669, 1969.
20. Linder-Horowitz, M., Knox, W. E., and Morris, H. P. Glutaminase activities and growth rates of rat hepatomas. Cancer Res. 29: 1195, 1969. 21. Sauer, L. A., and Dauchy, R. T. Keton body, glucase, lactic acid, and amino acid utilization by tumors in vivo in fasted rats. Cancer Res. 43: 3497, 1983. 22. Welbourne, T. C., Childress, D., and Givens, G. Renal regulation of interorgan glutamine flow in
metabolic acidosis. Amer. J. Physiol. 251: R858, 1986. 23. Bergmeyer, H. U. (Ed.) Methods ofEnzymaticAna/ysis. New York: Academic Press, 1974. 24. Marl& E. B., Aoki, T. T., Pozefsky, T., et al. Muscle and splanchnic glutamine and glutamate metabolism in postabsorptive and starved man. J. Clin. Invest. 50: 814, 1971. 25. Schworer, C. M., and Mortimore, G. E. Glucagoninduced autophagy and proteolysis in rat liver: Mediation by selective deprivation of intracellular amino acids. Proc. Natl. Acad. Sci. USA 76: 3169, 1979. 26. Weber, F. L., Maddrey, W. C., and Walser, M. Amino acid metabolism of dog jejunum before and during absorption of keto-analogues. Amer. J. Physiol. 232: E263, 1977. 27. Souba, W. W. Glutamine metabolism in catabolic states. Infect Surg. 5(11): 641, 1986. 28. Baskerville, A., Hambleton, P., and Benbough, J. E. Pathologic features of glutaminase toxicity. &if. J. Exp. Pathof. 61: 132, 1980. 29. Okabe, S., Honda, II, Takeuchi, K., ef al. Inhibitory effect of L-glutamine on gastric irritation and back diffusion of gastric acid in response to aspirin in the rat. Dig. Dis. 20: 626, 1975. 30. Souba, W. W., Scott, T. E., and Wilmore, D. W. Gastrointestinal regulation of parenterally administered nutrients. Surg. Forum 35: 176, 1984. 31. O’Dwyer, S., and Wilmore, D. W. Preservation of small bowel mucosa using glutamine-enriched parenteral nutrition. Surg. Forum 37: 56, 1986.
OF SURGICAL
Interorgan
RESEARCH
4,
720-726 (1988)
Glutamine Metabolism
in the Tumor-Bearing
Rat
WILEY W. SOUBA, M.D., Sc.D., FREDERICK R. STREBEL, B.S., JOAN M. BULL, M.D., EDWARD M. COPELAND, M.D., HEINRICH TEAGTMEYER, M.D., PH.D., AND KAREN CLEARY, M.D. Department of Surgery and Biochemistry, University of Florida School of Medicine, Gainesville, Florida 32610, Departments of Surgery and Pathology, M. D. Anderson Hospital and Tumor Institute, Houston, Texas; and Department of Surgery and Medicine, University of Texas Medical School, Houston, Texas
Presentedat the Annual Meeting of the Association of Academic Surgery, Orlando, Florida, November l-4, 1987 The effectsof progressive malignant diseaseon gut/liver glutamine metabolism were studied in order to gain further insight into the altered glutamine metabolism that characterizes the host with cancer and to further elucidate the causes and consequences of glutamine depletion in tumor-bearing rats. Rats were inoculated on Day 0 with 2 X lo6 viable fibrosarcoma cells and blood glutamine was measured every 6 days. On Day 24 the animals underwent laparotomy and sampling of arterial, portal venous, and hepatic venous blood. Arterial glutamine fell by more than one-third in tumor-bearing rats and the arterial-portal venous concentration difference for glutamine across the intestinal tract was diminished by 50% (P i 0.01). Simultaneously the fractional extraction of glutamine by the gut was reduced from 2 1 to 15%(P < 0.05). The liver switched from an organ of near glutamine balance in control rats to one of marked glutamine output in tumor-beating rats (P < 0.01). The wet weight ofthe small intestine was diminished by 15% in tumor-bearing rats and villous height was uniformly decreasedin tumor-bearing rats with an average reduction in villous height of 26% (P < 0.05). The causes of glutamine depletion in this tumor-bearing rat model remain unclear. The growing tumor may behave as a glutamine trap but also appears to alter glutamine metabolism in vital metabolic processing centers such as the gut and liver. Malignant cells may compete with gut mucosal cells for glutamine resulting in a diminished gut glutamine utilization and detrimental changes in mucosal architecture. The altered glutamine metabolism that characterizes these tumor-bearing rats may be one component of cancer cachexia. 0 1988 Academic Press,Inc.
Cancer cachexia is commonly observed in patients with advanced malignant disease.The etiology of this syndrome of weight loss, anorexia, and negative nitrogen balance remains obscure [l-3]. While loss of appetite and diminished food intake are likely to contribute to the development of cancer cachexia, it is also likely that certain growing tumors alter normal host metabolism by behaving as a nitrogen trap or by regulating nitrogen flux via some, as yet undefined, distant metabolic effect.The accelerated muscle catabolism and loss of lean body tissue characteristic of many cancer patients may be a reflection of the malignant tumor’s ability to alter amino acid metabolism in vital metabolic processing centers such as the intestine and liver and change the balance of flow of amino acids from skeletal muscle to the splanchnic viscera. 0022-4804/88 $1.50 Copyright 0 1988 by Academic Press,Inc. All rights of reproduction in any form reserved.
Tumors and other rapidly replicating tissues such as the intestinal epithelium are major glutamine consumers [4-81. Glutamine is the most abundant amino acid in blood and tissues and comprises nearly twothirds of the free amino acid pool in skeletal muscle [9-l I]. Glutamine is the principal amino acid consumed by tumor cells and often servesas their principal respiratory fuel [5]. With progressive tumor growth, altered glutamine metabolism becomes a characteristic feature of the tumor-bearing host. In rats bearing large fast-growing malignancies, plasma and hepatocyte intracellular glutamine concentrations fall at a time when glutamine utilization by the tumor may be excessive [ 121. Studies on skeletal muscle glutamine metabolism in tumor-bearing rats demonstrate accelerated glutamine release
720
SOUBA
ET AL.: INTERORGAN
[ 131 and probable depletion of intracellular glutamine levels. The resultant depletion of tissue glutamine stores and persistent catabolism in skeletal muscle that characterize progressive malignant disease may be etiologic factors in the development of cancer cachexia. Previous studies have demonstrated that the altered skeletal muscle glutamine metabolism that characterizes catabolic states is intimately related to alterations in splanchnic glutamine metabolism [9, 11, 141.This study was designed to examine glutamine metabolism by the intestine and liver in tumorbearing rats to seewhether there are splanchnit alterations in glutamine metabolism that parallel and reflect the presumed changes observed in skeletal muscle. We hypothesized that as the tumor grows it alters normal interorgan glutamine relationships. We suggest that with advanced malignant disease the principal tissues of glutamine utilization “outconsume” the glutamine producers leading to a depletion of glutamine stores in blood and tissues and further tissue catabolism. The purpose of this study was to (a) compare arterial glutamine levels in pair-fed tumor-bearing and control rats, (b) compare the flux of glutamine across the intestine and liver in control and tumor-bearing rats, and (c) examine the consequences of glutamine depletion on small intestinal morphology. MATERIALS
AND METHODS
Selectionand Maintenance of Rats Female inbred Fischer 344 rats weighing 160- 170 g were utilized for the studies. The rats were obtained from a nearby breeder and maintained in standard cages (5 rats maximum/cage) in the animal laboratories. The rats were fed a standard laboratory rat chow and water ad libitum and subjected to alternate 12-hr periods of dark and light. Following tumor cell inoculation, control and study rats were pair-fed since previous studies have demonstrated that rats bearing relatively fast-growing tumors may have a depressedfood intake relative to ad libitum fed
GLUTAMINE
METABOLISM
721
nontumor-bearing rats [ 151.The daily ration of the pair-fed nontumor-bearing animals was the same as that amount of food consumed by the tumor-bearing animals on the preceding day.
Transplantation and Choice of the Fibrosarcoma Cell Line Eighteen rats were randomly selected to undergo either subcutaneous injection on the back with a single cell suspension of 2 X 1O6viable fibrosarcoma cells or to undergo sham injection with an identical volume of sterile saline. This number of cells resulted in the growth of a palpable 3- to 4-cm-diameter tumor in 3-4 weeks. This tumor model has been used by other investigators to study tumor-host metabolism alterations [ 161. This tumor cell line is locally aggressive,metastasizes rarely, and never regressesspontaneously. This cell line is a relatively fastgrowing tumor; faster growing tumors alter host amino acid metabolism significantly and may behave as a nitrogen trap [ 1, 17, 181.Glutamine utilization rates are high for such tumors and we have found the concentration of glutamine to be decreased in the blood of our tumor-bearing rats. Glutamine utilization by a number of tumors correlates with mitochondrial glutaminase activity [ 18-201 and glutamine supply to the tumor [15, 211. These observations suggest that early changes in intestinal and hepatic glutamine metabolism may be altered to a greater degree in fast-growing tumors.
Study Procedure Control and tumor rats were weighed on the day of tumor cell inoculation or saline injection (Day 0), placed in individual metabolic cages for paired-feeding, and weighed every 6 days thereafter. Tumor diameters were measured regularly. Arterial blood (0.5 cc) (by heart puncture under light ether anesthesia) was drawn on Days 6, 12 and 18 post tumor cell or saline injection and analyzed for whole blood glutamine.
722
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988
On Day 24 (post inoculation) control and tumor-bearing rats were anesthetized with ether and underwent laparotomy for blood sampling, tissue sampling and processing, and measurement of tumor weight. Blood (0.5 cc) was removed from the aorta, portal vein, and hepatic vein as described by Welboume et al. [22]. Briefly, the abdomen was opened, the sternum was gently elevated with a hemostat, and the central lobe of the liver was depressed, exposing the hepatic venous drainage of the middle hepatic lobe. This vein was carefully cannulated by directing a 26-gauge needle fitted to heparized Silastic tubing into the vein about 6-8 mm, which allows sampling of hepatic venous blood. The intestines were then gently displaced exposing the portal vein which was surrounded with a 5-O silk ligature. Flow to the liver was momentarily occluded and blood was sampled by cannulation of the portal vein with a 26-gauge needle fitted to Silastic tubing attached to a heparinized 1-cc syringe. Arterial blood was withdrawn directly from the abdominal aorta. All studies were performed in the postabsorptive state. Arteriovenous concentration differences were calculated from paired data, that is, the arterial and venous samples came from the same rat. Following blood sampling, the small intestine was removed, gently rinsed free of debris, and placed in 10% buffered formalin so multiple jejunal sections could later be prepared for morphometric (histologic) analysis. The tumor was removed from the flank of each tumor-bearing rat, measured, and weighed.
2. Morphometric analysis. Villous height was examined using the light microscope. 3. Tumor measurements. Maximum tumor diameters were measured on Days 6, 12, 18, and 24. Tumor weight was obtained on Day 24 when the animals were sacrificed. RESULTS
Relationship between Tumor Growth, Body Weight, Food Intake, and Jejunal Morphology
Changes in tumor size and weight, body weight, and wet weight of the small intestine are shown in Table 1. Food intake remained fairly constant (approximately 11 g/24 hr/ rat) until Day 14 post tumor implantation at which time there was a gradual decrease in food consumption to approximately 7 g/24 hr/rat on Day 24. This decrease in food intake in control rats explains the stabilization of body weight and failure to gain weight between Days 14 and 24. Body weight also remained stable in tumor-bearing rats, though by Day 24 the animals were beginning to show signs of cachexia (decreased hindquarter skeletal muscle mass on physical exam, thinning of abdominal wall musculature). The latter observation can be explained, at least in part, by the growing malignancy which by Day 24 demonstrated an averageweight of 15 g and measured approximately 3.5 X 3.5 cm. Thus, carcass weight (total body weight minus tumor weight) in the tumor-bearing rats was 158 f 3 g on Day 24, representing a 5% decrease in body weight. Control rats, on the other hand, demonstrated a 4% increase in body weight despite having to control food intake (P < 0.05). Analytical Procedures Wet weight of the small intestine (Table 1) 1. Whole blood glutamine. Heparinized and jejunal villous height were measured on whole blood was mixed with an equal vol- Day 24. Tumor-bearing rats had small intesume of 10% ice-cold perchloric acid, vor- tines that weighed significantly less than texed, and centrifuged. The supematant was those of the control animals (3.10 f 0.10 vs neutralized to pH 4.75 and stored at -70°C. 3.58 f 0.09 g, P < 0.05). Jejunal epithelial Glutamine was measured employing micro- morphology was examined by light microsfluorometric enzymatic assaysadapted from copy in six control and six tumor-bearing the method described by Bergmeyer [23]. rats. Villous height was reduced by an aver-
SOUBA ET AL.: INTERORGAN
GLUTAMINE
723
METABOLISM
TABLE 1 Days post tumor implantation
Control (n = 9) Gut weight (g) (small bowel) Body weight (g) Tumor (n = 9) Gut weight (g) (small bowel) Body weight (g) Approximate tumor size (mm X mm)
0
6
12
165 + 3
168 253
171 k 3
173k3
3.58 f 0.09 172+3
166 * 3
169k 3
172+3
173+2
3.10 * 0.10* 172 + 3
10 x 10
20 x 20
35 x 35 (15 g)
-
1x1
Body weight minus tumor weight
18
163 f 4
24
158 + 3*
Note. Data are expressed as means + SEM. * P K 0.05 compared to controls.
glutamine by the intestinal tract (A-PV concentration difference/arterial concentration) was also diminished in tumor-bearing rats (2 1% in controls vs 15% in tumor-bearing rats, P < 0.05). Arterial Glutamine Concentration Significant alterations in liver glutamine metabolism also occurred in the tumor rats. Arterial glutamine (GLN) concentration The arterial-hepatic venous (A-HV) concenshowed a slight decreasing but not statistitration difference was positive in controls but cally significant reduction in the control rats switched to slightly negative with tumor imover time (Table 2). Tumor-bearing rats, on plantation (78 f 17 vs -9 f 4 pmoles/liter, P the other hand, demonstrated a fall in whole < 0.01). The PV-HV concentration differblood glutamine over time which was signifience was significantly more negative in the cantly lower than control levels on Day 24 tumor group (-39 + 9 vs -67 f 11 pmoles/ (4 16 f 36 vs 58 1 + 32 pmoles/liter, P < 0.05, liter, P < 0.05). Table 2). age of 26% (P < 0.05) in tumor-bearing rats and was consistently diminished in all six TBR rats compared to controls. In addition, the villi were blunted and thickened.
Arteriovenous Concentration D@erences and Glutamine Flux on Day 24 There was a significant decreasein the arterial-portal venous (A-PV) concentration difference for glutamine in tumor-bearing rats (63 f 9 vs 120 f 10 pmoles/liter in controls, P < 0.01) (Table 3). This reduction in uptake by the small intestine occurred at a time when jejunal villous height and mucosal cellularity were reduced. Extraction of
DISCUSSION
Advanced malignant diseaseand other catabolic disease states are characterized by a reduction in tissue stores and plasma concentrations of glutamine [9, 10, 12, 131. In this research project we hypothesized that with progressive tumor growth circulating glutamine stores become depleted and alterations in gut/liver glutamine metabolism develop. Such was the case.
724
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988 TABLE 2 ARTERIAL GLUTAMINE CONCENTRATIONDAYS POSTTUMOR IMPLANTATION
Control Arterial GLN (rmol/l) Tumor Arterial GLN (pmole/liter)
6
12
18
24
647 2 17
636 + 32
578 f 38
581 f 32
652+31
605 + 26
523 + 21
416 + 36*
Note. Data are expressed as means + SEM; n = 6 in each group for Days 6, 12, 18; n = 9 in each group on Day 24. * P < 0.0 1 compared to controls.
The tumor cell line utilized in these studies, the methylcholanthrene-induced fibrosarcoma, has been used by others [ 161. Although it is a fairly rapidly growing tumor, it is representative of the rates of growth of some tumors observed in the clinical setting. This cell line was chosen for initial studies since previous studies have demonstrated that fast-growing tumors are avid glutamine consumers and alterations in glutamine metabolism develop in rats bearing these tumors. It was felt that these rapidly growing tumors would alter gut-liver glutamine relationships fairly quickly and, hence, be appropriate for initial investigations in the area of interorgan glutamine metabolism. Food intake was controlled with the daily ration of the pair-fed nontumor-bearing rats being equivalent to that amount of food consumed by the tumor-bearing animals on the preceding day. The modest decrease in arterial glutamine (10%) in control rats over
the 24-day period is most likely due to the diminished food intake. Partial starvation has been shown to result in decreased levels of circulating glutamine [lo, 241. The 36% fall in arterial glutamine in tumor-bearing rats by Day 24 supports the concept that the growing tumor contributes to a reduction in plasma glutamine. Whether this occurs from excessiveglutamine utilization by the tumor, alterations in glutamine synthetic pathways in other tissues, or some other mechanism is not clear from our study. Other studies demonstrate that as tumors grow they become significant glutamine consumers; the rate of glutamine utilization parallels the rate of tumor growth [6, 181. Eventually the arteriovenous concentration differences across gut and liver become altered (Table 3). The net result may be that the glutamine utilization is in excessof glutamine production resulting in glutamine depletion. Skeletal muscle glutamine release
TABLE 3 ARTERIOVENOUSCONCENTRATIONDIFFERENCES FORGLN (rmole/liter) ON DAY 24
Control (n = 9) Tumor (n = 9)
A
PV
HV
A-PV
A-HV
PV-HV
581 + 32 416 rf: 26**
458 + 23 348 + 18*
502 * 16 420 f 16*
121* 10 63 2 9**
7s* 17 -9 f 4**
-39 _+9 -67 f 1l**
Note. Data are expressed as means + SEM. A, arterial; PV, portal vein; HV, hepatic vein; A-PV, arterial-portal venous concentration difference; A-HV, arterial-hepatic venous concentration difference; PV-HV, portal venous-hepatic venous concentration difference; - = release; + = uptake. * P < 0.05 compared to controls. ** P < 0.01 compared to controls.
SOUBA ET AL.: INTERORGAN
is accelerated in the tumor-bearing rat [ 131as it is in other catabolic disease states [9, 1l] and intracellular stores and circulating levels of this key amino acid become depleted. In the current study, when blood glutamine levels fell, the liver switched to an organ of net glutamine release, a phenomenon we have observed in other catabolic states [9, 141. This may occur in part in an effort to normalize circulating levels of glutamine. This may be of significance since it has been demonstrated that the intracellular concentration of glutamine is a regulator of protein catabolism in skeletal muscle [ 111 and liver [25]. Low intracellular glutamine levels appear to mediate net protein catabolism in these tissues. In the nontumor-bearing animal, the mucosa of the small intestine is the principal site of glutamine utilization [8, 261. In the rat, approximately 20-30% of circulating glutamine is removed by the enterocytes with each pass through the intestinal vasculature [8]. Metabolism of glutamine by the intestinal tract is an integral aspect of the intestine’s role as a regulator of nitrogen metabolism in normal and catabolic states [9, 10, 14, 271. The growing tumor may “compete” with the small intestine for glutamine. Both tumor cells and gut mucosal cells demonstrate a striking similarity in substrate utilization and glutamine metabolism [ 15, 2 11. Abnormal intestinal glutamine metabolism (perhaps resulting from distant effects of the tumor) may result in a diminished utilization by glutamine by the enterocytes which may be linked to the alterations in intestinal morphology noted in the jejunum of the tumorbearing rats. Our findings in this study may be consistent with other studies which suggest that glutamine is essential for maintenance of normal intestinal structure and function. For example, in one study, the reduction of plasma glutamine concentrations to undetectable levels in monkeys and rabbits resulted in vomiting, diarrhea, mild villous atrophy, mucosal alterations, and intestinal necrosis [28]. In a study in rats, Okabe and colleagues [29] demonstrated that gluta-
GLUTAMINE
METABOLISM
725
mine protects against aspirin-induced gastric ulcerations. Others [3 I] have demonstrated that the provision of glutamine-enriched parenteral solutions to rats resulted in an increasein jejunal mucosal weight, DNA content, and villous height when compared to animals fed isonitrogenous glutamine-depleted intravenous diets. These studies suggest that the delivery of adequate amounts of glutamine from exogenous or endogenous sources may be a requirement for maintaining the integrity of the mucosa of the small intestine during periods of glutamine depletion. REFERENCES 1. Kawamura, I., Moldawer, L. L., Keenan, R. A., et al. Altered amino acid kinetics in rats with progressive tumor growth. Cancer Res. 4t: 824, 1982. 2. Bennegard, K., Lindmark, L., Eden, E., et al. Flux of amino acids acrossthe leg in weight-losing cancer patients. Cancer Rex 44: 386, 1984. 3. Popp, M. B., Morrison, S. D., and Brennan, M. F. Total parenteral nutrition in a methylcholanthreneinduced rat sarcoma model. Cancer Treat. Rep. 65: 137, 1981. 4. Coles, N. W., and Johnstone, R. M. Glutamine metabolism in ehrlich ascites-carcinoma cells. Biothem. J. 83: 284, 1962. 5. Kovacevic, Z. The pathway of glutamine and glutamate oxidation in isolated mitochondria from mammalian cells. Biochem. J. 125: 757, 1971. 6. Kovacevic, Z., and Morris, H. P. The role of glutamine in the oxidative metabolism of malignant cells. Cancer Res. 32: 326, 1972. 7. Windmueller, H. G., and Spaeth, A. E. Uptake and metabolism of plasma glutamine by the small intestine. J. Biol. Chem. 249: 5070, 1974. 8. Windmueller, H. G. Glutamine utilization by the small intestine. Adv. Enzymol. 53: 202, 1982. 9. Souba, W. W., Smith, R. J., and Wilmore, D. W. Glutamine metabolism by the intestinal tract. JPEN 9(5): 608, 1985. 10. Souba, W. W. Glutamine Metabolism in Catabolic States: Role of the Intestinal Tract. Thesis, Department of Nutritional Biochemistry, Harvard School of Public Health, Cambridge, MA, 1984. 11. Muhlbacher, F., Kapadia, C. R., Colpoys, M. F., et al. Effects of glucocorticoids on glutamine metabolism in skeletal muscle. Amer. J. Physiol. 247: E75, 1984. 12. Wu, C., and Gauer, J. M. A study of free amino acids and of glutamine synthesis in tumor-bearing rats. Cancer Res. 20: 848, 1960. 13. Yamamoto, H., Aikawa, T., Matsutka, H., and
726
JOURNAL OF SURGICAL RESEARCH: VOL. 44, NO. 6, JUNE 1988
Ishikawa, E. Relative uptake of plasma amino acids by fetal and tumor tissues. Metabolism 23: 1017, 1974. 14. Souba, W. W., Smith, R. J., and Wilmore, D. W. Effect of glucocotticoids on glutamine metabolism in visceral organs. Metabolism 34(5): 450, 1985. 15. Sauer, L. A., Stayman, J. W., III, and Dauchy, R. T. Amino acid, glucose and lactic acid utilization in vivo by rat tumors. Cancer Rex 42: 4090, 1982. 16. Popp, M. B., Waganer, S. C., and Brito, 0. J. Host and tumor responsesto increasing levels of intravenous nutritional support. Surgery 94: 300, 1983. 17. Carrascosa, J. M., Martinez, P., and decastro, I. N. Nitrogen movement between host and tumor in mice inoculated with ehrlich ascitic tumor cells. Cancer Res. 44: 3831, 1984. 18. Knox, W. E., Linder, M., and Friedell, G. H. A series of transplantable rat mammary tumors with graded differentiation, growth rate, and glutaminase content. Cancer Res. 30: 283, 1970. 19. Knox, W. E., Horowitc, M. L., and Friedell, G. H. The proportionality of glutaminase content to growth rate and morphology of rat neoplasma. Cancer Res. 29: 669, 1969.
20. Linder-Horowitz, M., Knox, W. E., and Morris, H. P. Glutaminase activities and growth rates of rat hepatomas. Cancer Res. 29: 1195, 1969. 21. Sauer, L. A., and Dauchy, R. T. Keton body, glucase, lactic acid, and amino acid utilization by tumors in vivo in fasted rats. Cancer Res. 43: 3497, 1983. 22. Welbourne, T. C., Childress, D., and Givens, G. Renal regulation of interorgan glutamine flow in
metabolic acidosis. Amer. J. Physiol. 251: R858, 1986. 23. Bergmeyer, H. U. (Ed.) Methods ofEnzymaticAna/ysis. New York: Academic Press, 1974. 24. Marl& E. B., Aoki, T. T., Pozefsky, T., et al. Muscle and splanchnic glutamine and glutamate metabolism in postabsorptive and starved man. J. Clin. Invest. 50: 814, 1971. 25. Schworer, C. M., and Mortimore, G. E. Glucagoninduced autophagy and proteolysis in rat liver: Mediation by selective deprivation of intracellular amino acids. Proc. Natl. Acad. Sci. USA 76: 3169, 1979. 26. Weber, F. L., Maddrey, W. C., and Walser, M. Amino acid metabolism of dog jejunum before and during absorption of keto-analogues. Amer. J. Physiol. 232: E263, 1977. 27. Souba, W. W. Glutamine metabolism in catabolic states. Infect Surg. 5(11): 641, 1986. 28. Baskerville, A., Hambleton, P., and Benbough, J. E. Pathologic features of glutaminase toxicity. &if. J. Exp. Pathof. 61: 132, 1980. 29. Okabe, S., Honda, II, Takeuchi, K., ef al. Inhibitory effect of L-glutamine on gastric irritation and back diffusion of gastric acid in response to aspirin in the rat. Dig. Dis. 20: 626, 1975. 30. Souba, W. W., Scott, T. E., and Wilmore, D. W. Gastrointestinal regulation of parenterally administered nutrients. Surg. Forum 35: 176, 1984. 31. O’Dwyer, S., and Wilmore, D. W. Preservation of small bowel mucosa using glutamine-enriched parenteral nutrition. Surg. Forum 37: 56, 1986.