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Alterations in Carbohydrate Metabolism in Canine Lymphoma

David M. Vail, DVM, Gregory K. Ogilvie, DVM, Steven L. Wheeler, DVM, MSc, Martin J. Fettman, DVM, PhD, Shirley D. Johnston, DVM, PhD, and Rebecca L. Hegstad, BA

Following an overnight fast, blood samples were obtaiied from 14 dogs with previously untreated lymphoma before and 5, 15, 30, 45, 60, and 90 minutes following an intravenous challenge with 500 mg/kg dextrose. Samples were assayed for glucose, lactate, and insulin concentrations and compared statistically with ten control dogs of similar weight and age undergoing an identical dextrose challenge. Dogs with lymphoma had similar glucose tolerance curves when compared with controls. Lactate concentrations were significantly higher ( P < 0.001) at baseline and all time periods of the glucose tolerance test in dogs with lymphoma when compared with controls. Rise in lactate concentrations over baseline levels in the first 30 minutes of the glucose tolerance test were significantly higher in dogs with lymphoma ( P = 0.011). Insulin concentrations were significantly higher ( P < 0.001) at baseline and at the 5-, 45-,60-, and 90-minute time periods of the glucose tolerance test in dogs with lymphoma. Rise in insulin concentrations over baseline in the first 5 minutes of the glucose tolerance test were also significantly greater in dogs with lymphoma ( P = 0.021). These results indicate carbohydrate metabolism is altered in dogs with lymphoma. Many of these alterations parallel those observed in human patients suffering from cancer cachexia making canine lymphoma a potential model for further study of the pathogenesis and therapy of cancer cachexia. (Journal of Veterinary Internal Medicine

1990; 4:8-11)

IN PEOPLE and laboratory animals, altered endogenous metabolism including metabolic derangements in carbohydrate metabolism have been identified as the principle cause of cancer cachexia, a complex syndrome of progressive, involuntary weight loss observed in cancer patients often in the face of adequate nutritional intake.'-" Considerable evidence exists that the tumor and the host have different nutritional requirements. In essence, the tumor acts like an obligatory parasite growing at its own genetically determined rate, competing effectively with the host for limited available nutrients. Several studies have documented abnormal carbohyFrom the Comparative Oncology Unit (Vail. Ogilvie. Wheeler) and the Department of Pathology (Fettman), College of Veterinary Medicine and Biomedical Sciences, Colorado State University. Fon Collins, Colorado, and the Depanments of Small Animal Clinical Sciences (Johnston) and Large Animal Clinical Sciences (Hegstad). College of Veterinary Medicine, University of Minnesota. St. Paul, Minnesota. Supponed in pan by the Moms Animal Foundation: PHs grant #I CA 29582 awarded by the National Cancer Institute, Depanment of Health and Human Services; and the Minnesota Agricultural Experiment Station Project MIN-62-055 (Animal Hormone Experimental and Development Laboratory). Reprint requests: David M. Vail. DVM. Comparative Oncology Unit, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523.

drate metabolism in human cancer patients. More specifically, abnormalities have been noted in peripheral tissue glucose disposal, hepatic glucose production, and whole body glucose oxidation and t ~ r n o v e r . ' ~ - ' ~ Tumors preferentially metabolize glucose-using anaerobic glycolysis for energy, forming lactic acid as an end p r o d ~ c t . ~ . " . ' ~ , ' ~ lactate produced by the tumor is The then converted back into glucose via the Cori cycle in the host's liver. Overall, rather than the host using glucose in the Krebs cycle to produce 38 adenosine triphosphate (ATP), the tumor consumes the mole of glucose via anaerobic glycolysis with a net gain of only 2 ATP. The host then loses six high-energy phosphate bonds converting the lactate back to glucose through the Cori ~ y c l e . ~ , ~ . ' ~ Relative insulin resistance and/or insulin deficiency has been reported in people with cancer and in tumorbearing laboratory animal^.^^^^'^.'^-'^ Glucose tolerance tests have revealed a diabetic-like response in both cachectic and noncachectic cancer patient^.^'^^." This response is not due entirely to decreased insulin production, but also due in part to insulin resistance as the glucose intolerance is often resistant to exogenous in~ulin.~

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Vol. 4 . No.

METABOLISM ALTERATIONS IN CANINE LYMPHOMA

It is important to recognize that metabolic alterations that occur in human cancer patients are not confined to one or a few specific tumor types but have been reported in various forms of carcinoma, sarcoma, lymphoma, and leukemia.'-l6 T o date, no spontaneously occumng animal tumor models have been characterized for the study of metabolic alterations occumng in cancer cachexia. This study was undertaken to determine whether alterations in carbohydrate metabolism occur in dogs with lymphoma and lends itself as a model for further study of the pathogenesis and therapeutic strategies of cancer cachexia.
Materials and Methods

Dogs with Lymphoma


Fourteen client-owned dogs with histologically confirmed lymphoma were used in this study and were taken from the patient population presented to the Veterinary Teaching Hospital at the Colorado State University between October 1987 and June 1988. Dogs were excluded from the study if they had received chemotherapy, exogenous steroids, or surgery in the 30 days before presentation. In addition, dogs with non-neoplastic induced diseases (i.e., renal failure, hepatic cirrhosis, etc), clinical evidence of endocrinopathies, or hypercalcemia secondary to lymphoma were excluded. T o ensure a more uniform patient population, only those dogs with stage 111 or IV lymphoma, according to the World Health Organization classification scheme, were eligible for study." Nine dogs were stage 111 and five dogs were stage IV. All dogs had complete blood counts, biochemical profiles, bone marrow aspirates, and thoracic and abdominal radiographs performed for staging. Median and mean weights were 3 1 kg and 33.8 kg, respectively, with a range from 12.2 kg to 50.7 kg. Ages ranged from 4 to 13 years, with a median of 7 years and a mean of 7.5 years. Eight of the dogs were females (6 spayed) and 6 were males (5 neutered). The two intact female dogs were not exhibiting signs of clinical estrus.

5, 15, 30, 45, 60, and 90 minutes following 500 mg/kg dextrose* given intravenously as a 25% solution over 30 seconds. Glucose concentrations were determined on serum by a semiautomated glucose oxidase meth0d.t Lactic acid concentrations were determined spectrophotometrically using a lactic dehydrogenase method.$ Serum insulin concentrations were determined in duplicate by radioimmunoassay techniques using a commercially available kit.$ Samples were stored at -20C immediately after collection and assayed in large batches a t a later time. Glucose and lactate concentrations were determined at all sample times from all dogs with lymphoma and all con'trol dogs. Insulin concentrations were determinea on all samples from 10 dogs with lymphoma and 10 control dogs. Insulin concentrations on 4 dogs with lymphoma were not available for analysis.

Statistical Analysis
Data from each time period tested (i.e., 0, 5, 15, 30, 45. 60, 90 minutes) as well as variations from the zero time baseline values were compared between lymphoma an& control dogs using the Wilcoxin Rank-Sum test for twc g r o ~ p s . 'When P < 0.05 was present, data was consid~ ered significantly different.
Results

Serum glucose concentrations during all time periods of the glucose tolerance test did not differ significantly between lymphoma and control dogs (Fig. I). Serum lactate concentrations were significantly higher at time zero (baseline) ( P = 0.001) and all time periods ( P < 0.00 1 ) of the glucose tolerance test in d o g with lymphoma when compared with controls (Fig. 2). Lactate concentrations increased by a mean of 4.72 m u d l over baseline concentrations in the first 30 minutes of the glucose tolerance test in dogs with lymphoma. This is a significantly ( P = 0.01 1) larger rise
-

Control Dogs
Control dogs consisted of 10 dogs with no known medical problems and no prior exposure to exogenous steroids. Median and mean weights were 35.9 kg and 35.2 kg respectively, with a range from 26.8 kg to 42.7 kg. Ages ranged from 5 to 14 years with a median of 7 years and a mean of 7.8 years. Six of the control dogs were spayed females and four were neutered males.

Glucose Tolerance Testing with Concomitant Lactate and Insulin Levels


A modification of the protocol used by Mattheeuws was followed for glucose tolerance testing." Following an overnight ( 12- 1 8 hour) fast, blood was taken before and

Dextrose 50% solution diluted to 25% in sterile water, Vedco Inc.. St. Joseph, MO. t Glucose Analyzer 2, Beckman Co., Fullerton, CA. $ Lactate dehydrogenase reagent, Sigma Chemical Co., St. Louis. MO. 5 ICN Biomedicals, Inc., Carson, CA. catalog # 07-160102. Assa? sensitivity determined as 2 standard deviations from the mean of 6 samples of assay buffer, was 1.3 rU/ml. Within assay coefficients of variation in three canine serum pools were 4.W (mean, 94 f 3.7 pU/ml: n = 5), 4.0% (mean, 47 f 1.9; n = 5), and 8.6% (mean, 18.7 2 1.6; n = 5). Between assay coefficients of variation in two KIUm pools measured in five different assays were 10.6% (mean, 37.6 rt 4.0). and 8.2% (mean, 18.2 2 1.5). Dilutional parallelism was demonstrated by assaying two pools of canine serum at three dilutions and correcting the measured result for dilution; corrected values for pool I were 3 1.7 f 4.5, 39.6 f 4.8, and 33.5 f 5.0 rU/rnl, and for pool 2 were 121.7 ? 10.4, 94 f 3.7 and 74.8 2 6.4. Results obtained for crystalline porcine insulin added to canine serum (25, 50, 100 rU/rnl) were linear and quantitative ( r = 0.99).

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VAlL ET AL.
Glucose mgldl Insulin uUlml

Journal of Veterinary Internal Medicine

400

- -

140

15

30

45

80

90

15

30

45

80

90

Minutes Post Dextrose Challenge


Lymphoma (11.14) Control (n-10)

Minutes post Dextrose Challenge


Control (n-10) Lymphoma (n-10)

FIG. I. Serum glucose concentrations (mean 2 SD) in dogs with and without lymphoma before and after iv administration of 500 m u k g dextrose.

FIG. 3. Serum insulin concentrations (mean ? SD) in dogs with and without lymphoma before and after i.v. administration of 500 mglkg dextrose. = values from dogs with lymphoma differ significantly (P < 0.001) fwm control dogs at the same time period.

than that which occurred in control dogs (mean, 0.41 mg/dl). Serum insulin concentrations were significantly higher at time zero (P < 0.00 1) and at the 5-(P < 0.001), 45-(P < 0.00 1 ), 60-(P < 0.00 l), and 90-(P < 0.00 1)minute time periods of the glucose tolerance test in dogs with lymphoma when compared with controls (Fig. 3). Insulin/glucose ratios were also significantly higher at the baseline (P < 0.00 1 ), 5-(P = 0.0 17), 45-(P = 0.006), 60-(P = 0.005), and 90-(P = 0.004)minute time periods in dogs with lymphoma when compared with controls. Insulin concentrations increased by a mean of 74.78 pU/ml over baseline concentrations in the first 5 minutes of the glucose tolerance test in dogs with lymphoma. This is a significantly (P = 0.021) larger rise than the 40.93 pU/ml rise that occurred in control dogs. Comparison of the nine dogs with stage 111 lymphoma with the 5 dogs with stage IV lymphoma failed to reveal

significant differences in either glucose, lactate, or insulin concentrations at any time during the dextrose challenge.

Discussion
No significant difference was noted in glucose concentrations between dogs with lymphoma and controls throughout the course of the glucose challenge. This is unlike the glucose intolerance noted in human patients suffering from cancer ~ a c h e x i a . ~ . ~ ' . ~ ~ Typically, glucose intolerance is noted before overt weight loss, correlates with tumor volume, and is thought to occur as a result of reduced tissue sensitivity to i n s ~ l i n . ~ Lactate concentrations were significantly higher in dogs with lymphoma at all points of the glucose challenge. While a case could be made for decreased hepatic lactate use in dogs with stage IV lymphoma, who by definition have liver involvement, they were no more hyperlactatemic than dogs with stage 111 lymphoma. Hyperlactatemia is known to occur in the majority of human patients suffering from cancer cachexia as a result of increased anaerobic glycolysis.2.'1.15,16 signifiThe cantly greater increase in lactate concentrations from baseline observed in lymphoma dogs during the first 30 minutes of the glucose challenge is indirect evidence of conversion of glucose to lactate via anaerobic glycolysis. Isotope studies using specifically radiolabelled glucose would be required to confirm this supposition. Whether the observed conversion of glucose to lactate was due to tumor glycolytic activity, host glycolytic activity, or a combination of the two cannot be determined from this data. It is known that neoplastic cells may use embryonic forms of key enzymes of anaerobic metabolism. e.g., hexokinase, 6-phosphofructokinase, and pyruvate kinase, which are less subject to host feedback mechanisms thereby allowing the production of significant

Lactate m9ldl 25

15

30

45

80

B O

Minutes Post Dextrose Challenge


Control (n-10) Lymphoma (11-14)

FIG. 2. Serum lactate concentrations (mean 2 SD) in dogs with and without lymphoma before and after iv administration of 500 mglkg dextrose. = values from dogs with lymphoma differ significantly ( P < 0.00 1 ) from control dogs at the same time period.

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VOI. 4

. NO.

METABOLISM ALTERATIONS IN CANINE LYMPHOMA

1
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quantities of lactate.I7 However, it is also well documented that tumor derived or tumor induced circulating factors, e.g., tumor necrosis factor. interleukin- 1, etc.. can alter host endogenous metab~lism'.~ allowing for the possibility of host-derived lactate production. Regardless of the source, the net result of anaerobic glycolysis would be less energy available to the host. The observed increased lactate concentrations in dogs with lymphoma are clinically significant in that they will increase hepatic Con cycle load, resulting in further energy consumption by the host. It has been theorized that an increase of daily energy needs of up to 30% could occur secondary to such increased Con cycle activity in human cancer patient^.^.'.^.'^ Increased lactate concentrations in dogs with lymphoma may also be clinically significant in that Lactated Ringers solution may be an inappropriate fluid of choice for hydration. Lactic acidosis could also be of concern and has been reported in human patients suffering from cancer ~ a c h e x i a . Hy~,~ perinsulinism was observed in dogs with lymphoma as significantly more insulin per unit of glucose was present at baseline, early, and late in the glucose challenge. Whether this was due to decreased insulin sensitivity (receptor defect), decreased insulin responsiveness (postreceptor defect), or a combination of the two cannot be stated without supporting data derived from euglycemic clamp technique^.'^.^^ There is ample evidence of postreceptor nonresponsiveness to insulin in human and laboratory animal models of cancer cachexia resulting in part from increased serum concentrations of free fatty acids. lipoproteins, and various other metabolic defeCts.21.6. 17-20 Dogs with lymphoma in this study also 14. demonstrated insulin hypersecretion as evidenced by the significantly greater increase in insulin concentrations from baseline during the first 5 minutes of the dextrose challenge. Whether this was due to beta-cell hypersensitivity to glucose alone or hypersensitivity to some other insulin secretagogue cannot be resolved from these data. Hyperglycemic clamp techniquesz4and perhaps a lactate challenge would be helpful in determining the basis of the observed insulin hypersecretion. The observed hyperinsulinism and insulin hypersecretion may be necessary to overcome insulin resistance and maintain the normal glucose tolerance observed in these dogs. In summary, carbohydrate metabolism was shown to be altered in dogs with lymphoma as evidenced by hyperlactatemia, increased lactate production in response to exogenous glucose challenge, hyperinsulinism, and insulin hypersecretion observed in this study. Many of these alterations parallel those observed in human patients suffering from cancer cachexia. This, in combina-

tion with the frequency with which canine lymphom: occurs in the population and the relative ease witt which remissions can be achieved, makes spontaneou. canine lymphoma a potential model for further study o the pathogenesis and therapy of cancer cachexia.
References
I. Brennan NF. Uncomplicated starvation Venus cancer cachexiz Cancer Res 1977: 37:2359-2364. 2. Heber D. Byerley LO, Chi J. et al. Pathophysiology of malnutn tion in the adult cancer patient. Cancer 1986; 58: 1867-1 873 3. Chlebowski RT, Heber D. Metabolic abnormalities in cancer pa tients: Carbohydrate metabolism. Surg Clin North Am 1986 66:957-968. 4. Heber D, Byerley LO. Chlebowski RT. Metabolicabnormalities ii the cancer patient. Cancer 1958; 55:225-229. 5. Bouetti F. Pagnoni AM, Del Vecchio M. Excessive caloric ex penditure as a cause of malnutrition in patients with cancer Surg Gynecol Obstet 1980; 150:229-234. 6. Kern ICA. Norton JA. Cancer cachexia. Journal of Parentera Enteral Nutrition 1988: 12286-298. 7. Bray GA. Campfield LA. Metabolic factors in the control of er: ergy stores. Metabolism 1975: 24:99- 1 17. 8. DeWys WD. Pathophysiology of cancer cachexia: Current under standing and areas of future research. Cancer Res 1981 42:72 1-726. 9. Shein PS, Kisner D. Haller D, et al. Cachexia of malignant! Cancer 1976; 43:2070-2076. 10. Hansel1 DT. Davies JWL, Shenkin A. et al. The oxidation ofbod. fuel stores in cancer patients. Ann Surg 1986; 204:637-642. I I . Dempsey DT, Mullen JL. Macronutrient requirements in th. malnourished cancer patients. Cancer 1985; 55:290-294. 12. Young VR. Energy metabolism and requirements in the cance, patient. Cancer Res 1977: 37:2336-2347. 13. Burt ME. Lowry SF, Gorschboth C. et al. Metabolic alterations ir a non-cachectic animal tumor system. Cancer 198 I; 47:2 1382146. 14. Norton JA. Maher M. Wesley R, et al. Glucose intolerance ir sarcoma patients. Cancer 1984; 54:3022-3027. 15. Richtsmeier WJ. Dauchy R. Sauer LA. In vivo nutrient uptake b: head and neck cancen. Cancer Res 1987; 475230-5233. 16. Holroyde CP, Skutches CL, Boden G, et al. Glucose metabolism in cachectic patients with colorectal cancer. Cancer Res 19844~59 10-59 13. 17. Lawson DH, Richmond A. Nixon DW, et al. Metabolic ap proaches to cancer cachexia. Annu Rev Nutr 1982; 2:277-30 1 18. Lundholm K. Edstrom S. Karlberg I, et al. Metabolism in periph era1 tissues in cancer patients. Cancer Treatment Repon 1981; 65:79-83. 19. Moley JF, Morrison SD. Norton JA. Insulin reversal of cancecachexia in rats. Cancer Res 1985: 45:4925-493 l. 20. Svaninger G. Drott C, Lundholm K. Role of insulin in develop ment of cancer cachexia in nongrowing sarcoma-bearing mice Special reference to muscle wasting. JNCl 1987; 78:943-950. 2 1. World Health Organization Classification of Tumon in Domestic Animals. 1st ed. Geneva, Switzerland, 1980. 22. Mattheeuws D, Rottier R, Kaneko JJ, et al. Diabetes mellitus ir dogs: Relationship of obesity to glucose tolerance and insulir response. Am J Vet Res 1984: 45:98-103. 23. Conover WJ. In: Practical Nonparametric Statistics. 2nd ed. Neu York: John Wiley and Sons. 1980: 215-228. 24. Rizza RA, Mandarino U. Gerich JE. Mechanisms of insulin resistance in man. Am J Med 1981; 70:169-176. 25. DeFronzo RA, Tobin JD. Andres R. Glucose clamp technique: C method for quantifying insulin secretion and resistance. Am i Physiol 1979; 237:E2 14-213.

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