In Vivo Assessment

of the Metabolic

Alterations

in Glucagonoma

Syndrome

Samuel Klein, Farook Jahoor, Hidefumi Baba, Courtney M. Townsend, Jr, Mary Shepherd, and Robert R. Wolfe Stable-isotope methodology and indirect calorimetry were used to evaluate metabolic abnormalities in a patient with glucagonoma syndrome manifested by 17% body weight loss, hypoaminoacidemia, and hyperglycemia. Energy expenditure (26 kcall kg) was the same as that predicted by the Harris-Benedict equation. The rate of appearance (Ra) of intracellular leucine (2.70 pmol/kg/min), an index of protein breakdown, was normal, although the percentage of leucine flux oxidized (31%). an index of amino acid catabolism, was 50% greater than the normal mean value. Glucose Ra in plasma (12.9 pmol/kg/min), representing hepatic glucose production, and glycerol Ra in plasma (3.04 pmol/kg/min), a measurement of whole-body lipolysis, were 15% and 25% greater, respectively, than mean values found in normal volunteers. These results suggest that long-term alterations in energy, leucine, glucose, and lipid metabolism in patients with glucagonoma are minimal. However, small long-term metabolic alterations caused by glucagon excess, in conjunction with chronic negative energy balance, could be responsible for the weight loss, hypoaminoacidemia, and hyperglycemia observed in this patient population.

G pancreas

LUCAGON-PRODUCING, islet-cell tumors of the (glucagonomas) have profound metabolic cffccts and cause weight loss, hyperglycemia, and hypoaminoacidemia.’ Although it has been assumed that these abnormalities are caused by the “catabolic effect” of excessive amounts of circulating glucagon, most of the metabolic processes responsible for catabolism have not been measured. Some of the presumed metabolic alterations contradict the available data or are difficult to explain on the basis of hyperglucagonemia alone. For example, glucagon-induced gluconeogenesis from amino acids has been proposed to be responsible for both hyperglycemia and hypoaminoacidemia.” However, the only study to measure basal glucose production found it to be below normal.’ Furthermore, if the mechanism for hypoaminoacidemia is accelerated gluconeogenesis, it is surprising that plasma concentrations of all amino acids (glucogenic and nonglucogenic) are decreased.4 Glucagon infusion in normal or diabetic humans does not decrease all plasma amino acid levek5J In addition. in contrast to the chronic hyperglycemia in patients with glucagonoma, glucagon infusion in normal volunteers causes a transient effect on glucose production, and glucose concentration is rapidly restored to normal despite continued glucagon infusion.’ ‘To explore the mechanisms responsible for metabolic alterations observed in patients with glucagonomas, we used stable-isotope methodology and indirect calorimetry to evaluate energy (metabolic rate), protein (leucine kinetics), glucose (glucose production), and lipid (lipolysis, triglyceride-fatty acid [TG-FA] substrate cycling, and triglyceride oxidation) metabolism in a patient with a glucagonproducing tumor and glucagonoma syndrome. CASE REPORT .I 46-year-old woman was admitted to The University of Texas Medical Branch in August 1989 for evaluation of a recurrent migratory skin rash that had first appeared 2 years earlier. The rash was present on the buttocks, lower abdomen, and face, but was most severe on the legs and feet. She had lost 10 kg (17% of body weight) in the last 12 months and was 83% of ideal body weight. based on the 1983 Metropolitan Life Insurance tables; the patient denied decreased food intake, abdominal pain, or diarrhea. Biopsy specimens of the skin lesions disclosed a superficial epidermal necrolysis. Laboratory values were as follows: plasma glucagon, 1.200 pg/mL: insulin, 29 kU/mL; erythrocyte sedimentation rate, Mefabolwn, Vol41, No 11 (November), 1992: pp 1171-1175

4.5 mm/h; hemoglobin, 8.4 g/dL; iron, 77 pg/dL; total iron-binding capacity. 300 pg/dL; glucose. 139 mg/dL; bilirubin, 0.3 mg/dL; alkaline phosphatase, 812 U/L; serum albumin. 2.6 g/dL; total protein, 5.3 g/dL; and all plasma amino acid levels were below normal. A computerized tomogram (CT) of the abdomen showed a 5-cm mass in the tail of the pancreas and multiple bilobar metastatic lesions throughout the liver. Celiac and hepatic artery angiography demonstrated that abnormalities observed on the CT scan were hypervascular. Results of a bone scan were normal. The patient was treated with intravenous amino acids, orally administered zinc sulfate, and subcutaneous injections of a long-acting somatostatin analogue. octreotide acetate (Sandostatin, Sandoz, Minneapolis, MN) in the hospital. Large doses of octreotide (400 PLgthree times daily) were required for resolution of her rash. She did well after discharge until she discontinued her medical therapy due to its cost in October 1989. In November 1989, the patient was readmitted to The University of Texas Medical Branch with recurrent and severe skin lesions, profound hypoaminoacidemia, and mild hyperglycemia. Body weight, serum albumin levels, and plasma amino acid levels were similar to values found on admission 3 months earlier. After 4 days of treatment with intravenous amino acids (1 L/d 10% Travasol. Baxter Healthcare, Deerfield, IL) and octreotide. she underwent surgical debulking of the tumor. A partial pancreatectomy with removal of the primary tumor and a complete left-hepatic-lobe resection with removal of most of the metastatic lesions were performed. Histological and immunohistochemical findings in the primary and metastatic lesions were consistent with a diagnosis of glucagonoma. METHODS This study was approved by the Institutional Review Board of The University of Texas Medical Branch. The experimental protocol was performed in the Clinical Research Center of The

From the Departments of Internal Medicine. Preventive Medicine and Community Health, Surgery, and Anesthesiology, The Universiry of Texas Medical Branch and The Shriners Burns Institute. Galveston, TX. Supported by National Institutes qf Health Grant No. CA 50330, Clinical Research Grant No. RR-00073. and a grantfrom the Shriners Hospitals. Address reprint requests to Samuel Klein, MD. Division of Gastroenferology G-64. The University of Texas Medical Branch, Galwsrootl, TX 77550. Copyright 0 1992 by W.B. Saunders Cornpar!? 0026-0495192/4111-0005$03.00l0

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1172

University of Texas Medical Branch after informed consent was obtained from the patient. Experimental Protocol Metabolic studies were performed 1 day before the patient’s surgery, 8 hours after discontinuation of intravenous octreotide therapy, and 5 hours after completion of an infusion of amino acids (1 L 10% Travasol). The study was started at 9:00 AM after the patient fasted overnight (12 hours). Intravenous lines were inserted into the antecubital vein of one arm for infusion of isotopes and into the contralateral dorsal hand vein, which was heated, for arterialized venous samp1ing.s Baseline blood samples were obtained to determine plasma glucagon,9 insulin,1° C-peptide,” glycerol, I2 free fatty acid,‘* P-hydroxybutyrate,13 amino acid,14 and glucoseL4 concentrations. Stable isotopes [lJ3C]leucine (16.8 kmol/kg priming dose followed by 0.28 pmol/kg/min continuous infusion), [6,6-*H2]glucose (26.7 pmolikg priming dose followed by 0.33 ~mol/kg/min continuous infusion), and [2Hs]glycerol (1.5 kmol/kg priming dose followed by 0.10 ~molikglmin continuous infusion) were infused for 120 minutes after giving a priming dose of each isotope and [13C]sodium bicarbonate (1.68 kmol/kg), as described previously. 14-16Blood and breath samples were taken before starting the isotope infusion to determine background isotopic enrichments, and at 90, 100, 110, and 120 minutes to measure leucine kinetics,‘j glucose production,14 lipolysis, and TG-FA substrate cycling (hydrolysis of triglyceride to fatty acids and glycerol and subsequent reesterification of fatty acids to trig1yceride),16 as described previously. Oxygen consumption, carbon dioxide production, and resting energy expenditure (REE) were measured by indirect calorimetry using a metabolic cart (Sensor Medics, Anaheim, CA). Urinary nitrogen excretion during the 2-hour infusion study was measured with an automated nitrogen analyzer (Fisons, Danvers, MA). Calculations The rates of appearance (Ra) of glycerol in plasma and of intracellular leucine were calculated using Steele’s equation” modified for physiological and isotopic steady state, Ra (kmolikgl min) = F/IE. F is the isotope infusion rate (~molikglmin) and IE is the isotopic enrichment of glycerol or cY--ketoisocaproic acid (aKICA) at plateau. Determination of plasma aKICA enrichment allows an estimation of leucine released from intracellular protein breakdown, because aKICA is derived directly from and is in equilibrium with intracellular leucine. Because the infusion of stable isotopes contributed to the mass of the substrate pool, the first equation is modified to Ra (kmol/kg/min) = (F/IE) - F. The rate of TG-FA cycling represents the rate of reesterification of hydrolyzed triglycerides. It is calculated as the difference between rate of triglyceride oxidation, measured by indirect calorimetry, and rate of triglyceride lipolysis measured as glycerol Ra. Glycerol Ra is a good index of whole-body lipolysis because released glyceroi cannot be directly reincorporated into triglyceride by adipose tissue.18 Therefore, whole-body triglyceride recycling was calculated as Total TG-FA cycling (pmolikglmin) = glycerol Ra (~molikgimin) - total triglycerideoxidation (kmolikgi min), where palmityl-stearyl-oleyl-triglyceride (C55H10406, 7,740 kcal/mol) was considered to be a typical triglyceride.19 The energy cost of TG-FA cycling was estimated by calculating the number of “high-energy” phosphate bonds (adenosine triphosphate [ATP] + adenosine diphosphate [ADP]) required for reesterification. It was assumed that eight high-energy phosphate bonds were required/m01 triglyceride recycled.20 Because it is estimated that 18 kcal heat are released/m01 ATP hydrolyzed and

KLEIN ET AL

synthesized,** the total energy cost is approximately 144 kcal/mol triglyceride recycled. Triglyceride oxidation rates and energy expenditure were calculated from measurements of oxygen consumption, carbon dioxide production, and urinary nitrogen excretion.22 RESULTS

Data obtained in our patient with glucagonoma were compared with values obtained from normal volunteers studied in our laboratory14,‘6,23.24(Tables 1, 2, and 3). In contrast to our patient, a 46-year-old woman, volunteer subjects were men of normal weight whose ages ranged from 21 to 42 years. Plasma concentrations of hormones and substrates are shown in Table 1. Plasma glucagon concentration was markedly elevated. Plasma insulin, glucose, free fatty acid, glycerol, and P-hydroxybutyrate levels were also increased compared with mean values obtained in normal volunteers. Plasma levels of most amino acids were markedly below normal (Table 2). Leucine, glucose, and lipid kinetics are shown in Table 3. The Ra of intracellular leucine, an index of protein breakdown rate, and nonoxidative leucine disposal, an index of protein synthesis rate, were within 2 SD of mean values found in normal volunteers. Leucine oxidation, an index of net protein loss, was 40% greater than but still within 2 SD of normal mean values. The percentage of leucine Ra oxidized (31%) was more than 2 SD above the normal mean value (21% 2 2%). The rate of glucose production was only 15% greater than normal, but was more than 2 SD above the normal mean. Glycerol Ra was 25% and more than 2 SD above the mean found in normal volunteers, but was similar to that of cachectic patients who had the same percentage of loss in body weightzs Rates of triglyceride oxidation and TG-FA cycling in our patient were numerically greater than but within 2 SD of mean values in normal volunteers. Approximately 60% of fatty acids released were subsequently reesterified to triglyceride. Measured REE (25 kcal/kg) was similar to that predicted by the Harris-Benedict equation (26 kcal/kg).26 The energy cost of TG-FA cycling was approximately 20 kcal/d and accounted for less than 2% of REE. Oxygen consumption was 182 mL/min, carbon dioxide production was 140 mL/min, and nitrogen excretion in urine during the 2-hour study was 0.8 g.

Table 1. Metabolic Factors in a Patient With Glucagonoma and in Normal Volunteers Patient

With

Glucagonoma

Glucagon (pg/mL) Insulin (@J/mL) Glucose (mg/dL) Glycerol (KmoliL) Free fatty acids (kmol/L) 6-hydroxybutyrate (wmol/L)

10,040t 13t 161t 80

Normal Volunteers*

109 + 6 6 -t 0.01 96 * 3 6Ok

10

1,450t

420 ? 90

170t

50 + 10

*Data

(mean + SE) from normal volunteers studied in our labora~ory.‘w&H tValue greater than 2 SD above mean value for normal volunteers.

METABOLIC ALTERATIONS

IN GLUCAGONOMA

Table 2. Plasma Amino Acid Concentrations With Glucagonoma

SYNDROME

(pmol/L)

in a Patient

and in Normal Volunteers Patient With Glucagonoma

Normal Volunteers’

Glucogenic amino acids Alanine

34t

255 + 13

Arginine

19t

110 + 7

Aspartate

2t

10 + 1

Glutamate/glutamine

63t

546 + 56

Glycine

73t

226 + 32

Histidine

38t

80 + 3

Methionine

16

Proline

18 + 4

2t

213 + 16 119 + 14

Serine

29t

Threonine

17t

127 + 14

Valine

32t

220 5 9

Glucogenic and ketogenic amino acids lsoleucine

40t

59 * 3

Lysine

34t

167 + 9

Phenylalanine

36t

50 III 2

Tyrosine

34t

50 * 4

27t

113 t 10

Ketogenic amino acid Leucine *3ata

(mean t SE) from normal volunteers studied in our labora-

tory.‘4 Walue less than 2 SD below mean value for normal volunteers.

DISCUSSION

This is the first study in which isotope tracers have been used to characterize metabolic abnormalities in a patient with a glucagon-secreting, islet-cell tumor. Leucine kinetics were similar to normal, whereas glucose and lipid kinetics were slightly elevated. Because of the need for ongoing medical therapy in our patient, metabolic studies were not performed under ideal research conditions. The studies were performed after the patient fasted for 12 hours overnight, 8 hours after discontinuation of intravenous octreotide therapy, and 5 hours after completion of an amino acid infusion. These values were compared with those obtained in normal volunteers who had fasted for 12 hours overnight. Although the medical therapy could have affected the study results, it seems unlikely for several reasons. The half-life (tliz) of octreotide is biexponential, with the first tl12 at 10 minutes and the second at 90 minutes.Z7 Therefore, little if any octreotide should have Table 3. Leucine, Glucose, and Lipid Kinetics in a Patient With -

Glucagonoma and in Normal Volunteers [pmol/kg/min) Patient With Glucagonoma

NO~llld Volunteers*

Intracellular leucine Ra

2.70

2.79 t- 0.17

Leucine oxidation

0.83

0.59 ? 0.08

1.87

2.20 5 0.11

Nonoxidative leucine disposal Glucose production Glycerol Ra

12.9t 3.04t

11.1 t- 0.03 2.41 ? 0.13

Triglyceride oxidation

1.35

1.15 ? 0.30

TG-FA cycling

1.69

1.30 + 0.11

*Data (mean t SE) from normal volunteers studied in our laboratorv.‘4.‘6.23 Walue greater than 2 SD above mean value for normal volunteers.

1173

been circulating in the bloodstream at the time of the study. In fact, the plasma concentration of glucagon was quite high (10,400 pg/mL), suggesting that even if residual octreotide was present the tumor was still secreting large amounts of glucagon. That infused amino acids should have been cleared completely before the infusion study began is supported by low plasma amino acid concentrations found in our patient. Therefore, our patient should have been in a postabsorptive state. We have previously found that even during amino acid infusion, the rate of glucose production does not change. I4 In addition, the presence of an amino acid-induced metabolic effect would strengthen our findings that lipolysis was increased, but not protein catabolism. If an amino acid infusion had an effect. it would likely have reduced glycerol Ra and increased leucine flux. The presence of a IO-kg loss in body weight in our patient reflects an imbalance between energy intake and energy expenditure. Measured REE appeared to be normal because it was similar to that predicted by the Harris-Benedict equation. However, without knowing our patient’s preillness REE, we cannot exclude the possibility that her metabolic rate had increased. Furthermore, the Harris-Benedict equation may not be reliable in persons who have experienced significant weight loss. Studies of patients with anorexia nervosaZx and of food-restricted normall’ and obe&’ subjects have found that REE was below that predicted by body size, age, and sex. Even in persons of normal weight, validity of the Harris-Benedict equations has been questioned by studies demonstrating that these equations overestimated measured REE by 10% to 1.5%.31Therefore. it is possible that “normal” energy expenditure as judged by the Harris-Benedict equation in our patient represents glucagon stimulation of metabolic rate. Nevertheless, REE was not excessive (1,250 kcal/d), suggesting that total energy intake was low; octreotide therapy-induced malabsorption3? could also have contributed to her weight loss. The considerable amount of weight loss in our patient could have influenced metabolic variables evaluated in this study; therefore, it is difficult to separate metabolic effects of chronic energy deficit from chronic glucagon excess. The effect of short-term glucagon infusion on protein33J4 and glucose7 metabolism in normal volunteers is opposite to that of chronic negative energy balance,35-37 so data from our patient may represent a balance between these two divergent metabolic influences. Because both glucagon infusion3x-40 and inadequate energy intake’” stimulate lipolysis, lipid kinetics in our patient may reflect an additive effect of glucagon excess and energy deficiency. Values for leucine kinetics in our patient suggest that abnormalities in protein metabolism were minimal. Protein and energy restriction causes a decrease in rates of protein synthesis, protein breakdown, and urea production.35J6 In contrast, glucagon excess has been found to increase proteolysis, amino acid oxidation, and urea production.33.Q In our patient, leucine Ra (an index of protein breakdown) and nonoxidative leucine disposal (an index of protein synthesis) were normal. The possible increase in percentage of leucine flux that was oxidized may reflect the effect of glucagon excess. These results suggest that muscle-wasting

1174

KLEIN ET AL

observed in our patient was related to net protein loss caused by increased amino acid oxidation and decreased protein synthesis, and was not caused by an increase in the absolute rate of protein breakdown. The profound hypoaminoacidemia observed in our patient and in others with glucagonoma4 cannot be explained by glucagon-stimulated gluconeogenesis alone, because both glucogenic and nonglucogenic plasma amino acid levels were depressed. Our data suggest that an increased clearance of plasma amino acids by the liver, caused by glucagon-stimulated glucose production and amino acid oxidation, may be responsible for the decrease in all plasma amino acid levels. The basal rate of glucose production in our patient was approximately 15% greater than the mean value reported in normal volunteersI In normal postabsorptive humans, approximately 35% of glucose Ra is from the breakdown of hepatic glycogen and approximately 65% is from gluconeogenesis from plasma precursors such as amino acids, glycerol, and lactate.41 Because both inadequate energy intake and glucagon deplete hepatic glycogen stores, it is likely that an even greater percentage of glucose produced in our patient came from gluconeogenesis. An earlier study of substrate splanchnic balance in a patient with glucagonoma found that plasma amino acids, lactate, and glycerol could account for all glucose produced by the liver.3 In normal volunteers, glucagon infusion increases the rate of gluconeogenesis from amino acids.42 The relatively small increase in the rate of hepatic glucose production suggests that peripheral insulin resistance must also contribute to hyperglycemia in patients with glucagonoma.

Whole-body lipolytic rates were increased in our patient when compared with rates in previously studied, normal volunteers.16 However, lipolytic rates were similar to those in patients with chronic weight 10~s~~which suggests that energy imbalance in conjunction with glucagon excess may be an important factor in increased mobilization of fat. The increased rate of lipolysis was not accompanied by a concomitant increase in fat oxidation, so that the rate of released fatty acids reesterified to triglyceride (TG-FA cycling) increased. The increased rate of TG-FA cycling did not consume a significant amount of energy and accounted for less than 2% of the measured REE. The present study underscores the complex nature of metabolic abnormalities in patients with glucagonoma. It is difficult to separate effects of inadequate food intake from effects of glucagon excess on energy, protein, carbohydrate, and lipid metabolism; both are probably responsible for observed changes in body mass and plasma amino acid and glucose levels. The metabolic effects of chronic glucagon excess may counteract many normal adaptive responses to inadequate food intake, and thereby accelerate development of a malnourished state and help to maintain it. Even small glucagon-associated metabolic alterations could have dramatic clinical and physiological effects if these alterations continue for long periods of time. ACKNOWLEDGMENT The authors thank Dr Suzanne McClure for referring her patient for study; Betty Krumholz, LeAnne Romano, and Susan Fons for their technical assistance; and Billie Roach for preparation of the manuscript.

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METABOLIC

ALTERATIONS

IN GLUCAGONOMA

SYNDROME

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