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Is profound peripheral insulin resistance in patients with pancreatic cancer caused by a tumor-associated factor?
Is Profound Peripheral Insulin Resistance in Patients With Pancreatic Cancer Caused by a Tumor-Associated Factor? Johan Permert, MD, Thomas E. Adrian, PhD, Per Jacobsson, MD, Lennart Jorfelt, MD, A. Brent Fruin, BS, J6rgen Larsson, MD, Omaha,Nebraska
Diabetes in patients with pancreatic cancer occurs in 70% to 80% of the patients and is characterized by high plasma levels of insulin. In type II diabetes that is not associated with pancreatic cancer, peripheral insulin resistance and impaired muscle glycogen synthesis are major pathogenic factors. We investigated peripheral insulin sensitivity in patients with pancreatic cancer before and after tumor removed. The effects of pancreatic tumor extracts on glycogen synthesis in skeletal muscle in v i t r o and the tumor content of pancreatic islet hormones were also investigated. Marked peripheral insulin resistance was found in the patients with pancreatic cancer and was more pronounced in the diabetic patients than in the nondiabetic patients. Insulin sensitivity was not correlated with weight loss, tumor size, or bflirubin levels but improved after surgery. Tumor extracts from diabetic patients with pancreatic cancer caused a marked reduction of glycogen synthesis in skeletal muscle in vitro. All tumors contained islet hormones but not in concentrations sufficient to explain the effect on glycogen synthesis. These findings indicate that a diabetogenic factor associated with pancreatic adenocarcinomas could be involved in the development of the profound peripheral insulin resistance and thereby could contribute to the high incidence of diabetes observed in patients with pancreatic cancer.
Fromthe Departmentsof Surgery(JP, JL), PulmonaryMedicine(PJ), and ClinicalPhysiology(LJ), UniversityHospital,Link6ping,Sweden, and the Department of Biomedical Sciences (JP, TEA, ABF), CreightonUniversitySchoolof Medicine,Omaha,Nebraska.Supported by grants fromthe SwedishCancerSociety(2870-B91-01XAC)and the National Cancer Institute (CA 44799). Requests for reprints should be addressedto Johan Permert,MD, Departmentof Surgery,UniversityHospital,S-581 85 Link6ping,Sweden. Presentedat the 33rdAnnualMeetingof the Societyfor Surgeryof the AlimentaryTract, San Francisco,California,May 11-13, 1992.
iabetes or impaired glucose tolerance occurs in 70% to 80% of patients with pancreatic cancer. DiaD betes has previously been proposed as a predisposing factor for pancreatic cancer [1]. However, recent studies suggest that diabetes is more likely to be a consequence of the tumor [2-4]. Generally, the diabetic state occurs in a close temporal relationship to the cancer diagnosis. After surgery, glucose tolerance improves, and, in some patients, diabetes can even disappear [5]. The diabetic state in patients with pancreatic cancer is characterized by high plasma levels of insulin and by reduced body insulin sensitivity [2,3,6]. In addition, plasma concentrations of several other pancreatic peptides, such as islet amyloid polypeptide (IAPP), glucagon, and somatostatin, are increased [7]. Several studies have reported an altered ~-cell response to hyperglycemia in patients with pancreatic cancer [2,3,6], and a recent report suggested that there is an alteration in IAPP secretion by islet tissue close to the tumor [8]. Endocrine cells are frequently found in exocrine pancreatic adenocarcinomas, suggesting an endocrine component in these tumors that may secrete hormonal peptides [9]. After surgery, when the influence of the tumor is removed, the secretory profiles of the pancreatic peptides are normalized, and glucose tolerance improves [5,7,8]. In patients with type II diabetes, peripheral insulin resistance is a major pathogenic factor, and reduced glycogen synthesis in the skeletal muscles plays a major role in this metabolic abnormality [10]. In the present study, peripheral insulin sensitivity in patients with pancreatic cancer was investigated before and after surgery, in relation to their diabetic status and to associated diabetogenic factors, such as jaundice and weight loss. The effects of pancreatic tumor extracts on glycogen synthesis in skeletal muscle in vitro were also investigated, and the hormonal peptide content in these same extracts was measured by radioimmunoassay. PATIENTS AND M E T H O D S Clinical studies: Study design. Sixteen patients who were considered as candidates for radical surgery because of pancreatic ductal adenocarcinoma arising from the head or uncinate process and 7 age-matched healthy volunteers were examined in the surgical department at University Hospital in Link6ping, Sweden. Clinical data of patients and control subjects are given in Table I. Patients with signs of infection, renal failure, or cardiac failure, as assessed by medical history, clinical examination, and routine laboratory investigations, were excluded. The diagnosis of pancreatic cancer was verified histo-
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TABLE I
Characteristics of Patients and Control Subjects
Pancreatic cancer Controls
No. of Patients
Sex (M/F)
Age (y)
Weight (kg)
Height (cm)
SerumBilirubin (mmol/L)
Weight* Loss (%)
Tumor Size (cm)
16 7
9/7 4/3
65.7 _+ 2.8 66.1 _+ 1.2
68.1 _+ 3.5 79.1 _+ 4.7
171 _+ 4.2 176 + 3.3
102.0 _+ 17.2 12.1 _+ 1.7
5.8 -+ 1.4 0
3.2 _+ !.2 --
*Weight loss during the last 3 months in percentof basal weight.
logically or cytologically in all patients. Computed tomography and ultrasonography were performed to evaluate tumor size and resectability. All patients had abnormal bilirubin levels (Table I) but had been treated endoscopically with a biliary stent at least 48 hours before the investigations. No dietary restrictions were imposed, and all patients had normal physical activity in the hospital ward. The control subjects had no history of diabetes and were not receiving any medical treatment known to interfere with glucose metabolism. In 5 of the 16 patients, the tumor was considered to be resectable, and a subtotal pancreatectomy was performed using the method of Gall et al [11]. In this operation, 10% to 15% of the tissue from the tail of the pancreas is retained after surgery. The operation was radical in all patients, according to macroscopic findings and to the pathology report. The patients were examined again 3 months postoperatively. At this time, no patient had any sign of recurrence, according to clinical examination and ultrasonography. Methods. A detailed diabetic history was recorded, and an oral glucose tolerance test was performed in all patients and control subjects. Assessment was made using the diagnostic criteria of the World Health Organization (WHO) [12]. The glucose metabolic rate was determined using a hyperinsulinemic euglycemic clamp. After a 12-hour overnight fast, the subjects arrived in the laboratory in the morning. During this period, the patients had been given a standardized intravenous infusion of glucose at a rate of 30 mg/kg/h. For insulin-requiring diabetics, insulin was added in the amount required to maintain blood glucose between 5 and 6 mmol/L (equivalent to 90 to 108 mg/dL). Plastic eannulas were inserted into the radial artery of one arm for blood sampling and an antecubital vein of the contralateral arm for the infusions. The arterial eannula was flushed with 0.15 M saline, which contained 10 U/mL of heparin. A total of less than 1,000 U of heparin was given to each patient. The basal glucose and insulin levels were determined from two samples taken 5 minutes apart. A primed infusion of insulin (Actrapid Human, Novo, Copenhagen, Denmark) at a level of 0.5 mU/kg/min, chosen according to the protocol of De Fronzo, to obtain a plasma level of about 50 mU/L, was started [13]. After 90 minutes of infusion, the rate was increased to 1.0 mU/kg/min in
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order to reach a plasma level of about 100 mU/L, and the insulin infusion was continued at this rate for another 90 minutes. Insulin was dissolved in 0.15 M saline and 3 mL of the patient's own blood to give a final concentration of 300 mU/mL. Glucose (1.1 M glucose and 40 mM NaCI, Kabi Pharmacia, Stockholm, Sweden) was infused to maintain blood glucose at each patient's euglycemic (basal) level. The maintenance glucose dose was varied in order to obtain this euglycemic glucose level 4- 0.25 mmol/L. Blood glucose was measured every fifth minute, and the rate of glucose infused to maintain euglycemia was adjusted accordingly. For each insulin concentration, total body glucose uptake was calculated from the glucose infusion rate (mg/kg/min) averaged over the entire 90-minute infusion period. Samples for measurement of serum insulin were drawn every 10 minutes during the hyperinsulinemic state. /n dtro studies: Study design. The study utilized tumor tissue and pancreatic tissue adjacent to the tumor from eight patients with pancreatic ductal adenocarcinoma. In addition, normal pancreatic tissue was obtained from a patient with normal glucose tolerance who had surgery for a benign duodenal disorder. Five of the eight pancreatic tumors were from diabetic patients with cancer, and three were from patients with normal glucose tolerance. The effect of extracts from the different pancreatic tumors and from the sample of normal pancreas on glycogen synthesis in skeletal muscle was studied. Peptide concentrations, in all the extracts from tumors and pancreatic tissues adjacent to each tumor, were measured by radioimmunoassay. Tissue collection and extraction. The tissues were frozen in liquid nitrogen immediately after resection and stored at - 7 0 ~ for subsequent extraction. The tissues were weighed while still frozen and then plunged into boiling 0.5 M acetic acid (10 mL/g wet weight) and boiled for 10 minutes. The extracts were then partially purified on C- 18, reverse phase, Sep-Pak cartridges (Waters, Milford, MA), using multichannel syringe ram pumps (Harvard Instruments, Cambridge, MA). After washing, the Sep-Paks were eluted with 3 mL of 70% acetonitrile with 0.1% trifluoroacetic acid. These eluates were lyophilized and reconstituted in buffer (60 mM phosphate, pH 7.4, with 0.1% Triton-X 100 and 0.1% bovine serum albumin [BSA, Sigma Chemical Co., St. Louis, MO]) prior to assay and investigation of biologic activity.
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Determination of glycogen synthesis. The effects of the tumor extracts were investigated at two different concentrations (corresponding to 0.75 and 2.5 #g tumor tissue/mL incubation medium). As a positive control in the bioassay system, incubations with calcitonin gene-related peptide (CGRP) were performed at a concentration (10 nM) that had been previously shown to cause a marked decrease in muscle glycogen synthesis using this method
0.5 mU/kg/min
3'
[1,1.
r---***--7 I-*'7
I
**
F
Male Wistar rats (250 to 300 g) were guillotined, and strips (25 to 35 mg) of the soleus muscles were dissected from the hind legs, as previously described [15]. The muscle strips were attached at resting length to stainless steel clips. After the strips were weighed, they were transferred to silicon-treated Warburg flasks containing 4 mL of pre-oxygenated (95% 02:5% CO2) Krebs Ringer bicarbonate buffer [14] with 1.5% (w/vol) BSA (essential fatty acid free, Sigma Chemicals Co.) at pH 7.4. After 30 minutes of oxygenated preincubation, the strips were transferred to flasks containing fresh oxygenated buffer. D-(U-14C) glucose (0.5 #Ci/mL, Sigma), insulin (1.0 M), and the experimental agent (tissue extract or CGRP) were added, and the strips were incubated for 45 minutes. After 15 minutes of oxygenation, the flasks were sealed. At the end of the incubation, the muscle strips were instantly frozen on dry ice, and glycogen was extracted as previously described [16]. After three separate washings in 70% ethanol, the glycogen was redissolved in 1 mL of water and mixed with 5 mL of scintillation cocktail (Safety Solve, Research Products International, Mount Prospect, IL). The incorporation of 14C in the glycogen was measured on a beta counter. For each experimental incubation, a control incubation using a comparable muscle strip obtained from the same soleus muscle was performed in the absence of the experimental factor. Glycogen synthesis in the experimental incubations was expressed as the percentage of the synthesis in the control incubations. Radioimmunoassay: The concentrations of islet hormonal peptides (C-peptide, glucagon, somatostatin, pancreastatin, pancreatic polypeptide, and islet amyloid polypeptide) were measured by radioimmunoassay. Insulin was not measurable because the tissues were extracted by boiling in acid, thereby breaking the interchain disulfide bonds. Instead, the level of C-peptide was determined, as this peptide is not affected by boiling and is produced in equimolar concentrations to insulin. C-peptide was assayed using a commercially available antiserum (Novo, Copenhagen, Denmark), and the other peptides were measured by specific assays as previously described [8,17]. Plasma insulin was measured using a commercially available kit (Novo). Statistical analysis: All values presented in the text are given as mean 4- SEM. Comparison between the groups was carried out with Wilcoxon's signed rank test, using paired analysis for the groups studied before and after surgery. Correlations between glucose metabolic rate and weight loss, serum bilirubin levels, and tumor size were calculated by regression analysis.
1.0 mU/kg/min
mg/kg/min. 5.
I
*** - 1 F *** -I
// // // // // // .-/ //
1'
0
// // // // // // // //
NDDND +D
PC
ND D ND +D
C
PC
C
Figure 1. The glucose metabolic rate at two different insulin infusion levels (0.5 and 1.0 mU/kg/min) during hyperglycemic clamp in pancreatic cancer patients (PC, n = 16) and in controls (C, n = 7). The results are given for subgroups of nondlabetic patients (ND, n = 5) and diabetic patients (D, n = 11), and for all pancreatic cancer patients (ND -I- D, n = 16). The asterisks denote significance between the groups (*p <0.05, **p <0.01, and * * * p <0.001).
RESULTS Clinical study: Four of 16 patients (25%) with pancreatic cancer had a previous history of diabetes. In all four, diabetes had developed less than 20 months prior to the cancer diagnosis. Oral glucose tolerance tests indicated that 11 of 16 (69%) patients were diabetic by WHO criteria. Of the 11 diabetics, 6 (54%) required insulin treatment. Four of the five patients with resectable tumors were diabetic prior to surgery, and the diabetic status of three of the four improved postoperatively. After surgery, glucose tolerance became normal in two of the patients who were diabetic before surgery. Insulin treatment could be withdrawn from a third patient postoperatively. The patient with normal glucose tolerance preoperatively remained normal after surgery. Blood glucose levels in the diabetic patients with pancreatic cancer (mean: 6.6 4- 0.3 retool/L) were significantly increased (p <0.01) compared with the controls (mean: 5.0 4- 0.3 mmol/L) but were not different in the nondiabetic patients (mean: 5.3 4- 0.3 mmol/L). Plasma insulin was also elevated (p <0.05) in the diabetic patients with pancreatic cancer (mean: 18.3 4- 2.7 mU/L) compared with the controls (mean: 9.3 4- 2.0 mU/L), but no significant difference was seen between nondiabetic patients (mean: 12.3 4- 3.6 mU/L) and the controls. During the hyperinsulinemic clamp, the insulin levels during the two different infusion rates in the patient group and in the control group were comparable. (Levels of the low-dose infusion in patients and controls were 43.3 4- 3.9 mU/L and 47.4 4- 4.9 mU/L, respectively; levels of the high-dose infusion in patients and controls were 87.9 4- 9.6 mU/L and 84.2 4- 3.6 mU/L, respectively.) The
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PERMERT ET AL
0,5 mU/kg/min
% ,5~-1
1,OmU/kg/min
500,
% lOO_
x
x
=
t
x
400.
75_
300.
-r
200'
50.
100,
!1
0
Pre-op
Post-op
Pre-op
Post-op
Figure 2. The glucose metabolic rate, expressed as the percentage of the preoperative levels, at two insulin infusion levels (0.5 and 1.0 mU/kg/min), before and after subtotal pancreatectomy in the five patients with resectable tumors. Pre-op = preoperatively; Post-op -- postoperatively.
C-peptide
I0,000_ pmoVg 7,500_
90-
5,000_
60-
2,500 _
500-
somatostatin
pmol/g
800 _
375 _
600
250_
400--
pmol/g
glucagon
-
o
8
125--
200 -
o
m
O
50~
~mol/g
PP
8 - pmol/g
pancreastatin
O 375 -.
6-
9
250_
4--
~
1252
2_
~
O
Pancreatic Tumor
Adjacent Pancreas
Pancreatic Tumor
Adjacent Pancreas
Figure 4. Concentrations of the pancreatic islet hormones in pancreatic tumor tissues and in pancreatic tissue adjacent to the tumor. Concentrations of individual tumors are given by circles ( o p e n circles = diabetic patients; solid circles = nondiabetic patients). Concentrations in the adjacent pancreas are given as means -I- SEM (solid IKlUaI~,~).lAPP = islet amyloid polypeptide; PP --- pancreatic polypeptide.
glucose metabolic rate was decreased in the patients with pancreatic cancer compared with the control group at both insulin levels (Figure 1), This decrease was more pronounced in the diabetic patients than in the nondiabetic patients at both insulin levels (both p <0.05). The nondiabetic patients had a significantly lower glucose 64
I
I
I
I
D
ND
NP
CGRP
Control experiments
Figure 3. The effect on muscle glycogen synthesis of pancreatic tumor extracts from diabetic patients (D, n -- 5) and nondiabetie patients (ND, n -- 3). Control incubations with normal pancreas (NP) and calcitonin gene-related peptide ( ~ P ) (10 nM) were performed. The effects of tumors are given individually, and the results of the control incubations are given as the mean 4- SEM of at least four incubations.
30-
C*
0
Pancreatic tumors
IAPP
120- pmol/g
25-
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metabolic rate at the low insulin level but not at the higher one (p = 0.011 and p = 0.14, respectively) (Figure 1). No significant correlation was observed between glucose metabolic rate and bilirubin levels (p = 0.16), weight loss (p = 0.96), or tumor size (p = 0.56). In all five patients with resectable tumors, glucose metabolic rates improved markedly after surgery at both insulin levels and reached rates that were not significantly different from those of the controls (Figure 2). /n fftro studies: Four of five pancreatic tumor extracts from patients with diabetes had decreased glycogen synthesis at the higher concentration that was investigated (2.5 ~g tissue/mL) (Figure 3). The effect observed was comparable with that caused by CGRP at the 10-nM concentration. No effect was seen with extracts of normal pancreatic tissue or tumors from nondiabetic patients (Figure 3). None of the extracts influenced glycogen synthesis at the low concentration. Each of the islet hormones measured was present in all the tumor extracts, but concentrations varied considerably (Figure 4). In a few cases, the concentration of a particular peptide was higher in the tumor tissue than in the surrounding pancreatic tissue, but there were no significant differences between these tissues for any of the peptides (Figure 4). No significant differences were seen between the extracts from diabetic and nondiabetic patients. COMMENTS In the clinical part of this study, peripheral insulin sensitivity was investigated in patients with pancreatic cancer. In the in vitro part of the study, the concentra-
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tions of pancreatic peptides in tumor extracts and the effect of the extracts on glycogen synthesis in skeletal muscle were studied. The glucose metabolic rate was investigated at two insulin infusion rates (0.5 and 1.0 mU insulin/kg/min). The lower infusion rate was selected to obtain a plasma level of insulin corresponding to the endogenous production of insulin observed in pancreatic cancer patients during mild hyperglycemia, as determined in a previous study [2]. The higher infusion rate was selected to obtain plasma insulin levels in the upper physiologic range in which glucose uptake would not be significantly affected by endogenous glucose production [13]. The decrease in the glucose metabolic rate in patients with pancreatic cancer demonstrated a state of peripheral insulin resistance, which was more severe in diabetic patients than in nondiabetic patients. In several of the nondiabetic patients, the glucose uptake returned to normal when insulin levels were elevated, whereas the glucose metabolic rate in the diabetic patients remained decreased. This indicates a reduced responsiveness of glucose metabolism to insulin in diabetic patients with pancreatic cancer but not in the nondiabetic patients, at least in the range of plasma insulin levels investigated. Since no correlation was found in this study between decreased insulin sensitivity and weight loss, bilirubin levels, or tumor size, the reduced peripheral insulin sensitivity could not be explained by associated diabetogenic factors. In patients with type II diabetes that is not associated with pancreatic cancer, peripheral insulin resistance is an important factor in the development of diabetes [10]. In most type II diabetic patients, insulin resistance is caused by obesity, but diabetes can also appear in subsets of normal weight individuals [18]. Metabolically, however, these normal weight, type II diabetic patients have been found to resemble overweight individuals. An underlying factor causing both the reduced insulin sensitivity and the metabolic disorder of diabetes has been hypothesized [18]. In patients with pancreatic cancer, the reduced peripheral insulin sensitivity cannot be explained by obesity because patients with pancreatic cancer, as in this study, are generally lean. The postoperative improvement in glucose metabolic rate, which was seen in all patients after tumor removal, indicates that pancreatic tumors may have a role in the development of the insulin resistance that is seen in pancreatic cancer. After pancreatic resection, glucose metabolism improved because of increased insulin sensitivity, despite the marked reduction in insulin secretion. The postoperative improvement of diabetic status in patients with pancreatic cancer, which was also seen in a previous group of patients [5], demonstrates the profound importance of insulin resistance in diabetes associated with pancreatic cancer. Glucose metabolism can be influenced by malignancy in general. Impaired glucose tolerance has been reported in up to 30% of patients with different types of tumors [19]. Generally, glucose intolerance occurs more frequently in cachectic patients with advanced tumors, but peripheral insulin resistance has also been reported in noncachectic patients with a limited tumor disease [20].
However, the frequency of diabetes and glucose intolerance is higher in patients with pancreatic cancer than in those with any other type of malignancy [19]. The severity of the disorder is also greater in patients with pancreatic cancer; nearly half of these patients are frankly diabetic. This suggests that pancreatic tumors may produce a specific factor that is important in the development of diabetes. The possible role of pancreatic tumors in the development of insulin resistance was further emphasized by the inhibitory effect of tumor extracts on muscle glycogen synthesis in vitro, seen in four of five diabetic patients evaluated. The results of the radioimmunoassay indicated the production of all the different islet hormones in exocrine pancreatic tumors, but concentrations of the individual peptides in the tumors were not sufficient to cause diabetogenic effects. Higher concentrations of IAPP, glucagon, and somatostatin are required to exhibit diabetogenic effects in vivo and in vitro, according to previous studies [14,15,21,22]. Therefore, the inhibition of glycogen synthesis observed in this study could not be explained on the basis of tumor production of any of the peptides measured. Whether the observed effect was the result of interactions of the measured peptides or was caused by some other diabetogenic factor cannot be determined from the results of this study. However, the lack of effect seen with the extracts from normal pancreatic tissue and from the tumors of nondiabetic patients with pancreatic cancer may be noteworthy. These tissues contained the diabetogenic peptides in concentrations that were comparable with, or higher than, the concentrations in the tumors that reduced glycogen synthesis. The production of a specific diabetogenic factor by pancreatic tumors is possible because, during the neoplastic event, structurally altered, ectopic, or yet unknown substances can be produced and released, as well as mature peptides [23,24]. However, further investigations, such as detection of mRNA for different peptides, are necessary before the production of peptides and hormones by exocrine pancreatic tumors can be explained. In patients with type II diabetes who do not have pancreatic cancer, a combination of insulin resistance and altered insulin secretion is often required for the development of frank diabetes [I0]. Patients with pancreatic cancer show changes in the secretory response of insulin to glucose and also exhibit alterations in the secretion of several other pancreatic peptides [7]. Therefore, it is likely that other disorders, in addition to a reduced peripheral insulin sensitivity, contribute to the high frequency of frank diabetes seen in patients with pancreatic cancer. For example, if the tumor, directly or indirectly, produces a factor that is responsible for the changes observed in the peripheral tissues, effects on liver metabolism are also likely, since the concentration of such a factor would be much higher in portal blood than in the peripheral tissues. In conclusion, profound peripheral insulin resistance was found in patients with pancreatic cancer, and this appeared to be associated with the tumor itself. The decrease in insulin sensitivity was more pronounced in the
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diabetic patients and was not correlated with weight loss, tumor size, or bilirubin levels. It is tempting to speculate that the observed resistance to insulin in these patients also influences other metabolic pathways, such as amino acid and fat metabolism, and therefore contributes to the pronounced cachectic state often seen in patients with pancreatic cancer.
REFERENCES
1. Kessler II. Cancer mortality among diabetics. J Natl Cancer Inst 1970; 44: 673-86. 2. Permert J, Ihse I, Jorfeldt L, Arnquist H, von Schenck H, Larsson J. Pancreatic cancer is associated with impaired glucose metabolism. Eur J Surg; In press. 3. Schwarz SS, Zeidler A, Moossa AR, et al. A prospective study of glucose tolerance, insulin, C-peptide, and glucagon responses in patients with pancreatic carcinoma. Dig Dis 1978; 23:1107-14. 4. Boyle P, Hsieh C, Maisonneuve P, et al. Epidemiology of pancreas cancer (1988). Int J Pancreatol 1987; 5: 327-46. 5. Permert J, Jorfeldt L, Arnquist H, von Schenck H, Larsson J. Tumor removal improved glucose metabolism in humans. Clin Nutr 1992; 11: 17. 6. Cerosimo E, Pisters P, Pesola G, McDermott K, Bajorunas D, Brennan MF. Insulin secretion and action in patients with pancreatic cancer. Cancer 1991; 67: 468-93. 7. Permert J, Larsson J, Jorfeldt L, von Schenk H, Adrian TE. Islet hormone secretion in pancreatic cancer [abstract]. Digestion 1992; 52: 111. 8. Adrian TE, Permert J, Larsson Ji Westermark GT, Pour PM, Westermark P. The diabetes in pancreatic cancer patients is associated with a marked increase in islet amyloid polypeptide secretion. Regul Pept 1992; 40: 105. 9. Permert J, Mogaki M, Andr6n-Sandberg ,~, Kazakoff K, Pour PM. Pancreatic mixed ductal-islet tumors--Is this an entity? Int J Pancreatol 1992; 2: 23-9. 10. Turner RC, Williamson DH. Control of metabolism and the alterations in diabetes. In: O'Riordan JLH, editor. Recent advances in endocrinology and metabolism. Vol 2. Edinburgh: Churchill Livingstone, 1982. 11. Gall FP, Hernanek P, Gebhardt CH, Meier H. Erweiterte Resektion der Pankreas und perieampull~iren Karzinome. Regionale, totale und partielle Duodenopankreatektomie. Lever Magen Darm 1981; 11: 179-84. 12. World Health Organization Expert Committee: Diabetes mellitus. Technical Report Series 1985; No. 741, Geneva, Switzerland. 13. De Fronzo RA, Tobin TD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237: E214-32. 14. Leighton B, Cooper GJS. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 1988; 335: 632-5. 15. Crettaz M, Prentki M, Zaninetti D, Jeanrenaud B. Insulin resistance in soleus muscle from obese Zucker rats. Biochem J 1980; 186: 525-34. 16. Cuedent G, Loten E, Jeanrenaud C, et al. Decreased basal, non-insulin stimulated glucose uptake and metabolism by skeletal muscle isolated from obese hyperglycemic mice. J Clin Invest 1976; 58: 1078-88. 17. Bloom SR, Long RG, editors. Radioimmunoassay of gut regulatory peptides. Philadelphia: WB Saunders, 1982: 2-100. 18. Caro J. Insulin resistance in obese and nonobese man. J Clin Endocrinol Metab 1991; 73: 691-7. 19. Glicksman AS, Rawson RW. Diabetes and altered carbohydrate metabolism in patients with cancer. Cancer 1956; 9:1127-34. 66
20. Copeland GP, Leinster S J, Davis JC, Hipkin LJ. Insulin resistance in patients with colorectal cancer. Br J Surg 1987; 74: 1031-5. 21. Unger RH, Orci L. Glucagon and the A cell. N Engl J Med 1981; 304: 1575-82. 22. Kahn SE, Klaff L J, Schwartz MW, et al. Treatment with a somatostatin analog decreases pancreatic B-cell and whole body sensitivity to glucose. J Clin Endocrinol Metab 1990; 71: 994-1002. 23. Artan-Spire S, Wolf B, Czernichow P. Developmental pattern of TRH-degrading activity and TRH content in rat pancreas. Acta Endocrinol 1984; 106: 102-8. 24. Bandram L, Hilstedt L, Rehfeld J. Progastrin expression in mammalian pancreas. Proc Natl Acad Sci U S A 1990; 87: 298-302. DISCUSSION D. K. Andersen (Chicago, IL): When patients have an initial diagnosis of pancreatic carcinoma, they have often been ill for some time with significant weight loss and altered nutrition. Your preoperative studies were probably performed close to the time of the operation, whereas your postoperative studies were probably performed when the patients had recovered from the operation and had presumably resumed normal weight-maintaining diets. How do you know whether these changes in insulin sensitivity are not merely a reflection of the nutritional status of your patients studied at two different time points and under two different nutritional circumstances? Your inference of insulin sensitivity from the glucose infusion rates during the clamp studies m a y be an accurate estimate of insulin sensitivity, provided that the hepatic glucose production is zero. Can you tell us whether you verified that hepatic glucose production was zero, and therefore that the glucose infusion requirements were truly a reflection of glucose uptake in the periphery? J o h a n Permert: These patients had lost weight before admission to hospital, but we didn't observe any correlation between the degree of weight loss and our results. In a previous study, we did not observe any difference in the frequency of diabetes between those patients who had lost considerable weight and those who had lost little weight. Weight loss protein caloric malnutrition can cause impaired glucose tolerance, but it is unlikely to explain the very high frequency of frank diabetes in these patients. Weight loss influenced the impaired glucose metabolism seen in these patients but cannot explain the impaired metabolism fully. To address the question of whether the insulin levels were sufficient to block hepatic glucose production, we adopted the protocol described by DeFronzo. In this study, we did not perform any investigations of endogenous glucose production. In previous studies that used this infusion level, we found a block of the hepatic glucose production. Daniel Elahi (Boston, MA): I would like to challenge the previous statement about hepatic glucose production. Just because you showed a blockage of hepatic glucose production previously, I presume, in normal individuals, does not mean that the same dose will suppress the level in those who are ill.
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The difference between glucose utilization that you show, although statistically significant, is not large. If you test any patient who is ill, you can dearly demonstrate glucose intolerance. In fact, if you perform an oral glucose tolerance in most hospitalized patients, you will often find indications of diabetes because of the stress of hospitalization. Is your control group adequate? You may want to study some patients without carcinoma who are hospitalized for other reasons. Johan Permert: That's a good suggestion about performing the study in patients with other diagnoses. Clinically, there was postoperative improvement in the diabetic status of these patients. Four patients were diabetic before surgery. In two, subsequent glucose tolerance tests were normal, and one patient who required insulin preoperatively did not postoperatively. R. H. Bell (Cincinnati, OH): Pancreatic cancers in the head of the gland often obstruct the pancreatic duct and cause pancreatitis in the tail of the gland, and we know that acute pancreatitis or chronic pancreatitis can cause abnormalities in glucose metabolism. Is it possible that some of the improvement that you observed in glucose tolerance after resection of the tumor related to improved function in the remaining gland? Could you tell us how many patients had a Whipple procedure and total pancreatectomy, and whether there were any differences between patients undergoing different operations?
J o h a n Permert: All of the patients underwent the same operative procedure, a subtotal pancreatectomy, during which approximately 85% of the pancreatic tissue was resected. M. F. Brennan (New York, NY): We have had experience using euglycemic clamps in these patients, and to answer Dr. Andersen's question, at 1 mU/kg/min, a serum insulin level of about 80 mU/L will completely suppress the endogenous glucose production by the liver as measured by tritiated glucose in the 3' position. More worrisome, as we and many others have shown, is that if you use euglycemic clamps, you will have a very significant decrease in the plasma amino acid concentration, and if you don't supplement the plasma amino acid concentration, then your interpretation of not only the glucose turnover, but also the protein turnover, is quite different. Many of these changes can be completely reversed in normal individuals by supplementing the plasma amino acid concentration, which will drop by at least 50% if you do not supplement the amino acids. Did you supplement your euglycemic clamp so that it was at least euleucinemic or euaminoacidemic, because if you did not, then the interpretation would be quite different? Johan Permert: No, we did not supplement for amino acids during the clamp. Our use of the original protocol of DeFronzo and our methods of interpreting the data are the same as those used previously by other groups.
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Diabetes in patients with pancreatic cancer occurs in 70% to 80% of the patients and is characterized by high plasma levels of insulin. In type II diabetes that is not associated with pancreatic cancer, peripheral insulin resistance and impaired muscle glycogen synthesis are major pathogenic factors. We investigated peripheral insulin sensitivity in patients with pancreatic cancer before and after tumor removed. The effects of pancreatic tumor extracts on glycogen synthesis in skeletal muscle in v i t r o and the tumor content of pancreatic islet hormones were also investigated. Marked peripheral insulin resistance was found in the patients with pancreatic cancer and was more pronounced in the diabetic patients than in the nondiabetic patients. Insulin sensitivity was not correlated with weight loss, tumor size, or bflirubin levels but improved after surgery. Tumor extracts from diabetic patients with pancreatic cancer caused a marked reduction of glycogen synthesis in skeletal muscle in vitro. All tumors contained islet hormones but not in concentrations sufficient to explain the effect on glycogen synthesis. These findings indicate that a diabetogenic factor associated with pancreatic adenocarcinomas could be involved in the development of the profound peripheral insulin resistance and thereby could contribute to the high incidence of diabetes observed in patients with pancreatic cancer.
Fromthe Departmentsof Surgery(JP, JL), PulmonaryMedicine(PJ), and ClinicalPhysiology(LJ), UniversityHospital,Link6ping,Sweden, and the Department of Biomedical Sciences (JP, TEA, ABF), CreightonUniversitySchoolof Medicine,Omaha,Nebraska.Supported by grants fromthe SwedishCancerSociety(2870-B91-01XAC)and the National Cancer Institute (CA 44799). Requests for reprints should be addressedto Johan Permert,MD, Departmentof Surgery,UniversityHospital,S-581 85 Link6ping,Sweden. Presentedat the 33rdAnnualMeetingof the Societyfor Surgeryof the AlimentaryTract, San Francisco,California,May 11-13, 1992.
iabetes or impaired glucose tolerance occurs in 70% to 80% of patients with pancreatic cancer. DiaD betes has previously been proposed as a predisposing factor for pancreatic cancer [1]. However, recent studies suggest that diabetes is more likely to be a consequence of the tumor [2-4]. Generally, the diabetic state occurs in a close temporal relationship to the cancer diagnosis. After surgery, glucose tolerance improves, and, in some patients, diabetes can even disappear [5]. The diabetic state in patients with pancreatic cancer is characterized by high plasma levels of insulin and by reduced body insulin sensitivity [2,3,6]. In addition, plasma concentrations of several other pancreatic peptides, such as islet amyloid polypeptide (IAPP), glucagon, and somatostatin, are increased [7]. Several studies have reported an altered ~-cell response to hyperglycemia in patients with pancreatic cancer [2,3,6], and a recent report suggested that there is an alteration in IAPP secretion by islet tissue close to the tumor [8]. Endocrine cells are frequently found in exocrine pancreatic adenocarcinomas, suggesting an endocrine component in these tumors that may secrete hormonal peptides [9]. After surgery, when the influence of the tumor is removed, the secretory profiles of the pancreatic peptides are normalized, and glucose tolerance improves [5,7,8]. In patients with type II diabetes, peripheral insulin resistance is a major pathogenic factor, and reduced glycogen synthesis in the skeletal muscles plays a major role in this metabolic abnormality [10]. In the present study, peripheral insulin sensitivity in patients with pancreatic cancer was investigated before and after surgery, in relation to their diabetic status and to associated diabetogenic factors, such as jaundice and weight loss. The effects of pancreatic tumor extracts on glycogen synthesis in skeletal muscle in vitro were also investigated, and the hormonal peptide content in these same extracts was measured by radioimmunoassay. PATIENTS AND M E T H O D S Clinical studies: Study design. Sixteen patients who were considered as candidates for radical surgery because of pancreatic ductal adenocarcinoma arising from the head or uncinate process and 7 age-matched healthy volunteers were examined in the surgical department at University Hospital in Link6ping, Sweden. Clinical data of patients and control subjects are given in Table I. Patients with signs of infection, renal failure, or cardiac failure, as assessed by medical history, clinical examination, and routine laboratory investigations, were excluded. The diagnosis of pancreatic cancer was verified histo-
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TABLE I
Characteristics of Patients and Control Subjects
Pancreatic cancer Controls
No. of Patients
Sex (M/F)
Age (y)
Weight (kg)
Height (cm)
SerumBilirubin (mmol/L)
Weight* Loss (%)
Tumor Size (cm)
16 7
9/7 4/3
65.7 _+ 2.8 66.1 _+ 1.2
68.1 _+ 3.5 79.1 _+ 4.7
171 _+ 4.2 176 + 3.3
102.0 _+ 17.2 12.1 _+ 1.7
5.8 -+ 1.4 0
3.2 _+ !.2 --
*Weight loss during the last 3 months in percentof basal weight.
logically or cytologically in all patients. Computed tomography and ultrasonography were performed to evaluate tumor size and resectability. All patients had abnormal bilirubin levels (Table I) but had been treated endoscopically with a biliary stent at least 48 hours before the investigations. No dietary restrictions were imposed, and all patients had normal physical activity in the hospital ward. The control subjects had no history of diabetes and were not receiving any medical treatment known to interfere with glucose metabolism. In 5 of the 16 patients, the tumor was considered to be resectable, and a subtotal pancreatectomy was performed using the method of Gall et al [11]. In this operation, 10% to 15% of the tissue from the tail of the pancreas is retained after surgery. The operation was radical in all patients, according to macroscopic findings and to the pathology report. The patients were examined again 3 months postoperatively. At this time, no patient had any sign of recurrence, according to clinical examination and ultrasonography. Methods. A detailed diabetic history was recorded, and an oral glucose tolerance test was performed in all patients and control subjects. Assessment was made using the diagnostic criteria of the World Health Organization (WHO) [12]. The glucose metabolic rate was determined using a hyperinsulinemic euglycemic clamp. After a 12-hour overnight fast, the subjects arrived in the laboratory in the morning. During this period, the patients had been given a standardized intravenous infusion of glucose at a rate of 30 mg/kg/h. For insulin-requiring diabetics, insulin was added in the amount required to maintain blood glucose between 5 and 6 mmol/L (equivalent to 90 to 108 mg/dL). Plastic eannulas were inserted into the radial artery of one arm for blood sampling and an antecubital vein of the contralateral arm for the infusions. The arterial eannula was flushed with 0.15 M saline, which contained 10 U/mL of heparin. A total of less than 1,000 U of heparin was given to each patient. The basal glucose and insulin levels were determined from two samples taken 5 minutes apart. A primed infusion of insulin (Actrapid Human, Novo, Copenhagen, Denmark) at a level of 0.5 mU/kg/min, chosen according to the protocol of De Fronzo, to obtain a plasma level of about 50 mU/L, was started [13]. After 90 minutes of infusion, the rate was increased to 1.0 mU/kg/min in
62
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order to reach a plasma level of about 100 mU/L, and the insulin infusion was continued at this rate for another 90 minutes. Insulin was dissolved in 0.15 M saline and 3 mL of the patient's own blood to give a final concentration of 300 mU/mL. Glucose (1.1 M glucose and 40 mM NaCI, Kabi Pharmacia, Stockholm, Sweden) was infused to maintain blood glucose at each patient's euglycemic (basal) level. The maintenance glucose dose was varied in order to obtain this euglycemic glucose level 4- 0.25 mmol/L. Blood glucose was measured every fifth minute, and the rate of glucose infused to maintain euglycemia was adjusted accordingly. For each insulin concentration, total body glucose uptake was calculated from the glucose infusion rate (mg/kg/min) averaged over the entire 90-minute infusion period. Samples for measurement of serum insulin were drawn every 10 minutes during the hyperinsulinemic state. /n dtro studies: Study design. The study utilized tumor tissue and pancreatic tissue adjacent to the tumor from eight patients with pancreatic ductal adenocarcinoma. In addition, normal pancreatic tissue was obtained from a patient with normal glucose tolerance who had surgery for a benign duodenal disorder. Five of the eight pancreatic tumors were from diabetic patients with cancer, and three were from patients with normal glucose tolerance. The effect of extracts from the different pancreatic tumors and from the sample of normal pancreas on glycogen synthesis in skeletal muscle was studied. Peptide concentrations, in all the extracts from tumors and pancreatic tissues adjacent to each tumor, were measured by radioimmunoassay. Tissue collection and extraction. The tissues were frozen in liquid nitrogen immediately after resection and stored at - 7 0 ~ for subsequent extraction. The tissues were weighed while still frozen and then plunged into boiling 0.5 M acetic acid (10 mL/g wet weight) and boiled for 10 minutes. The extracts were then partially purified on C- 18, reverse phase, Sep-Pak cartridges (Waters, Milford, MA), using multichannel syringe ram pumps (Harvard Instruments, Cambridge, MA). After washing, the Sep-Paks were eluted with 3 mL of 70% acetonitrile with 0.1% trifluoroacetic acid. These eluates were lyophilized and reconstituted in buffer (60 mM phosphate, pH 7.4, with 0.1% Triton-X 100 and 0.1% bovine serum albumin [BSA, Sigma Chemical Co., St. Louis, MO]) prior to assay and investigation of biologic activity.
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Determination of glycogen synthesis. The effects of the tumor extracts were investigated at two different concentrations (corresponding to 0.75 and 2.5 #g tumor tissue/mL incubation medium). As a positive control in the bioassay system, incubations with calcitonin gene-related peptide (CGRP) were performed at a concentration (10 nM) that had been previously shown to cause a marked decrease in muscle glycogen synthesis using this method
0.5 mU/kg/min
3'
[1,1.
r---***--7 I-*'7
I
**
F
Male Wistar rats (250 to 300 g) were guillotined, and strips (25 to 35 mg) of the soleus muscles were dissected from the hind legs, as previously described [15]. The muscle strips were attached at resting length to stainless steel clips. After the strips were weighed, they were transferred to silicon-treated Warburg flasks containing 4 mL of pre-oxygenated (95% 02:5% CO2) Krebs Ringer bicarbonate buffer [14] with 1.5% (w/vol) BSA (essential fatty acid free, Sigma Chemicals Co.) at pH 7.4. After 30 minutes of oxygenated preincubation, the strips were transferred to flasks containing fresh oxygenated buffer. D-(U-14C) glucose (0.5 #Ci/mL, Sigma), insulin (1.0 M), and the experimental agent (tissue extract or CGRP) were added, and the strips were incubated for 45 minutes. After 15 minutes of oxygenation, the flasks were sealed. At the end of the incubation, the muscle strips were instantly frozen on dry ice, and glycogen was extracted as previously described [16]. After three separate washings in 70% ethanol, the glycogen was redissolved in 1 mL of water and mixed with 5 mL of scintillation cocktail (Safety Solve, Research Products International, Mount Prospect, IL). The incorporation of 14C in the glycogen was measured on a beta counter. For each experimental incubation, a control incubation using a comparable muscle strip obtained from the same soleus muscle was performed in the absence of the experimental factor. Glycogen synthesis in the experimental incubations was expressed as the percentage of the synthesis in the control incubations. Radioimmunoassay: The concentrations of islet hormonal peptides (C-peptide, glucagon, somatostatin, pancreastatin, pancreatic polypeptide, and islet amyloid polypeptide) were measured by radioimmunoassay. Insulin was not measurable because the tissues were extracted by boiling in acid, thereby breaking the interchain disulfide bonds. Instead, the level of C-peptide was determined, as this peptide is not affected by boiling and is produced in equimolar concentrations to insulin. C-peptide was assayed using a commercially available antiserum (Novo, Copenhagen, Denmark), and the other peptides were measured by specific assays as previously described [8,17]. Plasma insulin was measured using a commercially available kit (Novo). Statistical analysis: All values presented in the text are given as mean 4- SEM. Comparison between the groups was carried out with Wilcoxon's signed rank test, using paired analysis for the groups studied before and after surgery. Correlations between glucose metabolic rate and weight loss, serum bilirubin levels, and tumor size were calculated by regression analysis.
1.0 mU/kg/min
mg/kg/min. 5.
I
*** - 1 F *** -I
// // // // // // .-/ //
1'
0
// // // // // // // //
NDDND +D
PC
ND D ND +D
C
PC
C
Figure 1. The glucose metabolic rate at two different insulin infusion levels (0.5 and 1.0 mU/kg/min) during hyperglycemic clamp in pancreatic cancer patients (PC, n = 16) and in controls (C, n = 7). The results are given for subgroups of nondlabetic patients (ND, n = 5) and diabetic patients (D, n = 11), and for all pancreatic cancer patients (ND -I- D, n = 16). The asterisks denote significance between the groups (*p <0.05, **p <0.01, and * * * p <0.001).
RESULTS Clinical study: Four of 16 patients (25%) with pancreatic cancer had a previous history of diabetes. In all four, diabetes had developed less than 20 months prior to the cancer diagnosis. Oral glucose tolerance tests indicated that 11 of 16 (69%) patients were diabetic by WHO criteria. Of the 11 diabetics, 6 (54%) required insulin treatment. Four of the five patients with resectable tumors were diabetic prior to surgery, and the diabetic status of three of the four improved postoperatively. After surgery, glucose tolerance became normal in two of the patients who were diabetic before surgery. Insulin treatment could be withdrawn from a third patient postoperatively. The patient with normal glucose tolerance preoperatively remained normal after surgery. Blood glucose levels in the diabetic patients with pancreatic cancer (mean: 6.6 4- 0.3 retool/L) were significantly increased (p <0.01) compared with the controls (mean: 5.0 4- 0.3 mmol/L) but were not different in the nondiabetic patients (mean: 5.3 4- 0.3 mmol/L). Plasma insulin was also elevated (p <0.05) in the diabetic patients with pancreatic cancer (mean: 18.3 4- 2.7 mU/L) compared with the controls (mean: 9.3 4- 2.0 mU/L), but no significant difference was seen between nondiabetic patients (mean: 12.3 4- 3.6 mU/L) and the controls. During the hyperinsulinemic clamp, the insulin levels during the two different infusion rates in the patient group and in the control group were comparable. (Levels of the low-dose infusion in patients and controls were 43.3 4- 3.9 mU/L and 47.4 4- 4.9 mU/L, respectively; levels of the high-dose infusion in patients and controls were 87.9 4- 9.6 mU/L and 84.2 4- 3.6 mU/L, respectively.) The
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PERMERT ET AL
0,5 mU/kg/min
% ,5~-1
1,OmU/kg/min
500,
% lOO_
x
x
=
t
x
400.
75_
300.
-r
200'
50.
100,
!1
0
Pre-op
Post-op
Pre-op
Post-op
Figure 2. The glucose metabolic rate, expressed as the percentage of the preoperative levels, at two insulin infusion levels (0.5 and 1.0 mU/kg/min), before and after subtotal pancreatectomy in the five patients with resectable tumors. Pre-op = preoperatively; Post-op -- postoperatively.
C-peptide
I0,000_ pmoVg 7,500_
90-
5,000_
60-
2,500 _
500-
somatostatin
pmol/g
800 _
375 _
600
250_
400--
pmol/g
glucagon
-
o
8
125--
200 -
o
m
O
50~
~mol/g
PP
8 - pmol/g
pancreastatin
O 375 -.
6-
9
250_
4--
~
1252
2_
~
O
Pancreatic Tumor
Adjacent Pancreas
Pancreatic Tumor
Adjacent Pancreas
Figure 4. Concentrations of the pancreatic islet hormones in pancreatic tumor tissues and in pancreatic tissue adjacent to the tumor. Concentrations of individual tumors are given by circles ( o p e n circles = diabetic patients; solid circles = nondiabetic patients). Concentrations in the adjacent pancreas are given as means -I- SEM (solid IKlUaI~,~).lAPP = islet amyloid polypeptide; PP --- pancreatic polypeptide.
glucose metabolic rate was decreased in the patients with pancreatic cancer compared with the control group at both insulin levels (Figure 1), This decrease was more pronounced in the diabetic patients than in the nondiabetic patients at both insulin levels (both p <0.05). The nondiabetic patients had a significantly lower glucose 64
I
I
I
I
D
ND
NP
CGRP
Control experiments
Figure 3. The effect on muscle glycogen synthesis of pancreatic tumor extracts from diabetic patients (D, n -- 5) and nondiabetie patients (ND, n -- 3). Control incubations with normal pancreas (NP) and calcitonin gene-related peptide ( ~ P ) (10 nM) were performed. The effects of tumors are given individually, and the results of the control incubations are given as the mean 4- SEM of at least four incubations.
30-
C*
0
Pancreatic tumors
IAPP
120- pmol/g
25-
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metabolic rate at the low insulin level but not at the higher one (p = 0.011 and p = 0.14, respectively) (Figure 1). No significant correlation was observed between glucose metabolic rate and bilirubin levels (p = 0.16), weight loss (p = 0.96), or tumor size (p = 0.56). In all five patients with resectable tumors, glucose metabolic rates improved markedly after surgery at both insulin levels and reached rates that were not significantly different from those of the controls (Figure 2). /n fftro studies: Four of five pancreatic tumor extracts from patients with diabetes had decreased glycogen synthesis at the higher concentration that was investigated (2.5 ~g tissue/mL) (Figure 3). The effect observed was comparable with that caused by CGRP at the 10-nM concentration. No effect was seen with extracts of normal pancreatic tissue or tumors from nondiabetic patients (Figure 3). None of the extracts influenced glycogen synthesis at the low concentration. Each of the islet hormones measured was present in all the tumor extracts, but concentrations varied considerably (Figure 4). In a few cases, the concentration of a particular peptide was higher in the tumor tissue than in the surrounding pancreatic tissue, but there were no significant differences between these tissues for any of the peptides (Figure 4). No significant differences were seen between the extracts from diabetic and nondiabetic patients. COMMENTS In the clinical part of this study, peripheral insulin sensitivity was investigated in patients with pancreatic cancer. In the in vitro part of the study, the concentra-
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tions of pancreatic peptides in tumor extracts and the effect of the extracts on glycogen synthesis in skeletal muscle were studied. The glucose metabolic rate was investigated at two insulin infusion rates (0.5 and 1.0 mU insulin/kg/min). The lower infusion rate was selected to obtain a plasma level of insulin corresponding to the endogenous production of insulin observed in pancreatic cancer patients during mild hyperglycemia, as determined in a previous study [2]. The higher infusion rate was selected to obtain plasma insulin levels in the upper physiologic range in which glucose uptake would not be significantly affected by endogenous glucose production [13]. The decrease in the glucose metabolic rate in patients with pancreatic cancer demonstrated a state of peripheral insulin resistance, which was more severe in diabetic patients than in nondiabetic patients. In several of the nondiabetic patients, the glucose uptake returned to normal when insulin levels were elevated, whereas the glucose metabolic rate in the diabetic patients remained decreased. This indicates a reduced responsiveness of glucose metabolism to insulin in diabetic patients with pancreatic cancer but not in the nondiabetic patients, at least in the range of plasma insulin levels investigated. Since no correlation was found in this study between decreased insulin sensitivity and weight loss, bilirubin levels, or tumor size, the reduced peripheral insulin sensitivity could not be explained by associated diabetogenic factors. In patients with type II diabetes that is not associated with pancreatic cancer, peripheral insulin resistance is an important factor in the development of diabetes [10]. In most type II diabetic patients, insulin resistance is caused by obesity, but diabetes can also appear in subsets of normal weight individuals [18]. Metabolically, however, these normal weight, type II diabetic patients have been found to resemble overweight individuals. An underlying factor causing both the reduced insulin sensitivity and the metabolic disorder of diabetes has been hypothesized [18]. In patients with pancreatic cancer, the reduced peripheral insulin sensitivity cannot be explained by obesity because patients with pancreatic cancer, as in this study, are generally lean. The postoperative improvement in glucose metabolic rate, which was seen in all patients after tumor removal, indicates that pancreatic tumors may have a role in the development of the insulin resistance that is seen in pancreatic cancer. After pancreatic resection, glucose metabolism improved because of increased insulin sensitivity, despite the marked reduction in insulin secretion. The postoperative improvement of diabetic status in patients with pancreatic cancer, which was also seen in a previous group of patients [5], demonstrates the profound importance of insulin resistance in diabetes associated with pancreatic cancer. Glucose metabolism can be influenced by malignancy in general. Impaired glucose tolerance has been reported in up to 30% of patients with different types of tumors [19]. Generally, glucose intolerance occurs more frequently in cachectic patients with advanced tumors, but peripheral insulin resistance has also been reported in noncachectic patients with a limited tumor disease [20].
However, the frequency of diabetes and glucose intolerance is higher in patients with pancreatic cancer than in those with any other type of malignancy [19]. The severity of the disorder is also greater in patients with pancreatic cancer; nearly half of these patients are frankly diabetic. This suggests that pancreatic tumors may produce a specific factor that is important in the development of diabetes. The possible role of pancreatic tumors in the development of insulin resistance was further emphasized by the inhibitory effect of tumor extracts on muscle glycogen synthesis in vitro, seen in four of five diabetic patients evaluated. The results of the radioimmunoassay indicated the production of all the different islet hormones in exocrine pancreatic tumors, but concentrations of the individual peptides in the tumors were not sufficient to cause diabetogenic effects. Higher concentrations of IAPP, glucagon, and somatostatin are required to exhibit diabetogenic effects in vivo and in vitro, according to previous studies [14,15,21,22]. Therefore, the inhibition of glycogen synthesis observed in this study could not be explained on the basis of tumor production of any of the peptides measured. Whether the observed effect was the result of interactions of the measured peptides or was caused by some other diabetogenic factor cannot be determined from the results of this study. However, the lack of effect seen with the extracts from normal pancreatic tissue and from the tumors of nondiabetic patients with pancreatic cancer may be noteworthy. These tissues contained the diabetogenic peptides in concentrations that were comparable with, or higher than, the concentrations in the tumors that reduced glycogen synthesis. The production of a specific diabetogenic factor by pancreatic tumors is possible because, during the neoplastic event, structurally altered, ectopic, or yet unknown substances can be produced and released, as well as mature peptides [23,24]. However, further investigations, such as detection of mRNA for different peptides, are necessary before the production of peptides and hormones by exocrine pancreatic tumors can be explained. In patients with type II diabetes who do not have pancreatic cancer, a combination of insulin resistance and altered insulin secretion is often required for the development of frank diabetes [I0]. Patients with pancreatic cancer show changes in the secretory response of insulin to glucose and also exhibit alterations in the secretion of several other pancreatic peptides [7]. Therefore, it is likely that other disorders, in addition to a reduced peripheral insulin sensitivity, contribute to the high frequency of frank diabetes seen in patients with pancreatic cancer. For example, if the tumor, directly or indirectly, produces a factor that is responsible for the changes observed in the peripheral tissues, effects on liver metabolism are also likely, since the concentration of such a factor would be much higher in portal blood than in the peripheral tissues. In conclusion, profound peripheral insulin resistance was found in patients with pancreatic cancer, and this appeared to be associated with the tumor itself. The decrease in insulin sensitivity was more pronounced in the
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diabetic patients and was not correlated with weight loss, tumor size, or bilirubin levels. It is tempting to speculate that the observed resistance to insulin in these patients also influences other metabolic pathways, such as amino acid and fat metabolism, and therefore contributes to the pronounced cachectic state often seen in patients with pancreatic cancer.
REFERENCES
1. Kessler II. Cancer mortality among diabetics. J Natl Cancer Inst 1970; 44: 673-86. 2. Permert J, Ihse I, Jorfeldt L, Arnquist H, von Schenck H, Larsson J. Pancreatic cancer is associated with impaired glucose metabolism. Eur J Surg; In press. 3. Schwarz SS, Zeidler A, Moossa AR, et al. A prospective study of glucose tolerance, insulin, C-peptide, and glucagon responses in patients with pancreatic carcinoma. Dig Dis 1978; 23:1107-14. 4. Boyle P, Hsieh C, Maisonneuve P, et al. Epidemiology of pancreas cancer (1988). Int J Pancreatol 1987; 5: 327-46. 5. Permert J, Jorfeldt L, Arnquist H, von Schenck H, Larsson J. Tumor removal improved glucose metabolism in humans. Clin Nutr 1992; 11: 17. 6. Cerosimo E, Pisters P, Pesola G, McDermott K, Bajorunas D, Brennan MF. Insulin secretion and action in patients with pancreatic cancer. Cancer 1991; 67: 468-93. 7. Permert J, Larsson J, Jorfeldt L, von Schenk H, Adrian TE. Islet hormone secretion in pancreatic cancer [abstract]. Digestion 1992; 52: 111. 8. Adrian TE, Permert J, Larsson Ji Westermark GT, Pour PM, Westermark P. The diabetes in pancreatic cancer patients is associated with a marked increase in islet amyloid polypeptide secretion. Regul Pept 1992; 40: 105. 9. Permert J, Mogaki M, Andr6n-Sandberg ,~, Kazakoff K, Pour PM. Pancreatic mixed ductal-islet tumors--Is this an entity? Int J Pancreatol 1992; 2: 23-9. 10. Turner RC, Williamson DH. Control of metabolism and the alterations in diabetes. In: O'Riordan JLH, editor. Recent advances in endocrinology and metabolism. Vol 2. Edinburgh: Churchill Livingstone, 1982. 11. Gall FP, Hernanek P, Gebhardt CH, Meier H. Erweiterte Resektion der Pankreas und perieampull~iren Karzinome. Regionale, totale und partielle Duodenopankreatektomie. Lever Magen Darm 1981; 11: 179-84. 12. World Health Organization Expert Committee: Diabetes mellitus. Technical Report Series 1985; No. 741, Geneva, Switzerland. 13. De Fronzo RA, Tobin TD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 237: E214-32. 14. Leighton B, Cooper GJS. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 1988; 335: 632-5. 15. Crettaz M, Prentki M, Zaninetti D, Jeanrenaud B. Insulin resistance in soleus muscle from obese Zucker rats. Biochem J 1980; 186: 525-34. 16. Cuedent G, Loten E, Jeanrenaud C, et al. Decreased basal, non-insulin stimulated glucose uptake and metabolism by skeletal muscle isolated from obese hyperglycemic mice. J Clin Invest 1976; 58: 1078-88. 17. Bloom SR, Long RG, editors. Radioimmunoassay of gut regulatory peptides. Philadelphia: WB Saunders, 1982: 2-100. 18. Caro J. Insulin resistance in obese and nonobese man. J Clin Endocrinol Metab 1991; 73: 691-7. 19. Glicksman AS, Rawson RW. Diabetes and altered carbohydrate metabolism in patients with cancer. Cancer 1956; 9:1127-34. 66
20. Copeland GP, Leinster S J, Davis JC, Hipkin LJ. Insulin resistance in patients with colorectal cancer. Br J Surg 1987; 74: 1031-5. 21. Unger RH, Orci L. Glucagon and the A cell. N Engl J Med 1981; 304: 1575-82. 22. Kahn SE, Klaff L J, Schwartz MW, et al. Treatment with a somatostatin analog decreases pancreatic B-cell and whole body sensitivity to glucose. J Clin Endocrinol Metab 1990; 71: 994-1002. 23. Artan-Spire S, Wolf B, Czernichow P. Developmental pattern of TRH-degrading activity and TRH content in rat pancreas. Acta Endocrinol 1984; 106: 102-8. 24. Bandram L, Hilstedt L, Rehfeld J. Progastrin expression in mammalian pancreas. Proc Natl Acad Sci U S A 1990; 87: 298-302. DISCUSSION D. K. Andersen (Chicago, IL): When patients have an initial diagnosis of pancreatic carcinoma, they have often been ill for some time with significant weight loss and altered nutrition. Your preoperative studies were probably performed close to the time of the operation, whereas your postoperative studies were probably performed when the patients had recovered from the operation and had presumably resumed normal weight-maintaining diets. How do you know whether these changes in insulin sensitivity are not merely a reflection of the nutritional status of your patients studied at two different time points and under two different nutritional circumstances? Your inference of insulin sensitivity from the glucose infusion rates during the clamp studies m a y be an accurate estimate of insulin sensitivity, provided that the hepatic glucose production is zero. Can you tell us whether you verified that hepatic glucose production was zero, and therefore that the glucose infusion requirements were truly a reflection of glucose uptake in the periphery? J o h a n Permert: These patients had lost weight before admission to hospital, but we didn't observe any correlation between the degree of weight loss and our results. In a previous study, we did not observe any difference in the frequency of diabetes between those patients who had lost considerable weight and those who had lost little weight. Weight loss protein caloric malnutrition can cause impaired glucose tolerance, but it is unlikely to explain the very high frequency of frank diabetes in these patients. Weight loss influenced the impaired glucose metabolism seen in these patients but cannot explain the impaired metabolism fully. To address the question of whether the insulin levels were sufficient to block hepatic glucose production, we adopted the protocol described by DeFronzo. In this study, we did not perform any investigations of endogenous glucose production. In previous studies that used this infusion level, we found a block of the hepatic glucose production. Daniel Elahi (Boston, MA): I would like to challenge the previous statement about hepatic glucose production. Just because you showed a blockage of hepatic glucose production previously, I presume, in normal individuals, does not mean that the same dose will suppress the level in those who are ill.
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INSULIN RESISTANCE IN PANCREATIC CANCER PATIENTS
The difference between glucose utilization that you show, although statistically significant, is not large. If you test any patient who is ill, you can dearly demonstrate glucose intolerance. In fact, if you perform an oral glucose tolerance in most hospitalized patients, you will often find indications of diabetes because of the stress of hospitalization. Is your control group adequate? You may want to study some patients without carcinoma who are hospitalized for other reasons. Johan Permert: That's a good suggestion about performing the study in patients with other diagnoses. Clinically, there was postoperative improvement in the diabetic status of these patients. Four patients were diabetic before surgery. In two, subsequent glucose tolerance tests were normal, and one patient who required insulin preoperatively did not postoperatively. R. H. Bell (Cincinnati, OH): Pancreatic cancers in the head of the gland often obstruct the pancreatic duct and cause pancreatitis in the tail of the gland, and we know that acute pancreatitis or chronic pancreatitis can cause abnormalities in glucose metabolism. Is it possible that some of the improvement that you observed in glucose tolerance after resection of the tumor related to improved function in the remaining gland? Could you tell us how many patients had a Whipple procedure and total pancreatectomy, and whether there were any differences between patients undergoing different operations?
J o h a n Permert: All of the patients underwent the same operative procedure, a subtotal pancreatectomy, during which approximately 85% of the pancreatic tissue was resected. M. F. Brennan (New York, NY): We have had experience using euglycemic clamps in these patients, and to answer Dr. Andersen's question, at 1 mU/kg/min, a serum insulin level of about 80 mU/L will completely suppress the endogenous glucose production by the liver as measured by tritiated glucose in the 3' position. More worrisome, as we and many others have shown, is that if you use euglycemic clamps, you will have a very significant decrease in the plasma amino acid concentration, and if you don't supplement the plasma amino acid concentration, then your interpretation of not only the glucose turnover, but also the protein turnover, is quite different. Many of these changes can be completely reversed in normal individuals by supplementing the plasma amino acid concentration, which will drop by at least 50% if you do not supplement the amino acids. Did you supplement your euglycemic clamp so that it was at least euleucinemic or euaminoacidemic, because if you did not, then the interpretation would be quite different? Johan Permert: No, we did not supplement for amino acids during the clamp. Our use of the original protocol of DeFronzo and our methods of interpreting the data are the same as those used previously by other groups.
THE AMERICANJOURNALOF SURGERY VOLUME165 JANUARY1993 67