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Mechanisms for skeletal muscle insulin resistance in patients with pancreatic ductal adenocarcinoma
Nutrition 27 (2011) 796–801
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Nutrition journal homepage: www.nutritionjrnl.com
Applied nutritional investigation
Mechanisms for skeletal muscle insulin resistance in patients with pancreatic ductal adenocarcinoma Thorhallur Agustsson M.D. a, Melroy A. D’souza M.S., D.N.B., M.R.C.S.Ed. a, Greg Nowak M.D., Ph.D. b, Bengt Isaksson M.D., Ph.D. a, * a
Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC) at Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC) at Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2010 Accepted 31 August 2010
Objective: Weight loss, glucose intolerance, and insulin resistance are seen in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral insulin resistance is decreased after tumor resection in patients with PDAC, which is consistent with the hypothesis that factors from the tumor may induce skeletal muscle insulin resistance. Our aim was to investigate the possible mechanisms for their skeletal muscle insulin resistance. Accordingly, the action of insulin on glucose metabolism and content of energy metabolites in muscle of patients with PDAC were investigated. To explore whether PDAC cells could influence muscle glucose uptake, myotubes were exposed to media conditioned by PDAC cells. Methods: Muscle biopsies from patients with PDAC (n ¼ 13), cancer of other sites (n ¼ 8), chronic pancreatitis (n ¼ 8), and controls with benign diseases (n ¼ 8) were assessed for glycogen, adenosine triphosphate, and phosphocreatine content. Basal and insulin-stimulated glucose transport and incorporation into glycogen were also assessed. Myotubes were treated with media conditioned by PDAC (MiaPaca 2) cells and glucose transport was monitored. Results: Insulin-stimulated glucose transport, muscle glycogen, and adenosine triphosphate content were decreased in patients with PDAC compared with controls, and insulin stimulation did not significantly increase glucose incorporation into glycogen in vitro in patients with PDAC. Adenosine triphosphate content correlated with glycogen content but not with glucose transport in skeletal muscle. Media conditioned with human PDAC cells did not affect basal or insulinstimulated glucose transport in L6 myotubes. Conclusion: In patients with PDAC, muscle insulin resistance is an early and specific finding unrelated to weight loss, plasma free fatty acid levels, and energy status of the cell. PDAC cellderived factors did not directly induce insulin resistance in myotubes, suggesting a lack of direct tumor-related effects. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Pancreatic cancer Skeletal muscle Glucose metabolism Glucose transport Insulin resistance Myotubes
Introduction Early and profound weight loss [1–3] and glucose intolerance [4–7] contribute to the dismal prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). In vivo and in vitro experiments have indicated the presence of skeletal muscle insulin resistance in these patients [7–10]. Specifically, an
This work was supported by a research grant from Stockholms Läns Landsting. * Corresponding author. Tel.: þ46-8-585-86995; fax: þ46-8-585-82340. E-mail address: [email protected] (B. Isaksson). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.08.022
impaired insulin action on phosphatidylinositol-3-kinase activity, glucose transport, and glycogen synthase activity has been shown [10,11]. Weight loss in PDAC can be attributed to decreased food intake and/or increased energy expenditure [12,13]. However, although it is possible to increase energy intake by enteral or parenteral means, this seems to have little impact on the progressive weight loss seen in patients with PDAC [14], which has led to the suggestion that there is a partial metabolic block to accretion of lean tissue in these patients [15,16]. Peripheral insulin resistance with a lack of its anabolic effects is consistent with such a metabolic block. This has been suggested to be
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caused by specific tumor-derived factors [17]. Many insulin actions are known to be dependent on the energy levels of target cells [18]. Insulin-stimulated glucose transport into the muscle cell and its further non-oxidative metabolism to glycogen are energy-requiring processes that are impaired in patients with PDAC [10,11], which could imply that skeletal muscle insulin resistance in these patients is associated with a decreased muscle content of energy metabolites. Because insulin resistance and weight loss coupled with increased energy expenditure are frequently seen in patients with PDAC, we hypothesized that decreased insulin action on skeletal muscle glucose transport and further metabolism into glycogen could be influenced by a decreased muscle content of adenosine triphosphate (ATP) and phosphocreatine. Previous observations have shown that peripheral insulin resistance is significantly decreased after tumour resection in patients with PDAC [8,19], and this is consistent with the hypothesis that factors from the tumor may induce skeletal muscle insulin resistance. The aim of the study was to investigate isolated skeletal muscle from patients with PDAC and measure basal and insulin-stimulated glucose transport, glucose incorporation into glycogen, and content of ATP and phosphocreatine. Control groups were patients with cancers of other sites (cancer), benign diseases with weight loss (chronic pancreatitis [CP]), and benign gastrointestinal diseases with minimal weight loss (control). We also investigated if PDAC cells per se could influence insulin action on glucose metabolism in skeletal muscle.
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Assessment of glucose transport activity in isolated muscle tissue Glucose transport was assessed in skeletal muscle using the glucose analogue [3H]3-O-methyl glucose, as previously described for rat epitrochlearis muscle [21], with modifications for human skeletal muscle [22]. The 3-O-methylglucose enters the muscle cells using the same transport carrier protein as glucose. This glucose analogue cannot be further metabolized, thus allowing direct assessment of transport activity without any influence of glucose metabolism. The incubated muscle specimens were processed as previously described [21] and sample aliquots were counted in a liquid scintillation counter with channels preset for simultaneous [3H] and [14C]. The amount of each isotope present in the sample was determined and used to calculate the extracellular space and the intracellular concentration of 3-O-methyl glucose. The intracellular water content of the muscle specimen was determined by subtracting the measured extracellular water space from total muscle water. Extracellular space was estimated using [14C]mannitol. Mannitol cannot pass through muscle cell membrane and was therefore used as an indirect measurement of muscle cell viability [22]. The basal rate of 3-O-methylglucose transport was expressed per milliliter of intracellular water. Because muscle biopsies from different subjects were analyzed on separate occasions, insulin-stimulated glucose transport was expressed as fold increase over basal. Glucose incorporation into glycogen Muscles were removed and stripped as described for assessment of glucose transport activity. After the equilibration period, the skeletal muscle strips were incubated without or with insulin (1000 mU/mL) for 2 h in 2 mL of KHB solution containing 5 mM unlabeled glucose and [U-14C]glucose (0.3 mCi/mL). After incubation, the muscle specimens were rapidly removed and frozen in liquid nitrogen. Briefly, the skeletal muscle strips were processed as described previously [23], glycogen was precipitated [24], and [U-14C]glucose was used to estimate the rate of glucose incorporation into glycogen. ATP, phosphocreatine, and glycogen content
Materials and methods Patients and plasma analyses The ethics committee for human studies at the Karolinska Institutet approved the study protocol and informed consent was obtained from each subject before participation. The patients with PDAC and other cancers were recruited consecutively and none of them were diagnosed as diabetic before referral. Patient with known diabetes were excluded. Patients with cancer were defined as those with cancers other than PDAC and included cholangiocarcinoma (n ¼ 2), gallbladder cancer (n ¼ 2), cancer of the papilla of Vater (n ¼ 1), duodenal cancer (n ¼ 1), gastric cancer (n ¼ 1), and colon cancer (n ¼ 1). The patients with CP were recruited consecutively among those who were scheduled for surgical treatment. The control patients were recruited consecutively from those who were scheduled for an open abdominal operation for benign diseases, including pancreatic cystadenoma (n ¼ 5), benign liver cyst (n ¼ 1), idiopathic thrombocytopenic purpura (n ¼ 1), and adenoma of the papilla of Vater (n ¼ 1). Postoperatively, diagnoses were confirmed in all patients by histopathology. Body weight was measured on a single set of scales in the surgical outpatient clinic. Patients underwent an oral glucose tolerance test the day before surgery after an overnight fast (without a preoperative drink). Blood was collected in chilled tubes containing aprotinin and ethylenediaminetetra-acetic acid (400 KIU and 5 mg/mL of blood, respectively) and immediately centrifuged. Plasma was separated and stored at 70 C until the assays were performed. Plasma glucose was measured enzymatically using a biochemical analyzer (Kebo, Stockholm, Sweden) and plasma insulin was determined by radioimmunoassay using Insulin-RIA-100 kits (Pharmacia-Upjohn, Uppsala, Sweden).
Muscle biopsy procedure and in vitro muscle strip preparation After induction of anesthesia, skeletal muscle biopsies (w500 mg) were surgically excised from the rectus abdominis muscle. The samples were immediately transported to the laboratory in Krebs-Henseleit bicarbonate buffer (KHB) solution containing 5 mM HEPES, 0.1% bovine serum albumin, 5 mM glucose and 15 mM mannitol. In the laboratory, 10 to 20 muscle strips were prepared from the sample and mounted on Plexiglas clamps [20]. For equilibration, these strips were incubated for 15 min in KHB in sealed vials. Subsequently, the strips were incubated in KHB containing 0 or 1000 mU/mL of insulin for 30 min. Then, [3H] 3-O-methylglucose (5 mM, 2.5 mCi/mmol) and [14C]mannitol (15 mM, 26.3 mCi/mmol; ICN Biomedical, Costa Mesa, CA, USA) were added to the buffers. The vials were then incubated for 20 min. All the serial incubations were maintained at 35 C with 95% O2/5% CO2.
ATP and phosphocreatine were extracted from 5 mg of ground freeze-dried muscle and measured fluorometrically by enzymatic methods [25]. Glycogen content in muscle was determined enzymatically as glucose (using hexokinase and glucose-6-phosphate dehydrogenase and measuring the production of reduced nicotinamide adenine dinucleotide spectrophotometrically) after alkaline destruction of free glucose and hydrolysis of glycogen with amyloglucosidase as previously described [26]. Glycogen content was expressed as millimoles of glycosyl units per kilogram of dry weight. Glucose transport activity in L6 myoblasts Rat L6 myoblasts, human PDAC-cells (MiaPaca 2) and fibroblasts (HFF1) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Human osteosarcoma cells (MG-63) were obtained from a fellow laboratory at the Karolinska Institutet. Rat L6 myoblasts were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 22.5 mM glucose, 10% (v/v) fetal calf serum (FCS), 3% (v/v) 100 U/mL of penicillin, and 3% (v/v) 100 mg/mL of streptomycin (PEST). Cells were seeded in 12-well plates. Differentiation of myoblasts was started at 80% confluence, using DMEM supplemented with 5.6 mM glucose, 2% (v/v) FCS, and 3% (v/v) PEST for 5 d with a medium change at days 2 and 4. The differentiation of myoblasts to myotubes was monitored by microscope. Starvation started on day 6, for 21 h, using DMEM supplemented with 5.6 mM glucose, 0% (v/v) FCS, 3% (v/v) PEST, and 6% (v/v) of 1 mg/mL of bovine serum albumin (unconditioned media) or DMEM supplemented with 5.6 mM glucose, 0% (v/v) FCS, 3% (v/v) PEST, and 6% (v/v) of 1 mg/mL of bovine serum albumin– conditioned media after 24 h of incubation with fibroblasts, osteoblasts, or pancreatic carcinoma cells. The myotubes were incubated with 0.4 mM 2deoxyglucose (2-DOG) without or with insulin (2000 nM Actrapid) for 60 min and thereafter with radioactive 2-DOG with or without insulin for 10 min. Glucose transport was terminated by ice-cold phosphate buffered saline and then NaOH was added for 90 min to lyse the cells. Protein concentrations were determined by a Bio-Rad colorimetric assay (Bio-Rad, Hercules, CA, USA). The radioactivity in the lysate was determined by liquid scintillation counting. The 2DOG uptake was expressed as nanomoles of 2-DOG per milligram of protein per minute. MiaPaca 2, HFF1, and MG-63 were grown in media according to instructions from the ATCC and the fellow laboratory. The cells had undergone 4 to 10 passages until the media was used in the experiment. Chemicals Human insulin (Actrapid) was purchased from Novo Nordisk (Bagsvaerd, Denmark). The radioisotopes used for glucose transport were purchased from ICN Biomedical. D-[U-14C] glucose and 2-deoxy-D-[1-14C]-glucose were from
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Amersham Life Sciences (Arlington Heights, IL, USA). Amyloglucosidase was from Sigma Chemical (St. Louis, MO, USA). Hexokinase and glucose-6-phosphate dehydrogenase were from Boehringer (Mannheim, Germany). Antibiotic– antimycotic solution was purchased from life Technologies, Inc. (Grand Island, NY, USA). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA, USA). DMEM, RPMI-1640 medium, trypsin, geneticin, trifluoroacetic acid, bovine serum albumin (radioimmunoassay grade), and all other standard chemicals and reagents were from Sigma Chemical. Statistical analysis Analysis of variance with Dunnet’s post hoc test was used for multiple comparisons between means. Linear regression analysis was used for correlations between continuous variables. P < 0.05 was considered to indicate statistical significance. Data were analyzed using JMP 3.2.2 (SAS Institute, Cary, NC, USA) and presented as mean standard error of the mean.
units/kg of dry weight, P < 0.05; Fig. 2A). Glycogen content in the CP and cancer groups were not different from the control group. There were no statistically significant differences between the PDAC and cancer groups. In all patients, glycogen content was significantly correlated with ATP content (R2 ¼ 0.74, P < 0.05). In the basal state, glucose incorporation into glycogen was not significantly different across groups, although the CP, cancer, and PDAC groups tended to have lower rates (Fig. 2B). Insulin stimulation resulted in a 1.3-fold increase of glucose incorporation into glycogen (P < 0.05) in the control group (Fig. 2C). In the PDAC, CP, and cancer groups, insulin stimulation did not result in a significant increase in glucose incorporation into glycogen. ATP and phosphocreatine content
Results Plasma glucose and insulin concentrations and oral glucose tolerance test Table 1 lists the clinical characteristics of these subjects. Glucose tolerance was normal in all control patients, whereas 11 of 13 patients with PDAC were diabetic or glucose intolerant (Table 1). Fasting plasma glucose concentrations were significantly increased in patients with PDAC compared with controls and patients with cancer (Table 1). Fasting plasma insulin concentrations were not statistically different across groups, although insulin levels tended to be increased in the cancer and PDAC groups and decreased in the CP group compared with controls. Results are presented in Table 1. Glucose transport Basal 3-O-methylglucose transport was not significantly different among groups (Fig. 1A). Insulin stimulation resulted in a significant fold increase in glucose transport in all groups, except PDAC (data not shown). Insulin-stimulated glucose transport was significantly decreased in the PDAC compared with the control group (Fig. 1B). Insulin-stimulated glucose transport in the CP and cancer groups were not different from the control group, although there was a tendency for decreased glucose transport in the cancer group. In all patients, there was no correlation between insulin-stimulated glucose transport and muscle ATP content (data not shown). Glycogen content and glucose incorporation into glycogen Glycogen content was significantly lower in the PDAC than in the control group (119 17 versus 213 24 mmol of glycosyl
The ATP content was significantly lower in the PDAC than in the control group (8.4 1.2 versus 15.8 0.8 mmol/kg of dry weight, P < 0.05). There were no statistically significant differences between the PDAC and cancer groups. The CP and cancer groups also had decreased muscle ATP content, but these did not reach statistical significance compared with the control group (Fig. 3A). The trend was similar for phosphocreatine content, although the differences between means across groups were not statistically significant (Fig. 3B). Glucose transport in L6 myotubes Transport of 2-DOG was determined with and without the presence of 2000 nM insulin. Basal glucose uptake was similar across groups (Fig. 4A), and exposure to insulin resulted in a significant increase in glucose transport in all groups (Fig. 4B). There were no significant differences in basal or insulinstimulated glucose transport in myotubes without or with exposure to medium conditioned by PDAC cells, fibroblasts, or osteoblasts. Discussion In the present study, insulin resistance for glucose uptake and decreased glycogen and ATP content were found in skeletal muscle from patients with PDAC. In addition, conditioned media from human PDAC cells did not affect insulin action on glucose uptake in L6 myoblasts. Most patients with PDAC were diabetic or glucose intolerant without hypoinsulinemia, as has been previously reported [5,10], indicating the presence of peripheral insulin resistance. Indeed, insulin-stimulated glucose transport was markedly decreased in isolated skeletal muscle from patients with PDAC, confirming
Table 1 Patient characteristics*
Number (women/men) Age (y) BMI (kg/m2) Weight loss (%)y OGTT (normal/impaired/diabetes) Fasting plasma glucose (mM) Fasting plasma insulin (pM) FFAs (mM)
Controls
Chronic pancreatitis
Cancer
Pancreatic cancer
8 (5/3) 53 4 25.7 1 22 8/0/0 5.0 0.1 40 4 0.72 0.06
8 (3/5) 52 3 21.3 0.7 63 3/1/4 7.0 1 30 8 1.50 0.48
8 (5/3) 68 3z 23.0 2 33 5/0/3 5.3 0.3 59 6 1.19 0.08
13 (10/3) 70 2z 23.6 1.3 63 2/3/8 8.1 0.6x 50 9 0.86 0.05
BMI, body mass index; FFAs, free fatty acids; OGTT, oral glucose tolerance test * Data are presented as mean SE. y During previous 3 mo. z P < 0.05 compared with controls. x P < 0.05 compared with controls and cancer group.
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Fig. 1. The 3-O-methylglucose transport in isolated muscle strips from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Muscle strips were incubated in the presence of 5 mM glucose in the absence (A) or presence (B) of insulin (1000 mU/mL). Basal glucose transport is expressed as micromoles per milliliter per hour, and insulinstimulated glucose transport is expressed as fold increase over basal glucose transport. Data are presented as mean SEM. * P < 0.05 compared with control. CP, chronic pancreatitis; PC, pancreatic cancer.
historical findings [10] and supporting the hypothesis that insulin resistance at the skeletal muscle cell level contributes to the high frequency of impaired glucose tolerance or diabetes in these patients [8]. Furthermore, decreased insulin-stimulated glucose transport in skeletal muscle was observed in 11 of 13 patients with PDAC, indicating that this metabolic disturbance is strongly associated with PDAC. Peripheral insulin resistance and impaired glucose tolerance have been reported in other cancers [27,28] and have been proposed to be a general malignant phenomenon. In the present study, three patients with cancers of other sites were diabetic according to their oral glucose tolerance test results. However, in these patients, insulin-stimulated glucose transport was not significantly decreased compared with the controls, suggesting that mechanisms other than skeletal muscle insulin resistance at the glucose transport level may contribute to hyperglycemia in these patients. Hence, decreased insulin responsiveness for glucose transport in skeletal muscle was not a general consequence of malignancy in the present study. Rather, it seems to be a metabolic disturbance that is more pronounced and frequently occurring in patients with PDAC. The use of the non-metabolizable glucose analogue 3-Omethylglucose allowed us to directly assess glucose transport
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Fig. 2. Glycogen contents (A) and glucose incorporation into glycogen in the absence (B) or presence (C) of insulin (1000 mU/mL) in skeletal muscle from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Glycogen content is expressed as millimoles of glucosyl units per kilogram of dry weight. Basal and insulin-stimulated glucose incorporations into glycogen are expressed as nanomoles per gram per hour and fold increase over basal glucose incorporation, respectively. Muscle strips were incubated in the presence of 5 mM glucose. Data are presented as mean SEM. * P < 0.05 compared with control. CP, chronic pancreatitis; PC, pancreatic cancer.
activity without the additional influence of cellular glucose metabolism. However, it is also possible that skeletal muscle insulin resistance in patients with PDAC is further aggravated by defects in intracellular glucose metabolism, such as decreased glycogen synthase activity and increased glycogen phosphorylase activity [11]. The significantly decreased muscle glycogen content observed in patients with PDAC in the present study is unlikely to be due to only weight loss, because patients with CP had a similar degree of weight loss without significantly decreased muscle glycogen content compared with controls. Furthermore, there was no significant correlation between weight loss and muscle glycogen content in the present study (data not shown). Therefore, factors other than weight loss may have contributed to the decreased muscle glycogen content in PDAC. In contrast to the findings in control patients, there was no significant response to insulin in the rate of incorporation of glucose into glycogen in patients with PDAC, CP, or cancer, suggesting decreased insulin-stimulated non-oxidative glucose metabolism in all these patients compared with controls. When comparing glycogen content with muscle content of ATP and phosphocreatine, the means of the different groups show the same reciprocal pattern, although only patients with PDAC had significantly lower contents of glycogen and ATP compared with
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Fig. 3. Contents of ATP (A) and phosphocreatine (B) in skeletal muscle from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Contents are expressed as millimoles per kilogram of dry weight and presented as mean SEM. * P < 0.05 compared with control. ATP, adenosine triphosphate; CP, chronic pancreatitis; PC, pancreatic cancer.
controls, possibly suggesting an association between nonoxidative glucose metabolism and the energy status of the muscle cell. This hypothesis is further supported by a significant correlation between glycogen and ATP content when analyzing all patients together. In contrast, there was no correlation between muscle glucose transport and energy content, suggesting that other mechanisms are likely to contribute to the decreased glucose transport seen in patients with PDAC. The decrease of insulin-stimulated glucose transport may be due to defective signaling steps proximal to glucose transport, such as decreased phosphatidylinositol-3-kinase activity, as has previously been shown [10]. Increased lipid use in muscle has been proposed to cause insulin resistance in muscle [29] and increased free fatty acid (FFA) uptake in muscle results in a decrease in phosphatidylinositol-3-kinase activity and glucose transport [30]. In the present study, weight loss was moderate and not significantly different among groups, suggesting that factors other than anorexia and weight loss could be responsible for the observed skeletal muscle insulin resistance in patients with PDAC. One such possible mechanism could be an increased flux of lipid substrates into the muscle cell, resulting in insulin resistance. However, in the present study, plasma levels of FFA were not increased in patients with PDAC and there was no correlation between plasma concentrations of FFA and glucose
Fig. 4. Basal and insulin-stimulated 2-DOG transport (A) including insulinstimulated fold increase over basal (B) in L6 myotubes after 5 d of differentiation, starvation in 5.6 mM glucose of UM for 21 h, or in one of three conditioned media: human pancreatic carcinoma cells (MP2), FB, or OB. Cells were incubated with 0.4 mM 2-DOG without or with (2000 nM) insulin for 60 min and then with radioactive 2-DOG without or with insulin for 10 min. 2-DOG, 2-deoxyglucose; FB, fibroblasts; MP2, MiaPaca 2; OB, osteosarcoma cells; UM, unconditioned medium.
transport. Overall, the only positive association found for glucose transport in the present study was the presence of PDAC. In the present study, medium conditioned from human PDAC cells did not affect basal or insulin-stimulated glucose transport in L6 myotubes. It could be argued that the lack of inhibition of glucose transport by medium conditioned by PDAC cells is explained by an insufficient exposure time or a concentration of a putative inhibitory substance. We believe that 24-h exposure should provide adequate time for transcriptional and translational events to have occurred. Furthermore, with regard to substance concentration, the experimental setting should provide concentrations of any substance in the incubation buffer that would far exceed concentrations in the “in vivo” situation. This finding does not support the hypothesis that skeletal muscle insulin resistance in patients with PDAC is directly caused by circulating factors from the tumor. Conclusion We have shown that the skeletal muscle of patients with PDAC is characterized by insulin resistance for glucose transport
T. Agustsson et al. / Nutrition 27 (2011) 796–801
in addition to depleted glycogen and ATP stores. Neither weight loss nor plasma FFA level was associated with decreased glucose transport. Medium conditioned by PDAC cells did not directly induce insulin resistance in myotubes.
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Applied nutritional investigation
Mechanisms for skeletal muscle insulin resistance in patients with pancreatic ductal adenocarcinoma Thorhallur Agustsson M.D. a, Melroy A. D’souza M.S., D.N.B., M.R.C.S.Ed. a, Greg Nowak M.D., Ph.D. b, Bengt Isaksson M.D., Ph.D. a, * a
Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC) at Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC) at Karolinska Institutet, Karolinska University Hospital, Huddinge, Stockholm, Sweden b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 June 2010 Accepted 31 August 2010
Objective: Weight loss, glucose intolerance, and insulin resistance are seen in patients with pancreatic ductal adenocarcinoma (PDAC). Peripheral insulin resistance is decreased after tumor resection in patients with PDAC, which is consistent with the hypothesis that factors from the tumor may induce skeletal muscle insulin resistance. Our aim was to investigate the possible mechanisms for their skeletal muscle insulin resistance. Accordingly, the action of insulin on glucose metabolism and content of energy metabolites in muscle of patients with PDAC were investigated. To explore whether PDAC cells could influence muscle glucose uptake, myotubes were exposed to media conditioned by PDAC cells. Methods: Muscle biopsies from patients with PDAC (n ¼ 13), cancer of other sites (n ¼ 8), chronic pancreatitis (n ¼ 8), and controls with benign diseases (n ¼ 8) were assessed for glycogen, adenosine triphosphate, and phosphocreatine content. Basal and insulin-stimulated glucose transport and incorporation into glycogen were also assessed. Myotubes were treated with media conditioned by PDAC (MiaPaca 2) cells and glucose transport was monitored. Results: Insulin-stimulated glucose transport, muscle glycogen, and adenosine triphosphate content were decreased in patients with PDAC compared with controls, and insulin stimulation did not significantly increase glucose incorporation into glycogen in vitro in patients with PDAC. Adenosine triphosphate content correlated with glycogen content but not with glucose transport in skeletal muscle. Media conditioned with human PDAC cells did not affect basal or insulinstimulated glucose transport in L6 myotubes. Conclusion: In patients with PDAC, muscle insulin resistance is an early and specific finding unrelated to weight loss, plasma free fatty acid levels, and energy status of the cell. PDAC cellderived factors did not directly induce insulin resistance in myotubes, suggesting a lack of direct tumor-related effects. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Pancreatic cancer Skeletal muscle Glucose metabolism Glucose transport Insulin resistance Myotubes
Introduction Early and profound weight loss [1–3] and glucose intolerance [4–7] contribute to the dismal prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). In vivo and in vitro experiments have indicated the presence of skeletal muscle insulin resistance in these patients [7–10]. Specifically, an
This work was supported by a research grant from Stockholms Läns Landsting. * Corresponding author. Tel.: þ46-8-585-86995; fax: þ46-8-585-82340. E-mail address: [email protected] (B. Isaksson). 0899-9007/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2010.08.022
impaired insulin action on phosphatidylinositol-3-kinase activity, glucose transport, and glycogen synthase activity has been shown [10,11]. Weight loss in PDAC can be attributed to decreased food intake and/or increased energy expenditure [12,13]. However, although it is possible to increase energy intake by enteral or parenteral means, this seems to have little impact on the progressive weight loss seen in patients with PDAC [14], which has led to the suggestion that there is a partial metabolic block to accretion of lean tissue in these patients [15,16]. Peripheral insulin resistance with a lack of its anabolic effects is consistent with such a metabolic block. This has been suggested to be
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caused by specific tumor-derived factors [17]. Many insulin actions are known to be dependent on the energy levels of target cells [18]. Insulin-stimulated glucose transport into the muscle cell and its further non-oxidative metabolism to glycogen are energy-requiring processes that are impaired in patients with PDAC [10,11], which could imply that skeletal muscle insulin resistance in these patients is associated with a decreased muscle content of energy metabolites. Because insulin resistance and weight loss coupled with increased energy expenditure are frequently seen in patients with PDAC, we hypothesized that decreased insulin action on skeletal muscle glucose transport and further metabolism into glycogen could be influenced by a decreased muscle content of adenosine triphosphate (ATP) and phosphocreatine. Previous observations have shown that peripheral insulin resistance is significantly decreased after tumour resection in patients with PDAC [8,19], and this is consistent with the hypothesis that factors from the tumor may induce skeletal muscle insulin resistance. The aim of the study was to investigate isolated skeletal muscle from patients with PDAC and measure basal and insulin-stimulated glucose transport, glucose incorporation into glycogen, and content of ATP and phosphocreatine. Control groups were patients with cancers of other sites (cancer), benign diseases with weight loss (chronic pancreatitis [CP]), and benign gastrointestinal diseases with minimal weight loss (control). We also investigated if PDAC cells per se could influence insulin action on glucose metabolism in skeletal muscle.
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Assessment of glucose transport activity in isolated muscle tissue Glucose transport was assessed in skeletal muscle using the glucose analogue [3H]3-O-methyl glucose, as previously described for rat epitrochlearis muscle [21], with modifications for human skeletal muscle [22]. The 3-O-methylglucose enters the muscle cells using the same transport carrier protein as glucose. This glucose analogue cannot be further metabolized, thus allowing direct assessment of transport activity without any influence of glucose metabolism. The incubated muscle specimens were processed as previously described [21] and sample aliquots were counted in a liquid scintillation counter with channels preset for simultaneous [3H] and [14C]. The amount of each isotope present in the sample was determined and used to calculate the extracellular space and the intracellular concentration of 3-O-methyl glucose. The intracellular water content of the muscle specimen was determined by subtracting the measured extracellular water space from total muscle water. Extracellular space was estimated using [14C]mannitol. Mannitol cannot pass through muscle cell membrane and was therefore used as an indirect measurement of muscle cell viability [22]. The basal rate of 3-O-methylglucose transport was expressed per milliliter of intracellular water. Because muscle biopsies from different subjects were analyzed on separate occasions, insulin-stimulated glucose transport was expressed as fold increase over basal. Glucose incorporation into glycogen Muscles were removed and stripped as described for assessment of glucose transport activity. After the equilibration period, the skeletal muscle strips were incubated without or with insulin (1000 mU/mL) for 2 h in 2 mL of KHB solution containing 5 mM unlabeled glucose and [U-14C]glucose (0.3 mCi/mL). After incubation, the muscle specimens were rapidly removed and frozen in liquid nitrogen. Briefly, the skeletal muscle strips were processed as described previously [23], glycogen was precipitated [24], and [U-14C]glucose was used to estimate the rate of glucose incorporation into glycogen. ATP, phosphocreatine, and glycogen content
Materials and methods Patients and plasma analyses The ethics committee for human studies at the Karolinska Institutet approved the study protocol and informed consent was obtained from each subject before participation. The patients with PDAC and other cancers were recruited consecutively and none of them were diagnosed as diabetic before referral. Patient with known diabetes were excluded. Patients with cancer were defined as those with cancers other than PDAC and included cholangiocarcinoma (n ¼ 2), gallbladder cancer (n ¼ 2), cancer of the papilla of Vater (n ¼ 1), duodenal cancer (n ¼ 1), gastric cancer (n ¼ 1), and colon cancer (n ¼ 1). The patients with CP were recruited consecutively among those who were scheduled for surgical treatment. The control patients were recruited consecutively from those who were scheduled for an open abdominal operation for benign diseases, including pancreatic cystadenoma (n ¼ 5), benign liver cyst (n ¼ 1), idiopathic thrombocytopenic purpura (n ¼ 1), and adenoma of the papilla of Vater (n ¼ 1). Postoperatively, diagnoses were confirmed in all patients by histopathology. Body weight was measured on a single set of scales in the surgical outpatient clinic. Patients underwent an oral glucose tolerance test the day before surgery after an overnight fast (without a preoperative drink). Blood was collected in chilled tubes containing aprotinin and ethylenediaminetetra-acetic acid (400 KIU and 5 mg/mL of blood, respectively) and immediately centrifuged. Plasma was separated and stored at 70 C until the assays were performed. Plasma glucose was measured enzymatically using a biochemical analyzer (Kebo, Stockholm, Sweden) and plasma insulin was determined by radioimmunoassay using Insulin-RIA-100 kits (Pharmacia-Upjohn, Uppsala, Sweden).
Muscle biopsy procedure and in vitro muscle strip preparation After induction of anesthesia, skeletal muscle biopsies (w500 mg) were surgically excised from the rectus abdominis muscle. The samples were immediately transported to the laboratory in Krebs-Henseleit bicarbonate buffer (KHB) solution containing 5 mM HEPES, 0.1% bovine serum albumin, 5 mM glucose and 15 mM mannitol. In the laboratory, 10 to 20 muscle strips were prepared from the sample and mounted on Plexiglas clamps [20]. For equilibration, these strips were incubated for 15 min in KHB in sealed vials. Subsequently, the strips were incubated in KHB containing 0 or 1000 mU/mL of insulin for 30 min. Then, [3H] 3-O-methylglucose (5 mM, 2.5 mCi/mmol) and [14C]mannitol (15 mM, 26.3 mCi/mmol; ICN Biomedical, Costa Mesa, CA, USA) were added to the buffers. The vials were then incubated for 20 min. All the serial incubations were maintained at 35 C with 95% O2/5% CO2.
ATP and phosphocreatine were extracted from 5 mg of ground freeze-dried muscle and measured fluorometrically by enzymatic methods [25]. Glycogen content in muscle was determined enzymatically as glucose (using hexokinase and glucose-6-phosphate dehydrogenase and measuring the production of reduced nicotinamide adenine dinucleotide spectrophotometrically) after alkaline destruction of free glucose and hydrolysis of glycogen with amyloglucosidase as previously described [26]. Glycogen content was expressed as millimoles of glycosyl units per kilogram of dry weight. Glucose transport activity in L6 myoblasts Rat L6 myoblasts, human PDAC-cells (MiaPaca 2) and fibroblasts (HFF1) were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Human osteosarcoma cells (MG-63) were obtained from a fellow laboratory at the Karolinska Institutet. Rat L6 myoblasts were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 22.5 mM glucose, 10% (v/v) fetal calf serum (FCS), 3% (v/v) 100 U/mL of penicillin, and 3% (v/v) 100 mg/mL of streptomycin (PEST). Cells were seeded in 12-well plates. Differentiation of myoblasts was started at 80% confluence, using DMEM supplemented with 5.6 mM glucose, 2% (v/v) FCS, and 3% (v/v) PEST for 5 d with a medium change at days 2 and 4. The differentiation of myoblasts to myotubes was monitored by microscope. Starvation started on day 6, for 21 h, using DMEM supplemented with 5.6 mM glucose, 0% (v/v) FCS, 3% (v/v) PEST, and 6% (v/v) of 1 mg/mL of bovine serum albumin (unconditioned media) or DMEM supplemented with 5.6 mM glucose, 0% (v/v) FCS, 3% (v/v) PEST, and 6% (v/v) of 1 mg/mL of bovine serum albumin– conditioned media after 24 h of incubation with fibroblasts, osteoblasts, or pancreatic carcinoma cells. The myotubes were incubated with 0.4 mM 2deoxyglucose (2-DOG) without or with insulin (2000 nM Actrapid) for 60 min and thereafter with radioactive 2-DOG with or without insulin for 10 min. Glucose transport was terminated by ice-cold phosphate buffered saline and then NaOH was added for 90 min to lyse the cells. Protein concentrations were determined by a Bio-Rad colorimetric assay (Bio-Rad, Hercules, CA, USA). The radioactivity in the lysate was determined by liquid scintillation counting. The 2DOG uptake was expressed as nanomoles of 2-DOG per milligram of protein per minute. MiaPaca 2, HFF1, and MG-63 were grown in media according to instructions from the ATCC and the fellow laboratory. The cells had undergone 4 to 10 passages until the media was used in the experiment. Chemicals Human insulin (Actrapid) was purchased from Novo Nordisk (Bagsvaerd, Denmark). The radioisotopes used for glucose transport were purchased from ICN Biomedical. D-[U-14C] glucose and 2-deoxy-D-[1-14C]-glucose were from
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Amersham Life Sciences (Arlington Heights, IL, USA). Amyloglucosidase was from Sigma Chemical (St. Louis, MO, USA). Hexokinase and glucose-6-phosphate dehydrogenase were from Boehringer (Mannheim, Germany). Antibiotic– antimycotic solution was purchased from life Technologies, Inc. (Grand Island, NY, USA). Fetal bovine serum was obtained from Atlanta Biologicals (Norcross, GA, USA). DMEM, RPMI-1640 medium, trypsin, geneticin, trifluoroacetic acid, bovine serum albumin (radioimmunoassay grade), and all other standard chemicals and reagents were from Sigma Chemical. Statistical analysis Analysis of variance with Dunnet’s post hoc test was used for multiple comparisons between means. Linear regression analysis was used for correlations between continuous variables. P < 0.05 was considered to indicate statistical significance. Data were analyzed using JMP 3.2.2 (SAS Institute, Cary, NC, USA) and presented as mean standard error of the mean.
units/kg of dry weight, P < 0.05; Fig. 2A). Glycogen content in the CP and cancer groups were not different from the control group. There were no statistically significant differences between the PDAC and cancer groups. In all patients, glycogen content was significantly correlated with ATP content (R2 ¼ 0.74, P < 0.05). In the basal state, glucose incorporation into glycogen was not significantly different across groups, although the CP, cancer, and PDAC groups tended to have lower rates (Fig. 2B). Insulin stimulation resulted in a 1.3-fold increase of glucose incorporation into glycogen (P < 0.05) in the control group (Fig. 2C). In the PDAC, CP, and cancer groups, insulin stimulation did not result in a significant increase in glucose incorporation into glycogen. ATP and phosphocreatine content
Results Plasma glucose and insulin concentrations and oral glucose tolerance test Table 1 lists the clinical characteristics of these subjects. Glucose tolerance was normal in all control patients, whereas 11 of 13 patients with PDAC were diabetic or glucose intolerant (Table 1). Fasting plasma glucose concentrations were significantly increased in patients with PDAC compared with controls and patients with cancer (Table 1). Fasting plasma insulin concentrations were not statistically different across groups, although insulin levels tended to be increased in the cancer and PDAC groups and decreased in the CP group compared with controls. Results are presented in Table 1. Glucose transport Basal 3-O-methylglucose transport was not significantly different among groups (Fig. 1A). Insulin stimulation resulted in a significant fold increase in glucose transport in all groups, except PDAC (data not shown). Insulin-stimulated glucose transport was significantly decreased in the PDAC compared with the control group (Fig. 1B). Insulin-stimulated glucose transport in the CP and cancer groups were not different from the control group, although there was a tendency for decreased glucose transport in the cancer group. In all patients, there was no correlation between insulin-stimulated glucose transport and muscle ATP content (data not shown). Glycogen content and glucose incorporation into glycogen Glycogen content was significantly lower in the PDAC than in the control group (119 17 versus 213 24 mmol of glycosyl
The ATP content was significantly lower in the PDAC than in the control group (8.4 1.2 versus 15.8 0.8 mmol/kg of dry weight, P < 0.05). There were no statistically significant differences between the PDAC and cancer groups. The CP and cancer groups also had decreased muscle ATP content, but these did not reach statistical significance compared with the control group (Fig. 3A). The trend was similar for phosphocreatine content, although the differences between means across groups were not statistically significant (Fig. 3B). Glucose transport in L6 myotubes Transport of 2-DOG was determined with and without the presence of 2000 nM insulin. Basal glucose uptake was similar across groups (Fig. 4A), and exposure to insulin resulted in a significant increase in glucose transport in all groups (Fig. 4B). There were no significant differences in basal or insulinstimulated glucose transport in myotubes without or with exposure to medium conditioned by PDAC cells, fibroblasts, or osteoblasts. Discussion In the present study, insulin resistance for glucose uptake and decreased glycogen and ATP content were found in skeletal muscle from patients with PDAC. In addition, conditioned media from human PDAC cells did not affect insulin action on glucose uptake in L6 myoblasts. Most patients with PDAC were diabetic or glucose intolerant without hypoinsulinemia, as has been previously reported [5,10], indicating the presence of peripheral insulin resistance. Indeed, insulin-stimulated glucose transport was markedly decreased in isolated skeletal muscle from patients with PDAC, confirming
Table 1 Patient characteristics*
Number (women/men) Age (y) BMI (kg/m2) Weight loss (%)y OGTT (normal/impaired/diabetes) Fasting plasma glucose (mM) Fasting plasma insulin (pM) FFAs (mM)
Controls
Chronic pancreatitis
Cancer
Pancreatic cancer
8 (5/3) 53 4 25.7 1 22 8/0/0 5.0 0.1 40 4 0.72 0.06
8 (3/5) 52 3 21.3 0.7 63 3/1/4 7.0 1 30 8 1.50 0.48
8 (5/3) 68 3z 23.0 2 33 5/0/3 5.3 0.3 59 6 1.19 0.08
13 (10/3) 70 2z 23.6 1.3 63 2/3/8 8.1 0.6x 50 9 0.86 0.05
BMI, body mass index; FFAs, free fatty acids; OGTT, oral glucose tolerance test * Data are presented as mean SE. y During previous 3 mo. z P < 0.05 compared with controls. x P < 0.05 compared with controls and cancer group.
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Fig. 1. The 3-O-methylglucose transport in isolated muscle strips from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Muscle strips were incubated in the presence of 5 mM glucose in the absence (A) or presence (B) of insulin (1000 mU/mL). Basal glucose transport is expressed as micromoles per milliliter per hour, and insulinstimulated glucose transport is expressed as fold increase over basal glucose transport. Data are presented as mean SEM. * P < 0.05 compared with control. CP, chronic pancreatitis; PC, pancreatic cancer.
historical findings [10] and supporting the hypothesis that insulin resistance at the skeletal muscle cell level contributes to the high frequency of impaired glucose tolerance or diabetes in these patients [8]. Furthermore, decreased insulin-stimulated glucose transport in skeletal muscle was observed in 11 of 13 patients with PDAC, indicating that this metabolic disturbance is strongly associated with PDAC. Peripheral insulin resistance and impaired glucose tolerance have been reported in other cancers [27,28] and have been proposed to be a general malignant phenomenon. In the present study, three patients with cancers of other sites were diabetic according to their oral glucose tolerance test results. However, in these patients, insulin-stimulated glucose transport was not significantly decreased compared with the controls, suggesting that mechanisms other than skeletal muscle insulin resistance at the glucose transport level may contribute to hyperglycemia in these patients. Hence, decreased insulin responsiveness for glucose transport in skeletal muscle was not a general consequence of malignancy in the present study. Rather, it seems to be a metabolic disturbance that is more pronounced and frequently occurring in patients with PDAC. The use of the non-metabolizable glucose analogue 3-Omethylglucose allowed us to directly assess glucose transport
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Fig. 2. Glycogen contents (A) and glucose incorporation into glycogen in the absence (B) or presence (C) of insulin (1000 mU/mL) in skeletal muscle from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Glycogen content is expressed as millimoles of glucosyl units per kilogram of dry weight. Basal and insulin-stimulated glucose incorporations into glycogen are expressed as nanomoles per gram per hour and fold increase over basal glucose incorporation, respectively. Muscle strips were incubated in the presence of 5 mM glucose. Data are presented as mean SEM. * P < 0.05 compared with control. CP, chronic pancreatitis; PC, pancreatic cancer.
activity without the additional influence of cellular glucose metabolism. However, it is also possible that skeletal muscle insulin resistance in patients with PDAC is further aggravated by defects in intracellular glucose metabolism, such as decreased glycogen synthase activity and increased glycogen phosphorylase activity [11]. The significantly decreased muscle glycogen content observed in patients with PDAC in the present study is unlikely to be due to only weight loss, because patients with CP had a similar degree of weight loss without significantly decreased muscle glycogen content compared with controls. Furthermore, there was no significant correlation between weight loss and muscle glycogen content in the present study (data not shown). Therefore, factors other than weight loss may have contributed to the decreased muscle glycogen content in PDAC. In contrast to the findings in control patients, there was no significant response to insulin in the rate of incorporation of glucose into glycogen in patients with PDAC, CP, or cancer, suggesting decreased insulin-stimulated non-oxidative glucose metabolism in all these patients compared with controls. When comparing glycogen content with muscle content of ATP and phosphocreatine, the means of the different groups show the same reciprocal pattern, although only patients with PDAC had significantly lower contents of glycogen and ATP compared with
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Fig. 3. Contents of ATP (A) and phosphocreatine (B) in skeletal muscle from control patients (n ¼ 8), patients with CP (n ¼ 8), patients with non-pancreatic cancer (n ¼ 8), and patients with PC (n ¼ 13). Contents are expressed as millimoles per kilogram of dry weight and presented as mean SEM. * P < 0.05 compared with control. ATP, adenosine triphosphate; CP, chronic pancreatitis; PC, pancreatic cancer.
controls, possibly suggesting an association between nonoxidative glucose metabolism and the energy status of the muscle cell. This hypothesis is further supported by a significant correlation between glycogen and ATP content when analyzing all patients together. In contrast, there was no correlation between muscle glucose transport and energy content, suggesting that other mechanisms are likely to contribute to the decreased glucose transport seen in patients with PDAC. The decrease of insulin-stimulated glucose transport may be due to defective signaling steps proximal to glucose transport, such as decreased phosphatidylinositol-3-kinase activity, as has previously been shown [10]. Increased lipid use in muscle has been proposed to cause insulin resistance in muscle [29] and increased free fatty acid (FFA) uptake in muscle results in a decrease in phosphatidylinositol-3-kinase activity and glucose transport [30]. In the present study, weight loss was moderate and not significantly different among groups, suggesting that factors other than anorexia and weight loss could be responsible for the observed skeletal muscle insulin resistance in patients with PDAC. One such possible mechanism could be an increased flux of lipid substrates into the muscle cell, resulting in insulin resistance. However, in the present study, plasma levels of FFA were not increased in patients with PDAC and there was no correlation between plasma concentrations of FFA and glucose
Fig. 4. Basal and insulin-stimulated 2-DOG transport (A) including insulinstimulated fold increase over basal (B) in L6 myotubes after 5 d of differentiation, starvation in 5.6 mM glucose of UM for 21 h, or in one of three conditioned media: human pancreatic carcinoma cells (MP2), FB, or OB. Cells were incubated with 0.4 mM 2-DOG without or with (2000 nM) insulin for 60 min and then with radioactive 2-DOG without or with insulin for 10 min. 2-DOG, 2-deoxyglucose; FB, fibroblasts; MP2, MiaPaca 2; OB, osteosarcoma cells; UM, unconditioned medium.
transport. Overall, the only positive association found for glucose transport in the present study was the presence of PDAC. In the present study, medium conditioned from human PDAC cells did not affect basal or insulin-stimulated glucose transport in L6 myotubes. It could be argued that the lack of inhibition of glucose transport by medium conditioned by PDAC cells is explained by an insufficient exposure time or a concentration of a putative inhibitory substance. We believe that 24-h exposure should provide adequate time for transcriptional and translational events to have occurred. Furthermore, with regard to substance concentration, the experimental setting should provide concentrations of any substance in the incubation buffer that would far exceed concentrations in the “in vivo” situation. This finding does not support the hypothesis that skeletal muscle insulin resistance in patients with PDAC is directly caused by circulating factors from the tumor. Conclusion We have shown that the skeletal muscle of patients with PDAC is characterized by insulin resistance for glucose transport
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in addition to depleted glycogen and ATP stores. Neither weight loss nor plasma FFA level was associated with decreased glucose transport. Medium conditioned by PDAC cells did not directly induce insulin resistance in myotubes.
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