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Department of Biomedical Sciences, Creighton University School of Medicine (U.A., R.D.R., T.E.A.), Omaha, Nebraska 68178; Research Service (151), Department of Veteran Affairs Medical Center (R.D.R.), Omaha, Nebraska 68105; and Arvid Wretlind Laboratory for Metabolic Research, Department of Surgery, Karolinska Institutet at Huddinge University Hospital (U.A., J.P., J.L., C.A.), S-141 86 Huddinge, Sweden
Address all correspondence and requests for reprints to: Johan Permert, M.D., Ph.D., Department of Surgery, Karolinska Institutet at Huddinge University Hospital, S-141 86 Huddinge, Stockholm, Sweden. E-mail: Johan.Permert{at}karo.ki.se
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Abstract |
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In a cross-over design, two sets of experiments in which conscious unrestrained rats received chronic IAPP infusions at 0 and 2 or 0 and 7 pmol/kg·min were performed. Peripheral glucose disposal was determined by means of the hyperinsulinemic euglycemic clamp technique. Chronic elevations of circulating IAPP at concentrations that reduced food intake [43.5 ± 6.2 g (control) vs. 35.7 ± 8.2 g (IAPP; P < 0.01) and 34.0 ± 2.2 g (control) vs. 28.8 ± 1.4 g (IAPP; P = 0.07) for the 7 and 2 pmol/kg·min experiments, respectively] had no effect on the glucose metabolic rate [GMR; 18.5 ± 0.6 mmol/kg·h (control) vs. 18.7 ± 0.9 mmol/kg·h (IAPP) and 14.4 ± 0.7 mmol/kg·h (control) vs. 15.6 ± 0.7 mmol/kg·h (IAPP) for the 7 and 2 pmol/kg·min experiments, respectively]. Thus, effects on glucose metabolism are unlikely to explain the anorectic effect of IAPP.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously reported markedly elevated fasting plasma concentrations of IAPP in patients with pancreatic cancer compared with healthy controls (10). Pancreatic cancer patients are severely cachectic and exhibit marked weight loss early in the course of the disease (11). Up to 80% have diabetes or abnormal glucose tolerance at the time of diagnosis, indicating the severity of their metabolic abnormality (12, 13). We have recently shown that chronic IAPP infusion, which produced plasma concentrations similar to those in pancreatic cancer patients, reduced food intake and caused a marked reduction in body weight gain in rats (14). The effects of chronic low dose infusions of IAPP on glucose metabolism have not been previously reported. The aim of the present study was to investigate the effect of chronic elevations of IAPP on glucose metabolism in rats when infused to mimic plasma concentrations seen in pancreatic cancer patients. This would indicate whether this peptide could play a role in the insulin resistance associated with this disease in addition to having an anorectic effect. The effects of IAPP on fasting blood glucose and the GMR during hyperinsulinemic euglycemic clamp were investigated in conscious rats.
The successful performance of the clamp procedure necessitated frequent and rapid blood sampling for glucose measurement. To accomplish this, we developed a novel aortic catheter procedure for use in our studies.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male Wistar rats (450–550 g) were individually housed in
stainless steel wire cages in an air- and temperature-controlled room
(22–23 C), with 12-h light (0600–1800 h) and dark (1800–0600 h)
cycles. Water and pelleted rodent feed (laboratory rodent diet 5001,
PMI Feeds, St. Louis, MO) were available ad libitum unless
otherwise stated. Daily food intake was determined by placing a weighed
amount of food pellets in the food container and subtracting the weight
of the remaining pellets 24 h later. Water and bedding were
changed twice weekly. Ethical aspects of the experiment were approved
by the Creighton University animal research committee.
Design of the indwelling aortic catheter
The catheter parts and dimensions are illustrated in Fig. 1
. The catheter was constructed from
polyethylene (PE) tubing (210 mm; PE-50; id, 0.58 mm; od, 0.96 mm;
Intramedic 7411, Clay Adams, Parsippany, NJ), silicone rubber tubing
(85- and 8-mm lengths of tubing; id, 0.51 mm; od, 0.94 mm; SILASTIC
508–002, Dow Corning, Midland, MI), 0.8-mm thick Dacron felt (5
x 7 mm; 54–6-032, Boston Felt, Rochester, NH), clear irradiated
heat-shrinkable tubing (20- and 8-mm lengths of tubing with id of 1.2
mm; 12 mm of tubing with id of 1.6 mm; Polyolefin, Alpha Wire Corp.,
Elisabeth, NJ). Catheter assembly used the techniques originally
described by Weeks (15) with modifications as follows. Briefly, one end
of the silicone tubing was expanded by dipping in xylene for
approximately 10 sec. The PE tubing was then inserted 8 mm into the
lumen. A stainless steel wire was inserted from the PE-50 end to
prevent occlusion of the tubing when the 8-mm heat-shrinkable tubing
collar was sealed around the SILASTIC-PE junction by rotation in a hot
air stream. A thickening of the PE tubing was created 75 mm from the
silicone/PE junction by rotating the tubing in a hot air stream, with
pressure applied on each side of the softened segment of tubing. An
8-mm silicone collar was then placed distal to the thickening. The
procedure was repeated at the other end of the collar, creating a
dumbbell shape on the PE tubing to keep the collar in place. On the PE
end of the catheter, a hybrid end was created by inserting a mandril
into the PE tubing lumen and then shrinking a piece of heat-shrinkable
tubing (20 mm; id, 1.2 mm) over the PE tubing. This was followed by a
second layer of heat-shrinkable tubing (12 mm; id, 1.6 mm) to form a
hybrid end designed to accept a 20-gauge hypodermic needle. Finally,
Dacron felt was attached with silicone glue both to the heat-shrinkable
tubing at the silicone/PE junction and to the silicone collar. Before
insertion, the catheters were sterilized using ethylene oxide gas.
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The ventral and dorsal cervical areas of the neck and ventral abdomen were shaved and swabbed with povidone-iodine. The rat was wrapped in a sterile translucent towel drape (no. 201-1000, Phoenix Medical Technology, Andrews, SC). A midline abdominal skin incision was made, and a hemostat was used to form a sc tunnel in the left flank. The tunnel opened at a point in the midline 3 cm distal to the occiput. The catheter was inserted in the tunnel, leaving the distal Dacron cuff just beneath the skin at the dorsum. A syringe containing heparinized 0.9 M saline (40 U/ml; heparin sodium, Lyphomed, Deerfield, IL) was attached to the hybrid end of the catheter, and the lumen was filled. The abdominal cavity was then opened in the midline, and the large and small intestines were retracted to the right. A small cut was made in the left flank to enable the silicone part of the catheter to be pulled into the abdominal cavity, where the proximal Dacron cuff was sutured to the muscle. The retroperitoneum was then opened in the midline over the distal abdominal aorta, which was freed from soft tissue on its anterior aspect from the iliac bifurcation to the left renal artery and vein by blunt dissection. The silicone part of the catheter was then tunnelled retroperitoneally and positioned in an inverted S-shape under the left kidney to allow movement, situating the tip of the catheter parallel to the aorta. During this dissection, care was taken to avoid damage to the ureters. The aorta was then temporarily occluded with a small artery clamp adjacent to the renal vessels. Ten units of heparin were administered through a 23-gauge needle inserted into the aorta halfway between the renal vessels and the bifurcation. The distal end of the catheter was bevelled and inserted 5 mm caudally through the puncture in the aorta. The catheter was then anchored to the tissue proximal to the aortic insertion point using a small drop of cyanoacrylate glue (Krazy Glue, Borden, Columbus, OH). After insertion of the catheter, the lumen was flushed. During ongoing injection, the external part of the catheter was clamped by a hemostat. The syringe was then removed, and the catheter was plugged with a metal stylet. The stylet was introduced 1 mm further after the hemostat was removed, thus ensuring that the catheter lumen was kept free of blood. The retroperitoneal edges were approximated and closed with a drop of cyanoacrylate glue. The abdominal wall was closed using two layers of uninterrupted 4/0 Vicryl sutures.
A catheter was placed in the right external jugular vein through a small skin incision on the ventral neck, as previously described (16). This catheter was filled with heparinized saline, as described above, and exteriorized with the aortic catheter through the incision at the back of the neck. This incision was then sutured. Leather saddles were used to protect the catheters throughout the experimental period, as previously described (17). To maintain catheter patency, all catheters were flushed every fifth day with heparinized saline.
IAPP and vehicle infusions
For both experiments, IAPP (Multiple Peptide Systems, San Diego,
CA) was dissolved in a vehicle of 75% dimethylsulfoxide-25% 0.9
M saline. IAPP or vehicle was continuously infused using
osmotic minipumps (Alzet, Alza Corp., Palo Alto, CA). For minipump
insertion each rat was anesthetized by ketamine (50 mg/kg, ip; Ketaset,
Aveco Co., Fort Dodge, IA). Lidocaine (2.5 mg in 0.5 ml; Elkins-Sinn,
Cherry Hill, NJ) was administered sc at the point of pump insertion.
The pump was implanted under aseptic conditions into a sc pocket on the
animal’s back. For the first experiment, IAPP (2 pmol/kg·min) or
vehicle was infused for 5 days using a model 1007D minipump before the
clamp procedure. For the second experiment, IAPP (7 pmol/kg·min) or
vehicle was infused for 2 days using a model 1003D minipump before the
clamp procedure. Each animal served as its own control, using a
cross-over experimental design in which half of the animals received
IAPP first, and half received vehicle first. The first pump was
replaced by the alternate treatment 2 days after the infusion from the
previous pump had ceased.
Insulin and glucose infusions were given through the venous catheter. All blood samples were collected from the aortic catheter. To ensure an iv route of administration, the jugular catheter was tested after each experiment. Catheter function was tested by an injection of ketamine (12 mg/kg; Ketaset, Aveco Co., Fort Dodge, IA). Rats that were not anesthetized within 30 sec were excluded from the experimental group.
Hyperinsulinemic euglycemic clamp
To study peripheral insulin sensitivity, the hyperinsulinemic
euglycemic clamp of DeFronzo was used (18). After an overnight, 8-h
fast, 1.8 ml blood were collected for quantification of fasting IAPP
and insulin. Fasting blood glucose was measured in 25 µl whole blood
using a YSI 2300 stat glucometer (Yellow Springs Instruments Co.,
Yellow Springs, OH). Both insulin and glucose infusions were
administered using syringe ram pumps (Harvard model 22 pump, Harvard
Apparatus, South Natick, MA). A constant infusion of insulin (Actrapid,
Novo Nordisk, Copenhagen, Denmark) at 10 µU/kg·min in 0.9
M saline with 0.1% rat albumin was administered. This dose
of insulin is the minimum required to abolish hepatic glucose output
(19). A variable infusion of glucose (dextrose, 300 mg/ml) was
administered to maintain euglycemia (blood glucose, 5.5 ± 0.5
mmol/liter). Blood glucose levels were measured with the glucometer
every 5–10 min. The glucose infusion pump rate was controlled by an
IBM computer programmed to record the volume of glucose administered
over each time period. At steady state (90 and 120 min), another 1.8 ml
blood were sampled for later determination of IAPP and insulin. The GMR
(mmol per kg/h) at steady state was calculated from the amount of
infused glucose and the weight of the rat. All clamps were performed
between 0900–1500 h. A total of eight animals in the first experiment
and nine animals in the second experiment underwent hyperinsulinemic
euglycemic clamp procedures after both treatments (IAPP and
vehicle).
Blood sampling and RIA
Arterial blood samples were collected into tubes chilled on ice
containing EDTA (1.5 mg/ml) and aprotinin (400 kallikrein inhibitor
units/ml; Trasyslol, Bayer, Leverkusen, Germany). After each sample,
the animal’s blood loss was replaced with an equal amount of
heparinized saline (4 U heparin/ml saline). Within 15 min, the blood
sample was centrifuged at 1700 x g for 10 min at 4 C.
The plasma was then decanted and stored at -80 C until extraction and
RIA. The extraction and RIA procedures for IAPP and insulin have been
previously described in detail (10, 14). Recovery during the Sep-Pak
extraction was more than 90%, and the within-batch coefficients of
variation for both assays were less than 8%. The IAPP and insulin
assays detect changes between adjacent samples of 2 and 4 pmol/liter,
respectively.
Statistics
All data are presented as the mean ± SEM.
Statistical analysis of cumulative food intake, body weight change,
fasting blood glucose levels, and GMR was carried out using paired
Student’s t test. For plasma levels of IAPP and insulin,
one-way ANOVA with Bonferroni post-hoc corrections was used.
P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Food intake on the day before the hyperinsulinemic euglycemic clamp during IAPP infusion at 7 pmol/kg·min (35.7 ± 8.2 g) was significantly lower than the control value (43.5 ± 6.2 g; P < 0.01). Food consumption during the 2 pmol/kg·min IAPP infusion (28.8 ± 1.4 g) was also lower than the control value (34.0 ± 2.2 g), but this difference just failed to reach statistical significance (P = 0.07). Body weights on the day before clamp were similar in all treatments groups [481.2 ± 15.2 g (IAPP) vs. 490.5 ± 12.5 g (control) for the 2 pmol/kg·min experiment and 446.5 ± 12.6 g (IAPP) vs. 452.6 ± 7.4 g (control) for the 7 pmol/kg·min experiment]. Although most animals lost weight due to surgery, the IAPP-treated animals lost more weight (-11.8 ± 2.1 g vs. -2.0 ± 9.8 g (control; P = 0.39) in the 2 pmol/kg·min experiment and significantly more in the 7 pmol/kg·min experiment: IAPP, -11.9 ± 3.8 g vs. 0.8 ± 2.7 g (control; P < 0.05)]. However, the cross-over design of the experiments is likely to have reduced the magnitudes of the IAPP-induced weight losses.
There were no significant differences in fasting blood glucose
concentrations [4.53 ± 0.10 mmol/liter (IAPP) vs.
4.72 ± 0.12 mmol/liter (control) and 3.99 ± 0.20 mmol/liter
(IAPP) vs. 4.24 ± 0.19 mmol/liter (control) for the 2
and 7 pmol/kg·min experiments, respectively]. The GMR data during
hyperinsulinemic euglycemic clamp are summarized in Fig. 2. There was no significant difference in
GMR between control and IAPP treatment at either dose. This indicates
no difference in peripheral insulin sensitivity during chronic IAPP
infusion at these doses. Fasting plasma IAPP concentrations were
significantly increased at both infusion doses of IAPP
(P < 0.001; Table 1).
Fasting insulin concentrations were not significantly different between
control and IAPP infusions at either dose. During hyperinsulinemic
euglycemic clamp, insulin concentrations increased by approximately
20-fold, but were not different between the control and IAPP-treated
groups (P < 0.001; Table 1). IAPP concentrations were
not significantly influenced by the hyperinsulinemic euglycemic clamp
procedure in any of the groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experiments of the present study were undertaken to evaluate whether chronically elevated circulating IAPP, at concentrations similar to those seen in pancreatic cancer patients, is sufficient to cause the insulin resistance frequently seen in these patients. Because pancreatic cancer patients have chronically elevated plasma IAPP concentrations and because some of the effects of metabolic hormones such as insulin are mediated at the transcriptional level and, therefore, require some time for an effect to be observed, we chose to study the effect of long term infusions. The lack of effect on glucose disposal at plasma IAPP concentrations that were sufficient to influence food intake suggests that IAPP on its own does not account for the insulin resistance that accompanies pancreatic cancer. In contrast, by inducing anorexia, IAPP is likely to contribute to the cachexia and severe weight loss associated with this disease (14).
The mechanism of the potent anorectic effect of IAPP is uncertain. It is not clear whether the effects of low dose infusions are mediated centrally or peripherally, as intrahypothalamic or intracerebroventricular administration of the peptide is equally effective as iv or ip administration (14, 17, 20, 21, 22). A recent report revealed that IAPP causes a marked inhibition of gastric emptying, providing one possible mechanism (23). However, the anorectic effect was not influenced by subdiaphragmatic vagotomy, ruling out this important neural pathway as the mediator of the effect (22).
Previous studies using a number of different models have demonstrated
an effect of IAPP on glucose metabolism in several species (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37).
However, in all of these studies, the doses of IAPP used were much
higher than those in the present study and were far above the
physiological range. Indeed, the authors of some of these previous
studies concluded that IAPP is unlikely to be of physiological
importance in peripheral glucose metabolism (26). One previous study
demonstrated a dose-responsive reduction in peripheral glucose
utilization in the dog in response to human IAPP. However, the effect
was only significant at a dose of 25 µg/kg·h (
108
pmol/kg·min), a dose 15-fold higher than the highest dose of the
present study. No effect was observed at doses in the range of those
used in the current experiment, although only one animal was used for
each dose (25). One other study addressed the issue of prolonged
elevation of circulating IAPP. The effects of 24-h iv IAPP infusions on
insulin sensitivity and glucose tolerance were studied in rats (27).
Glucose intolerance and insulin resistance were induced at 20 µg
IAPP/h (243 pmol/kg·min), an infusion rate more than 30 times
higher than that used in our present study, and the effects of this
higher dose were less prominent after 24 h compared with the
effects seen after 2 h of infusion (27). The results of the
present and previous studies demonstrate that IAPP does have effects on
peripheral glucose metabolism, but only at markedly supraphysiological
doses.
Despite the lack of effects of IAPP on glucose metabolism in the
present studies, the new aortic catheter was very successful. This
catheter permitted the frequent and rapid blood sampling necessary for
the hyperinsulinemic euglycemic clamp to be performed in conscious and
unrestrained animals. As 19 of 20 of these catheters remained patent
during the whole study period (2 months), this study demonstrates
that long term physiological studies can be carried out without the
influence of operative trauma. Aortic placement allows for a larger
catheter than previous techniques, such as catheterization of the
carotid artery. The silicone intravascular portion causes less
irritation, which minimizes vascular tissue reaction to foreign
material and avoids trauma to the aortic wall. The Dacron sleeves,
positioned where the catheter enters the skin and the abdominal cavity,
allow rapid formation of scar tissue, promoting healing and preventing
local infection. The hybrid end, which is designed to accept a 20-gauge
needle, allows easy injection and blood sampling.
In summary, the new aortic catheterization technique allowed rapid and repeated blood sampling during euglycemic hyperinsulinemic clamp studies for up to 2 months. Although a chronic low dose infusion of IAPP reduced food intake, no effect on peripheral glucose disposal was seen. These findings suggest that the elevations of IAPP seen in patients with pancreatic cancer are unlikely to contribute to the glucose intolerance seen in this disease, but may well contribute to the associated cachexia.
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Acknowledgments |
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Footnotes |
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Received April 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) in the fasted, anesthetized rat. Life Sci 52:1717–1726[CrossRef][Medline]
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