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Original Studies |
Department of Medicine, Division of Diabetes (S.M., H.Y.-J.), University of Helsinki, 00029 HUCH, Helsinki, Finland; and Amylin Pharmaceuticals, Inc. (M.S.F.), San Diego, California 92121
Address correspondence and requests for reprints to: Hannele Yki-Järvinen, M.D., Department of Medicine, Division of Diabetes, University of Helsinki, P.O. Box 340, 00029 HUCH, Helsinki, Finland. E-mail: ykijarvi{at}helsinki.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Circulating amylin is partly glycosylated (19). Glycosylation of amylin has been suggested to accelerate its aggregation. It is unknown whether excessive amounts of glycosylated amylin are found in plasma of diabetic patients. In the present study, we measured first and second phase plasma total and nonglycosylated amylin and serum free insulin concentrations during a hyperglycemic glucose clamp in individuals with normal, impaired, and diabetic glucose tolerance. The hyperglycemic clamp technique allows concentration measurements to be performed during maintenance of a stable glucose stimulus. To abolish any confounding effect of acute hyperglycemia on amylin and insulin secretion, the type 2 diabetic patients were rendered normoglycemic prior to the hyperglycemic clamp using an iv insulin infusion.
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Subjects and Methods |
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A total of 23 subjects were studied. Each subject participated
in an OGTT (20), to classify glucose tolerance, and in a hyperglycemic
clamp study, to characterize first and second phase insulin and amylin
concentrations. Clinical characteristics of the subjects are given in
Table 1
. Five patients were treated with
diet only. Three patients used glyburide, which was
discontinued 2 days before the study. The nature, risks, and potential
benefits of the study were explained to all subjects prior to obtaining
their written informed consent to participate. The experimental
protocol was designed and performed according to the principles of
Helsinki Declaration and was approved by the Ethical Committee of the
Helsinki University Central Hospital.
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The hyperglycemic clamp technique was used for the quantification of first and second phase insulin and amylin secretion (21). All subjects consumed a weight maintenance diet containing at least 200 g carbohydrate for 3 days prior the study. The study was started after a 12-h overnight fast. A hand vein was cannulated retrogradely with a 18 G catheter (Venflon; Viggo-Spectramed, Helsingborg, Sweden), and the hand was maintained in a thermoregulated box at 65 C to obtain arterialized blood samples (22). An ipsilateral antecubital vein was cannulated with a 18 G catheter for infusion of glucose. The arterialized venous plasma glucose concentration was raised to 5.6 mmol/L (100 mg/dL) over the measured basal glucose concentration and maintained at this level for 2 h (21). This was accomplished by infusing a 2-min iv priming dose of a 50% glucose solution (23). Thereafter, a 20% glucose solution was infused iv at the rate needed to maintain the plasma glucose concentration at the desired plateau and adjusted based on plasma glucose measurements, which were performed at 5-min intervals throughout the study. The type 2 diabetic patients received an iv infusion of insulin for normalization of plasma glucose concentrations prior to the study, as described previously (23). The insulin infusion was discontinued for 60 min prior to start of the hyperglycemic clamp, to avoid any suppressive effects of exogenous insulin on insulin or amylin secretion. Samples for measurements of serum free immunoreactive insulin and plasma amylin concentrations were taken at 0, 2.5, 5.0, 7.5, 10, 20, 30, 60, 70, and 120 min, and in addition at 15, 40, 50, 80, 90, and 100 min for insulin. The areas under the curve for the concentrations of insulin and amylin during 0–10 min and 10–120 min were defined as first and second phase insulin and amylin secretion.
Analytical procedures
Plasma samples for total and nonglycosylated amylin measurements were collected in ethylenediaminetetraacetate tubes and analyzed using two separate immunoassays that use monoclonal antibodies (Amylin Pharmaceuticals, Inc., San Diego, CA), as recently described in detail (24, 25). The cross-reactivity of these antibodies with calcitonin gene related peptides I and II, calcitonin, and insulin was less than 0.01%. The total amylin assay measures both nonglycosylated and glycosylated forms of amylin (19). These glycosylated forms have O-linked oligosaccharides attached at threonine residues near the N terminus (19). This type of glycosylation is enzymatic and occurs during biosynthesis of amylin. The other amylin assay measures specifically nonglycosylated amylin. Serum free insulin immunoreactivity was determined by double antibody RIA (Pharmacia Insulin RIA kit; Pharmacia, Uppsala, Sweden) after precipitation with polyethylene glycol (26). The cross-reactivity of insulin-antibody is, by weight, 41% with proinsulin, less than 0.1% for insulin-like growth factors I and II, and less than 0.1% for C-peptide. The minimum detective concentration is 0.5 pmol/L for both amylin assays, and the intra-assay and interassay coefficients of variation are less than 10% and less than 15%, respectively. For free insulin immunoreactivity, the minimum detectable concentration is 2.5 mU/L, and the intra-assay and interassay coefficients of variation are less than 5.3% and 7.6%, respectively. Plasma glucose was measured in duplicate using the glucose oxidase method (Glucose Analyzer II; Beckman Instruments, Inc./Hybritech, Fullerton, CA). HbA1c was measured by high-pressure liquid chromatography using the fully automated Glycosylated Hemoglobin Analyzer System (Bio-Rad Laboratories, Inc., Richmond, CA). Fasting serum C-peptide concentrations were determined by RIA (27).
Statistical analyses
Data between the study groups were analyzed using ANOVA, followed by pairwise comparison using Fischer’s least-significant-difference test. All calculations were made using the SYSTAT statistical package (SYSTAT Inc., Evanston, IL). Areas under curves were calculated using GraphPad Prism version 2.01 (GraphPad Software, Inc., San Diego, CA). All data are expressed as mean ± SEM.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Serum free immunoreactive insulin and plasma amylin concentrations
were both characterized by two distinct phases in subjects with normal
and impaired glucose tolerance. In individuals with normal and impaired
glucose tolerance, the maximal first phase serum free immunoreactive
insulin concentration was approximately two times as high as the
highest concentration during the second phase (Fig. 1, top left). This profile
differed from that of amylin, which peak concentrations in both groups
were approximately two times higher during the second phase than the
first phase (Fig. 1, bottom left). In patients with type 2
diabetes, no first phase peak could be discerned for either free
immunoreactive insulin or total amylin concentrations (Fig. 1). The
percentage of the first phase of the total area under the concentration
curve was significantly higher for free immunoreactive insulin than for
total amylin in both individuals with normal and impaired glucose
tolerance, as shown in Fig. 2.
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The first and second phase serum free immunoreactive insulin
and plasma amylin concentrations were significantly lower in patients
with type 2 diabetes than in those with normal or impaired glucose
tolerance (Fig. 1
, right). Subjects with impaired glucose
tolerance had a significantly lower first phase but not second phase
concentrations of both peptides than those with normal glucose
tolerance (Fig. 1, right).
Plasma total and nonglycosylated amylin concentrations
Fig. 3 depicts serum free
immunoreactive insulin, plasma total, and nonglycosylated amylin
concentrations during the hyperglycemic clamp. As with total amylin, a
first phase peak characterized glucose-induced secretion of
nonglycosylated amylin in individuals with normal and impaired glucose
tolerance but not those with type 2 diabetes (Fig. 3). The profile of
nonglycosylated amylin was comparable with that of total amylin, but,
again, different from that of free immunoreactive insulin (Fig. 3).
During the entire 120-min hyperglycemic clamp, plasma nonglycosylated
amylin concentrations averaged 28 ± 7, 35 ± 7, and 45
± 16% [not significant (NS) between groups] of total in
individuals with normal, impaired, and diabetic glucose tolerance (Fig. 3). The corresponding percentages for first phase were 34 ± 10,
37 ± 10, and 38 ± 12% (NS) and for the second phase
26 ± 5, 41 ± 9, and 24 ± 12% (NS), respectively.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and 2). The plasma concentrations of nonglycosylated amylin accounted for
25–45% of total amylin, with no differences between the groups (Fig. 3).
Circulating amylin and insulin concentrations, measured either under
basal conditions (6, 7, 13), after oral glucose (6, 7, 13, 14), after a
meal (6), or after an iv glucose bolus (17), have been shown to be
significantly correlated. Based on such correlations, it has been
thought that the peptides are cosecreted, and the circulatory molar
concentration ratios reflect the relative rates of secretion of the two
peptides. The hyperglycemic clamp technique used in the present study
revealed, however, differences in the magnitude of the first phase as
compared with the second phase of the two hormones. This could be due
to either lack of cosecretion or to clearance differences. Regarding
lack of cosecretion, some studies have suggested lack of cosecretion
in vitro from the isolated pancreas (28, 29). This finding,
however, may have been due to the use of either immature ß-cells (28)
or coculturing of islets with adenocarcinoma cells (29), which may have
altered ß-cell function. In other studies, where adult rat isolated
islets have been used (30) or in perfused pancreas studies where
perfusate has been extracted prior to amylin assay (31), such
dissociation of amylin and insulin secretion has not been seen.
Differences in amylin clearance are, therefore, more likely to explain
the differences in the magnitude of the first and second phase plasma
concentrations for the two hormones. In keeping with this, Clodi
et al. (18) determined the apparent volume of distribution,
fractional clearance, and the mean residence time of amylin in normal
subjects. The distribution space of amylin (173 mL/kg) was similar to
that previously reported for insulin (157 mL/kg), but fractional
clearance of amylin (0.38/min) was two to three times longer than that
reported for insulin. Also, the mean residence time of amylin (28 min)
was twice that of insulin (14 min). These data could explain, at least
in part, why the ratio of second to first phase amylin concentrations
were higher than those of insulin (Figs. 1 and 2) and support the
conclusion of Clodi et al. (18) that the molar insulin to
amylin ratios do not accurately reflect differences in the secretion of
the two peptides.
Assuming clearance of amylin is not dependent on glucose tolerance, first phase amylin secretion in subjects with impaired glucose tolerance, and first and second phase insulin and amylin secretion in patients with type 2 diabetes, were defective compared with those with normal glucose tolerance. The data in the impaired glucose tolerance subjects are consistent with previous data on insulin secretion in Caucasian subjects with impaired glucose tolerance (32). First and second phase secretory profiles of amylin have not been previously reported in these subjects. The data suggest that circulating hyperamylinemia does not contribute to impaired glucose tolerance in humans and that amylin and insulin secretion deteriorate in parallel in these subjects. This of course, does not exclude the possibility that amylin is continuously accumulating in the pancreas.
We also compared first and second phase insulin and amylin concentrations between type 2 diabetic and normal subjects, and these studies were performed under identical glycemic conditions as in the subjects with normal and impaired glucose tolerance. Both first and second phase secretion were severely impaired in the patients with type 2 diabetes. These data are consistent with those of Ludvik et al. (8), who also found subnormal insulin and amylin concentrations both after an iv glucose bolus and after oral glucose in type 2 diabetic patients, and with those of Kahn et al. (14), who found the increment in insulin and amylin concentrations to be reduced relative to prevailing glycemia in type 2 diabetes. Circulating amylin and insulin concentrations have also been found to be higher in type 2 diabetic subjects than in normal subjects after oral glucose (7). In the latter study, however, the type 2 diabetic patients were not matched with respect to age and not characterized with respect to body weight, and the increased concentrations were observed during hyperglycemic conditions. Also, it should be noted that most RIAs for insulin measure insulin immunoreactivity, which includes not only insulin but also proinsulin and possibly its split products (33). This will, as probably was also the case in the present study, underestimate the degree of insulin deficiency in both impaired glucose tolerance and diabetic subjects (33). Because proinsulin is cleared slower from plasma than insulin (34), the difference in the ratio of the first and second phase plasma amylin vs. insulin concentrations would have been even greater than was now observed. The very low circulating amylin concentrations in the present study, in view of the several reports of marked amyloid deposits in the pancreas of type 2 diabetic patients, suggest that worsening of glucose tolerance is characterized by a progressive impairment in amylin secretion to the plasma, which may be a consequence of retention in the pancreas or of progressive ß-cell destruction.
Nonenzymatic glycosylation of amylin has been shown to accelerate its
aggregation and shorten the nucleation period necessary to polymerize
unseeded amylin (4). Because it is possible that amylin glycosylated by
an enzymatic process may have similar amyloidogenic properties (35), we
sought to determine whether these amylin forms were increased in type 2
diabetic patients. We found the concentrations of nonglycosylated and
total amylin to be similarly reduced in type 2 diabetic patients as
compared with normal subjects (Fig. 3). This implies that increased
glycosylation of amylin does not characterize circulating amylin in
human diabetes. There are still no data to indicate the importance of
the glycosylated vs. nonglycosylated amylin. The fact that
it does not differ between normal subjects and patients with diabetes
indicates that it may play an undefined role in normal secretion or
function, but there is no data identifying the role. The present data
showing similar proportions of glycosylated and nonglycosylated
amylin both in first and second phase and in subjects with normal and
impaired glucose tolerance and in patients with type 2 diabetes implies
that these two forms of the peptide are not secreted or cleared
differently.
We conclude that the profile of plasma total and nonglycosylated amylin concentrations, as determined under similar steady-state hyperglycemic conditions in subjects with varying degrees of glucose tolerance, differ markedly from that of insulin. These differences could reflect either slower clearance of amylin than insulin (18, 36) or differences in the secretory dynamics of the two peptides. Regarding defects in amylin secretion, first phase insulin and amylin concentrations are lower in subjects with impaired glucose tolerance as compared with those with normal glucose tolerance and very low in patients with type 2 diabetes. These data, together with parallel reductions in total and nonglycosylated amylin, demonstrate that neither circulating hyperamylinemia nor an increase in glycosylated amylin characterize human type 2 diabetes or impaired glucose tolerance.
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Acknowledgments |
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Footnotes |
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Received November 10, 1999.
Revised March 1, 2000.
Revised April 26, 2000.
Accepted April 26, 2000.
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References |
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