1 Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia; and 2 School of Biological Sciences and 3 Department of Medicine, School of Medicine, University of Auckland, Auckland 1, New Zealand
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To clarify roles of
amylin, we investigated metabolic responses to rat amylin-(837), a
specific amylin antagonist, in normal and insulin-resistant, human
growth hormone (hGH)-infused rats. Fasting conscious rats were infused
with saline or hGH, each with and without amylin-(8
37) (0.125 µmol/h), over 5.75 h. At 3.75 h, a hyperinsulinemic (100 mU/l) clamp
with bolus
2-deoxy-D-[3H]glucose
and [14C]glucose was
started. hGH infusion led to prompt (2- to 3-fold) basal
hyperamylinemia (P < 0.02) and
hyperinsulinemia. Amylin-(8
37) reduced plasma insulin
(P < 0.001) and enhanced several
measures of whole body and muscle insulin sensitivity
(P < 0.05) in both saline- and
hGH-infused rats. Amylin-(8
37) corrected hGH-induced liver insulin
resistance, increased basal plasma triglycerides and lowered plasma
nonesterified fatty acids in both groups, and reduced muscle
triglyceride and total long-chain acyl-CoA content in saline-treated
rats (P < 0.05). In isolated soleus
muscle, amylin-(8
37) blocked amylin-induced inhibition of glycogen
synthesis but had no effect in the absence of amylin. Thus
1) hyperamylinemia accompanies
insulin resistance induced by hGH infusion;
2) amylin-(8
37) increases whole
body and muscle insulin sensitivity and consistently reduces basal
insulin levels in normal and hGH-induced insulin-resistant rats; and
3) amylin-(8
37) elicits a
significant alteration of in vivo lipid metabolism. These findings
support a role of amylin in modulating insulin action and suggest that
this could be mediated by effects on lipid metabolism.
human growth hormone; euglycemic clamp; muscle; liver; triglycerides; long-chain acyl-CoA
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AMYLIN, a 37-amino acid polypeptide, was first isolated
and characterized from islet amyloid deposits in patients with
non-insulin-dependent diabetes mellitus (NIDDM) (7).
Immunohistochemical studies have revealed that amylin is cosecreted
with insulin from pancreatic -cells (24). Despite considerable
investigation, the metabolic role of amylin is far from clear (7).
Amylin has the potential to exert an autocrine or paracrine effect on
islet insulin secretion (33), and in addition a number of
extrapancreatic actions of amylin have been reported, although
controversy remains as to their physiological importance. In muscle,
amylin has been shown to cause glycogenolysis and oppose glycogen
synthesis (8) and, as a result, increases lactate levels in plasma
(38). Amylin administration also results in increased hepatic glucose
production (16, 23).
Amylin has been implicated as a factor in insulin-resistant states (18). Evidence of increased circulating levels has been found in states such as obesity and NIDDM (7), suggesting that hyperamylinemia may accompany the hyperinsulinemia, which is a characteristic of such states.
We recently demonstrated that continuous infusion of human growth hormone (hGH) causes prompt hyperinsulinemia and insulin resistance in the rat (14). Here we reasoned that hGH-induced hyperinsulinemia may be accompanied by hyperamylinemia and that this may contribute to the generation of hGH-induced insulin resistance. Another factor that may contribute to the onset of hGH-induced (9, 14) and diet-induced (31) insulin resistance is increased systemic and muscle lipid availability. However, except for two negative reports (8, 21), related to adipose tissue lipolysis, we are unaware of any studies examining whether there is any link between amylin and lipid metabolism.
In the past, it has often proved difficult to demonstrate the
physiological effects of an endogenous hormone by administration to
biological systems of small quantities of the hormone itself (sufficient only to produce near-physiological increments in
concentration). Specific antagonists of hormone action have often
proved to be more reliable indicators of the physiological role played
by an endogenous hormone (4). Here we have used the principle of syntopic antagonism to probe the physiological role of endogenous amylin on fuel metabolism in the rat (4). The specific amylin antagonist amylin-(837) has previously been used to examine roles of
amylin (2, 10), and we have employed this antagonist in our own study.
Amylin-(8
37) is a truncated peptide of native rat amylin (35) . In
vivo (2) and in vitro (1, 3, 10, 35) studies suggest that it acts as a
specific amylin antagonist. Further data related to the specificity of
the action of amylin-(8
37) in muscle were obtained in the present
study.
The present study was designed to assess the effects of amylin-(837)
on glucose and lipid metabolism in normal and insulin-resistant rats
under insulin-stimulated and basal conditions. The studies demonstrate
profound effects favoring enhanced insulin action with concomitant
alteration of lipid metabolism in both normal and insulin-resistant
rats.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials for Infusion
Rat amylin-(8Animals
Ethical aspects. All surgical and in vivo experimental procedures performed were approved by the Garvan Institute of Medical Research-St. Vincent's Hospital Animal Experimentation Ethics Committee and were in accordance with the National Health and Medical Research Council of Australia guidelines for the use of animals in research. All in vitro muscle experiments were performed according to protocols approved by the University of Auckland Animal Ethics Committee.
In vivo animal preparation. Adult male Wistar rats (365 ± 8 g) were fed standard laboratory diet (Allied Feeds, Rhodes, NSW, Australia) with water ad libitum and were housed individually after surgery in enclosed, well-ventilated metabolic cages in a temperature controlled environment (22 ± 1°C) with 12:12 h light-dark cycle (lights on at 0600). Rats were accustomed to human contact to minimize stress associated with handling during studies.
Surgery
One week before the day of the clamp, rats were chronically cannulated via the right jugular vein and left carotid artery under ketamine hydrochloride (90 mg/kg)-xylazine (10 mg/kg) anesthesia. Cannulas were exteriorized, and patency was maintained as previously described (17). Subsequent recovery over a 7-day period was closely monitored with measurement of food intake and body weight gain.Study Protocols
Infusion studies.
Cannulated rats were randomly assigned to one of four infusion groups,
continuously receiving saline (control), saline plus amylin-(837),
hGH, or hGH plus amylin-(8
37) over a 5.75-h period. hGH (21 µg · kg
1 · h
1)
and amylin-(8
37) (0.125 µmol/h) were infused continuously in 0.9%
saline (vol 600 µl/h) via the carotid cannula using a syringe pump
(Harvard Instruments, S. Natick, MA). The in vivo amylin-(8
37) infusion rate was the same as previously used (2).
Extended basal studies.
Additional saline and saline plus amylin-(837) groups were used for
longer basal studies, extended for the whole 5.75-h infusion period.
Plasma samples were collected hourly. After collection and plasma
separation, erythrocytes were resuspended in saline and injected back
into the rat. Forty-five minutes before the end of the infusion, a
bolus dose of tracer was administered (as for clamp protocol), and
plasma and tissue samples were collected and processed as described
above.
Estimation of individual muscle glucose metabolic index. Frozen tissues (50-100 mg) were homogenized in 10-20 vol of ice-cold water for 30 s using an Ultra-Turrax (Janke and Kunkel, Ika-Werk, Germany). With 0.2-ml supernatant samples, free and phosphorylated 2-[3H]DG were separated by ion-exchange chromatography on Dowex 1-X8 (100-200 mesh, acetate form, Bio-Rad, Richmond, CA) (17). A further 0.2 ml of the supernatant was counted directly to provide the total tracer content. The accumulation of tissue phosphorylated 2-[3H]DG and the plasma 2-[3H]DG disappearance curve were used to calculate the glucose metabolic index (R'g), an estimate of the tissue-specific glucose uptake rate (17). Other analyses for tissue glycogen and triglyceride content were performed as described previously (5, 13).
Actions of amylin and amylin-(837) in isolated incubated soleus
muscle strips.
Male Wistar rats weighing 148 ± 11 g were anesthetized with
pentobarbital sodium (45-50 mg/kg body wt) and then killed by cervical dislocation. Soleus muscles were quickly dissected out under
carboxygenated Krebs-Henseleit buffer (KHB) and teased into two halves.
After a further washing step in KHB and a preincubation for 30 min in
Dulbecco's modified Eagle's medium containing 0.1% (wt/vol) bovine
serum albumin and no added peptides, insulin, or insulin with
increasing amounts of peptides, respectively, 0.5 µCi
D-[U-14C]glucose
was added, and the muscle strips were incubated for another 2 h at
30°C under a constant gas flow of 95%
O2-5%
CO2 in a shaking water bath.
Thereafter the strips were blotted dry on filter paper, the tendons
were excised, and strips were snap frozen in liquid nitrogen. The
frozen muscle strips were then freeze dried. About 5 mg of freeze-dried
muscle were solubilized in 0.4 ml 60% (wt/vol) KOH for glycogen
analysis. The glycogen was precipitated overnight at
20°C
with 1.2 ml absolute ethanol, centrifuged (8,000 g, 15 min, 4°C), and washed twice
with 1.2 ml ice-cold ethanol. The dried glycogen precipitate was then
dissolved in 0.6 ml of water. One aliquot (0.3 ml) was used to measure
[14C]glycogen
formation by liquid scintillation spectrometry as a measure of de novo
glycogen synthesis, and the other was hydrolyzed to
D-glucose by adding 1 U of
glucan 1,4-
-glucosidase enzyme activity from
Aspergillus niger (Sigma, St. Louis,
MO) and making up the volume to 0.5 ml with 0.2 M sodium acetate (pH
4.8). The glucose concentration was analyzed with an YSI 2300 Stat
glucose analyzer (Yellow Springs Instrument, Yellow Springs, OH). The total glycogen content in each muscle strip is expressed as micromoles of glucosyl units per gram of dry weight.
Other analytical methods for in vivo studies. Blood and plasma glucose concentrations were measured using a YSI 23AM glucose analyzer. Plasma nonesterified fatty acids (NEFA) were measured by an acyl-CoA oxidase-based colorimetric assay method (Wako Pure Chemical Industries, Osaka, Japan), and plasma triglycerides were estimated using a colorimetric assay (Triglyceride Procedure 336, Sigma Diagnostics). Plasma glycerol was measured using a colorimetric assay (GPO Trinder) from Sigma Diagnostics. Plasma insulin was measured using a double-antibody radioimmunoassay (RIA) kit (Linco, St. Louis, MO). Plasma hGH was measured as previously described (36), using an in-house RIA with an antiserum against pituitary-derived hGH raised in rabbits and a double-antibody polyethylene glycol-facilitated precipitation technique.
Tissue long-chain acyl-CoA measurement. The method for measurement of total tissue long-chain acyl-CoA is as previously described (14). In brief, the methodology involves solvent extraction of long-chain acyl-CoA from tissues, phase separation and purification by column elution, and injection of the samples onto a Novapak C18 reverse-phase high-performance liquid chromatography (HPLC) column (Waters Millipore) for ultraviolet detection. The identification of each species was from its respective retention times obtained by applying a known amount of each species to the HPLC column before analysis of the samples. The reported parameter is the sum of the six major species determined [palmitoyl (16:0)-, palmitoleoyl (16:1)-, linolenoyl (18:3)-, linoleoyl (18:2)-, oleoyl (18:1)-, and stearoyl (18:0)-CoA].
Plasma amylin assay.
Plasma amylin was extracted using an acetone-HCl (1 M)-water mixture
(40:1:5; vol/vol/vol) with a ratio of 1:2 (wt/vol) for plasma to
extraction solution (28). This gave a measured optimal recovery of 58%
in our hands. All plasma amylin concentrations were corrected for the
incomplete extraction using a multiplier of 1.0/0.58. Supernatants were
lyophilized and stored at 80°C until analysis. Peptide
concentrations after reconstitution in assay buffer were determined in
duplicate using a commercial RIA kit (Peninsular Laboratories, Belmont,
CA). Rat amylin-(8
37) gave <5% cross-reactivity (nonparallel) with
the rat amylin standard; for this reason, endogenous amylin could not
be measured in samples from rats infused with amylin-(8
37). The limit
of detection for plasma amylin was 3 pM, the linear range was
5-125 pM, and the intra-assay coefficient of variation was <3%
at the midrange.
Statistical Analysis
All results are expressed as means ± SE. Statistical comparisons between control and hGH-treated groups were performed using two-factor analysis of variance and the Student's unpaired (2 tail) t-test (Macintosh Statview SE + Graphics program, Abacus Concepts-Brain Power). ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In Vitro Studies of Amylin-(837) Specificity in Muscle
Amylin-(837) in absence of amylin.
Initial in vitro studies in isolated incubated soleus muscle strips
demonstrated that 10 µM amylin-(8
37) had no effect in the absence
of amylin on either the basal or insulin-stimulated net rate of
[14C]glucose
incorporation into glycogen (Fig. 1). In
contrast, 30 nM amylin significantly decreased insulin-stimulated
[14C]glucose
incorporation into glycogen (Fig. 1).
|
Amylin-(837) inhibition of amylin action.
As shown in Fig. 2, 10 nM amylin
significantly decreased net insulin-stimulated
[14C]glucose
incorporation rate into glycogen. This inhibitory effect was reversed
by addition of 10 µM amylin-(8
37) to the incubation medium. Final
glycogen mass among groups (Fig. 2) showed a similar pattern to the
[14C]glucose
incorporation measurements but was not significantly different among
groups.
|
In Vivo Studies
Preclamp responses.
Plasma amylin levels after 2 or 3.75 h of hGH infusion were increased
approximately threefold compared with saline-infused rats
(P < 0.02) but were not
significantly different from saline controls at the end of the glucose
clamp (Table 1). Amylin concentrations were
determined only for the saline- and hGH-infused groups, since amylin-(837) cross-reacted in the RIA for amylin. During preclamp infusions, plasma glucose levels were similar at ~7 mM in all the
groups (Table 2). Infusion of hGH alone
raised plasma insulin and NEFA levels, but when combined with
amylin-(8
37) infusion, insulin and NEFA remained at saline-infused
levels. Amylin-(8
37) infusion also suppressed plasma insulin and NEFA
levels in the saline plus amylin-(8
37) group compared with the saline
control (Table 2). However plasma triglycerides were significantly
elevated by amylin-(8
37) in both treated groups (Table 2).
|
|
Clamp responses.
Glucose clamps were performed at similar plasma glucose levels in all
four groups (Table 3). Clamp insulin levels
were also similar in the four groups (Table 3). Plasma triglycerides
were suppressed to equivalent levels in all groups during the clamp. Infusion of hGH for 5.75 h caused peripheral insulin resistance similar
to that shown previously (14), characterized by a decreased clamp
glucose infusion rate (GIR) and Rd
compared with the saline control (Table 3). Furthermore, elevated HGO
during the clamp indicated impaired insulin suppressibility of HGO in
the hGH-infused group. Infusion of amylin-(837) prevented hGH-induced
insulin resistance (i.e., GIR, Rd,
and HGO were not different from saline-infused levels). Furthermore,
amylin-(8
37) significantly increased insulin-stimulated Rd in the saline-infused plus
amylin-(8
37) group (Table 3).
|
Clamp R'g.
As shown in Fig. 3, insulin-stimulated red
quadriceps R'g was
significantly enhanced by amylin-(837), both in the hGH-infused and
saline control groups. Similar significant effects were also found in
red gastrocnemius muscle [saline 15.8 ± 1.8, saline + amylin-(8
37), 20.8 ± 1.5 (P < 0.05 vs. saline), hGH 8.9 ± 1.6, and hGH + amylin-(8
37) 15.0 ± 1.2 µmol · 100 g
1 · min
1
(P < 0.01 vs. hGH)]. This is
consistent with the enhancement of whole body insulin sensitivity
parameters GIR and Rd (Table 3).
The insulin-stimulated R'g of
white adipose tissue was also significantly enhanced by amylin-(8
37)
infusion in the hGH-treated group but not in the saline control group
(Fig. 3).
|
Insulin-stimulated glycogen synthesis.
Amylin-(837) infusion significantly increased insulin-stimulated
[14C]glucose
incorporation into glycogen in red gastrocnemius muscle in both hGH-
and saline-treated groups (Fig. 4).
Glycogen content at the end of the clamp (Fig. 4) followed a similar
pattern. The in vivo findings are consistent with the in vitro
responses in soleus muscle strips (Figs. 1 and 2).
|
Extended basal responses.
Because it appeared that there were significant effects of
amylin-(837) in saline-infused rats, further studies were performed to examine this in more detail. In these studies, infusion of saline or
saline plus amylin-(8
37) was performed for 5.75 h without a
hyperinsulinemic clamp. Plasma glucose remained at ~7 mM in both
groups. With amylin-(8
37) infusion, plasma insulin, NEFA, and
glycerol decreased, whereas plasma triglycerides increased (Table
4), each consistent with results presented
in Table 2. Plasma glycerol was additionally measured and was
significantly decreased compared with controls, suggesting that
decreased lipolysis is a contributor to the lowered NEFA levels (Table
4). Basal glucose turnover (HGO and
Rd) was significantly reduced in
the saline plus amylin-(8
37) group compared with saline alone (Table 4). Red muscle R'g measured
under basal conditions was not altered by amylin-(8
37) [saline
vs. saline + amylin-(8
37), 19.4 ± 1.4 vs. 17.5 ± 1.4 µmol · 100 g
1 · min
1].
|
Muscle and liver lipids.
As shown in Fig. 5, amylin-(837) infusion
resulted in elevated liver triglyceride content
(P < 0.05 vs. control), whereas red
muscle triglyceride content was significantly reduced
(P < 0.05 vs. control). Total
long-chain acyl-CoA (sum of all species) levels in liver and red muscle
were increased and decreased respectively, by amylin-(8
37) infusion,
reflecting the pattern in liver and muscle triglyceride measurements
(Fig. 5).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hyperamylinemia is usually present in insulin-resistant mammals
(reviewed in Ref. 7). However, although high concentrations of
exogenously administered amylin evoke insulin resistance in vivo and in
incubated skeletal muscle strips in vitro, the contribution to in vivo
insulin resistance made by increased circulating concentrations of
endogenously derived amylin remains unknown. To investigate this
relationship, secretion and action of amylin were studied in an animal
model of insulin resistance, the hGH-infused rat (14). Amylin action
was probed by infusion of the competitive amylin antagonist,
amylin-(837), a synthetic peptide whose structure was derived by
truncating the seven NH2-terminal
residues from rat amylin. Here, we report for the first time that
infusion of hGH into rats evoked hyperamylinemia. Second, infusion of
rat amylin-(8
37) into normal rats or those with hGH-evoked insulin resistance led to increased whole body insulin sensitivity. After treatment with amylin-(8
37), basal insulin concentrations in both
groups were significantly decreased compared with those in the
respective control groups. Furthermore, the extent of the fall in
plasma insulin concentration was consistent with the increase in
insulin sensitivity in both normal and insulin-resistant rats. Third,
infusion of amylin-(8
37) elicited changes in circulating and tissue
lipids in both normal and insulin-resistant rats. Of particular note is
our observation that the fall in plasma NEFA concentrations elicited by
amylin-(8
37) was the opposite of what could have been predicted on
the basis of decreased circulating insulin concentrations. We know of
no prior reports in the literature demonstrating effects of an amylin
antagonist on tissue lipid distribution.
In the current study, we confirmed that amylin-(837) acts as an
antagonist of the in vitro effects of amylin in skeletal muscle. In the
absence of amylin, amylin-(8
37) altered neither basal nor
insulin-stimulated rates of incorporation of
D-[U-14C]glucose
into glycogen. However, in the presence of amylin, it dose dependently
reversed amylin-evoked inhibition of insulin-stimulated incorporation
of
D-[U-14C]glucose
into glycogen. These findings are consistent with the previous report
of Deems et al. (10). There are further lines of evidence to support
the view that amylin-(8
37) acts as an antagonist at amylin receptors.
In one study it completely displaced bound
125I-labeled amylin from lung
membranes (3); in another, it reversed amylin-mediated inhibition of
secretion of insulin from rat pancreatic islets (35). The neuropeptide
calcitonin gene-related peptide (CGRP) is structurally similar to
amylin and mimics its effects in many biological systems (reviewed in
Ref. 7). The truncated peptide CGRP-(8
37) also acts as an amylin
blocker in some systems (11, 34). In a recent study, CGRP-(8
37), but
not amylin-(8
37), inhibited CGRP-induced vasodilation in rat kidney
(6), indicating that the former is specific for CGRP receptors in renal
tissues. These results, when taken together, indicate that the peptide amylin-(8
37) is selective for amylin receptors.
In our current in vivo studies, amylin-(837) not only reversed
effects evoked by hGH on carbohydrate and lipid metabolism but also
further enhanced the insulin sensitivity of normal rats. This effect on
insulin sensitivity in saline-treated rats indicates that this amylin
blocker either inhibits the action of basal amylin present under
nonstimulated conditions or that it can act independently of amylin
receptors to enhance whole body insulin sensitivity. The latter notion,
however, is not supported by the in vitro findings in isolated
incubated soleus muscle. Another possible mechanism for the enhancement
of insulin sensitivity could be via enhanced blood flow. Because amylin
and CGRP are structurally similar, CGRP is a potent vasodilator, and
amylin is likely to act at CGRP receptors (7), it is conceivable that,
by blocking the amylin acting on CGRP receptors, amylin-(8
37) might
increase blood flow and thereby enhance insulin-induced
Rd. However, this explanation cannot account for the action of amylin-(8
37) to restore insulin sensitivity in soleus muscle incubated in vitro. Furthermore, the
findings of Gardiner et al. (11) show that amylin can evoke insulin
resistance in vivo in the absence of hemodynamic effects.
Our finding that plasma insulin concentrations decrease after infusion
of amylin-(837) is at possible variance with the findings of Wang et
al. (35). However, a quite different experimental design was used, and
the concentrations of amylin-(8
37) at the level of the pancreatic
islet in their in vitro study were very much higher than could be
expected in our current in vivo studies. There is evidence that
amylin-(8
37) not only antagonizes amylin-evoked inhibition of insulin
secretion (27) but also is capable of increasing insulin secretion when
added by itself (in absence of amylin) to islets or to the isolated,
perfused pancreas. (It should be noted, however, that these
preparations secrete endogenous amylin.) In the light of these in vitro
findings, we suggest that the decrease in plasma insulin without an
observable change in plasma glucose concentrations, which we found in
our current studies, is likely to be a response to the extrapancreatic
actions of amylin-(8
37). The fact that altered basal insulin
secretion and peripheral insulin sensitivity were tightly coupled is
demonstrated in Fig. 6. The highly
significant negative correlation (r = 0.70, P < 0.0001) between clamp GIRs
and basal (preclamp) plasma insulin concentrations, derived from our
data for all groups, provides further evidence for a direct
relationship between increased insulin sensitivity and decreased
insulin secretion. The identity of the metabolic signals involved is,
however, currently uncertain. It has been suggested that circulating
NEFA (32) or pancreatic
-cell long-chain acyl-CoA (29) could play
important roles in determining the rate of insulin secretion. It is
thus possible that the fall in insulin secretion is mediated by the
fall in plasma NEFA, and we believe a similar situation may be
operative after administration of the insulin-sensitizer
thiazolidinedione, BRL-49653, which can reduce NEFA levels and basal
plasma insulin levels without a demonstrable change in plasma glucose
levels in rats (26). Alternatively, it could be argued that the
decreases in plasma insulin that we observed here could be due to
amylin-(8
37) acting as a partial agonist of amylin on insulin
secretion. We believe this is unlikely, however, since there is no
published evidence to indicate that amylin-(8
37) can act as a partial
amylin agonist in any tissue, and here amylin-(8
37) clearly acted as
an antagonist in muscle.
|
Infusion of amylin-(837) enhanced whole body as well as peripheral
insulin sensitivity, as indicated by the increased clamp GIR and
Rd in both hGH- and saline-treated
groups. In addition amylin-(8
37) enhanced insulin suppressibility of
HGO in hGH-infused rats. In line with the enhancement of peripheral
insulin sensitivity by amylin-(8
37) infusion, muscle and white
adipose tissue insulin-mediated glucose uptake was enhanced, as
indicated by R'g. Significant enhancement of net glycogen synthesis, as measured by
[14C]glucose
incorporation into glycogen during the clamp procedure in
amylin-(8
37)-infused rats, provides further evidence for improved peripheral insulin sensitivity. It is well documented that amylin has
antagonistic effects on net glycogen synthesis in vivo (19, 39) and
thus enhanced insulin-mediated net glycogen synthesis in the saline
plus amylin-(8
37)-infused group is in accord with a blockade of
endogenous amylin action. Regarding the liver, in other studies, it has
been demonstrated that amylin is capable of stimulating lactate
recycling in vivo (37). Therefore the improved insulin suppressibility
of HGO in our study could be related to diminishing lactate recycling
in the liver (direct Cori cycle).
It was interesting to find altered lipid distribution after
amylin-(837) infusion, particularly in the group treated with saline
plus amylin-(8
37) infusion, since there is little available evidence
to suggest that amylin influences lipid metabolism (8, 21). In the
present study, lowering of basal plasma NEFA and glycerol by
amylin-(8
37) infusion in saline-treated animals is consistent with a
decreased rate of lipolysis. The increase in plasma triglyceride
concentration indicates increased production or decreased clearance of
very low density lipoprotein-triglyceride or a combination of the two.
Very low density lipoprotein production and secretion from the liver
have been shown to be significantly affected by peripheral insulin
concentrations (20), and the reduced plasma insulin levels in the
current study after amylin-(8
37) infusion may be responsible for the
increased plasma triglyceride concentrations. Alternatively, a
decreased clearance of triglycerides into skeletal muscle may
simultaneously explain high plasma triglyceride levels and contribute
to reduced muscle triglyceride and long-chain acyl-CoA levels. Studies
of lipid turnover kinetics during amylin-(8
37) infusion are needed to
resolve these issues. Increased liver triglycerides and long-chain
acyl-CoA concentrations during amylin-(8
37) infusion indicate
possible de novo triglyceride synthesis or recycling of NEFA.
The finding that in vivo amylin-(837) infusion leads to a significant
reduction in muscle lipids adds a new dimension to possible mechanisms
by which amylin may influence muscle insulin sensitivity. We have
previously reported that there is a striking negative association
between muscle insulin sensitivity and local triglyceride content in
various rat diet models of altered muscle insulin sensitivity (31). In
the case of hGH-induced insulin resistance, increased lipid
mobilization from adipose tissue is a characteristic over the time
frame studied here (14). The mechanisms by which altered muscle lipid
availability could lead to altered potency of insulin action are
currently not clear but could include the classic Randle cycle as well
as the possibility of other mechanisms (22). In particular, a central
role for accumulation of cytosolic long-chain acyl-CoA has been
proposed (29); these could act as regulatory ligands for several
enzymes of glucose metabolism (30). Alternatively altered muscle lipid availability may influence insulin signaling via diacylglycerol-protein kinase C interactions (25). Further studies are required to examine
whether these interactions are important in the case of amylin (8
37)
infusion and, if so, whether they are a direct or indirect effect
within muscle.
Amylin-(837) reversed hGH-induced insulin resistance and
hyperinsulinemia, suggesting a possible association between amylin and
hGH-evoked insulin resistance. Hyperamylinemia was present throughout
much of the initial phase of hGH infusion. However, that amylin
concentrations had returned to normal by the end of the euglycemic
clamp and similar insulin-lowering effects were found in normal rats
and hGH-infused rats suggests that amylin-(8
37) might be inducing a
relatively independent but opposing effect to hGH infusion on
glucoregulation. Further work will be needed to resolve this. A
decrease in amylin concentration during the euglycemic clamp is
consistent with a previous report that hyperinsulinemia suppressed
blood amylin concentrations in normal humans (12).
In conclusion, the specific amylin antagonist, amylin-(837), enhances
whole body, liver, and muscle insulin sensitivity with a concomitant
decrease of basal plasma insulin in both normal and insulin-resistant,
hGH-infused rats. Furthermore amylin-(8
37) infusion was associated
with altered lipid distribution. These findings taken together support
a role for amylin in glucose homeostasis and link amylin for the first
time to the regulation of lipid metabolism.
![]() |
ACKNOWLEDGEMENTS |
---|
The expert assistance of Allan Watkinson, Kuet Li, Joanna Edema, Donna Wilks, and Vicki Theos is gratefully acknowledged.
![]() |
FOOTNOTES |
---|
This study was supported in part by research grants from The National Health and Medical Research Council of Australia and Diabetes Australia Research Trust (to E. W. Kraegen) and the Endocore Research Trust and the Health Research Council of New Zealand (to G. J. S. Cooper).
Address for reprint requests: E. W. Kraegen, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia.
Received 23 May 1997; accepted in final form 3 July 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aiyar, N.,
E. Baker,
J. Martin,
A. Patel,
J. M. Stadel,
R. N. Willette,
and
F. C. Barone.
Differential calcitonin gene-related peptide (CGRP) and amylin binding sites in nucleus accumbens and lung: potential models for studying CGRP/amylin receptor subtypes.
J. Neurochem.
65:
1131-1138,
1995[Medline].
2.
Bennet, W. M.,
C. S. Beis,
M. A. Ghatei,
P. G. Byfield,
and
S. R. Bloom.
Amylin tonally regulates arginine-stimulated insulin secretion in rats.
Diabetologia
37:
436-438,
1994[Medline].
3.
Bhogal, R.,
D. M. Smith,
and
S. R. Bloom.
Investigation and characterisation of binding sites for islet amyloid polypeptide in rat membranes.
Endocrinology
130:
906-913,
1992[Abstract].
4.
Black, J.
Nobel Lecture in Physiology or Medicine1988. Drugs from emasculated hormones: the principle of syntopic antagonism.
In Vitro Cell. Dev. Biol.
25:
311-320,
1989[Medline].
5.
Carr, T. P.,
C. J. Andresen,
and
L. L. Rudel.
Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.
Clin. Biochem.
26:
39-42,
1993[Medline].
6.
Chin, S. Y.,
J. M. Hall,
S. D. Brain,
and
I. K. Morton.
Vasodilator responses to calcitonin gene-related peptide (CGRP) and amylin in the rat isolated perfused kidney are mediated via CGRP1 receptors.
J. Pharmacol. Exp. Ther.
269:
989-992,
1994[Abstract].
7.
Cooper, G. J. S.
Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease.
Endocr. Rev.
15:
163-201,
1994[Medline].
8.
Cooper, G. J. S.,
B. Leighton,
G. D. Dimitriadis,
M. Parry-Billings,
J. M. Kowalchuk,
K. Howland,
J. B. Rothbard,
A. C. Willis,
and
K. B. Reid.
Amylin found in amyloid deposits in human type 2 diabetes mellitus may be a hormone that regulates glycogen metabolism in skeletal muscle.
Proc. Natl. Acad. Sci. USA
85:
7763-7766,
1988[Abstract].
9.
Davidson, M. B.
Effect of growth hormone on carbohydrate and lipid metabolism.
Endocr. Rev.
8:
115,
1987[Medline].
10.
Deems, R. O.,
F. Cardinaux,
R. W. Deacon,
and
D. A. Young.
Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.
Biochem. Biophys. Res. Commun.
174:
716-720,
1991[Medline].
11.
Gardiner, S. M.,
A. M. Compton,
P. A. Kemp,
T. Bennett,
C. Bose,
R. Foulkes,
and
B. Hughes.
Antagonistic effects of human alpha-calcitonin gene-related peptide (8-37) on regional hemodynamic actions of rat islet amyloid polypeptide in conscious Long-Evans rats.
Diabetes
40:
948-951,
1991[Abstract].
12.
Hanabusa, T.,
K. Kuba,
C. Oki,
Y. Nakano,
K. Okai,
T. Sanke,
and
K. Nanjo.
Islet amyloid polypeptide (IAPP) secretion from islet cells and its plasma concentration in patients with non-insulin-dependent diabetes mellitus.
Diabetes Res. Clin. Pract.
15:
89-96,
1992[Medline].
13.
Handel, E. V.
Estimation of glycogen in small amounts of tissue.
Anal. Biochem.
11:
256-265,
1965[Medline].
14.
Hettiarachchi, M.,
A. Watkinson,
A. B. Jenkins,
V. Theos,
K. K. Y. Ho,
and
E. W. Kraegen.
Growth hormone-induced insulin resistance and its relationship to lipid availability in the rat.
Diabetes
45:
415-421,
1996[Abstract].
15.
Ho, K. Y.,
A. J. Weissberger,
M. C. Stuart,
R. O. Day,
and
L. Lazarus.
The pharmacokinetics, safety and endocrine effects of authentic biosynthetic human growth hormone in normal subjects.
Clin. Endocrinol. (Oxf.)
30:
335-345,
1989[Medline].
16.
Koopmans, S. J.,
A. D. M. van Mansfeld,
H. S. Jansz,
H. M. J. Krans,
J. K. Radder,
M. Frolich,
S. F. De Boer,
D. K. Kreutter,
G. C. Andrews,
and
J. A. Maassen.
Amylin-induced in vivo insulin resistance in conscious rats: the liver is more sensitive than peripheral tissues.
Diabetologia
34:
218-224,
1991[Medline].
17.
Kraegen, E. W.,
D. E. James,
A. B. Jenkins,
and
D. J. Chisholm.
Dose-response curves for in vivo insulin sensitivity in individual tissues in rats.
Am. J. Physiol.
248 (Endocrinol. Metab. 11):
E353-E362,
1985
18.
Leighton, B.,
and
G. J. S. Cooper.
Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro.
Nature
335:
632-635,
1988[Medline].
19.
Leighton, B.,
and
E. Foot.
The effects of amylin on carbohydrate metabolism in skeletal muscle in vivo and in vitro.
Biochem. J.
269:
19-22,
1990[Medline].
20.
Lewis, G. F.,
and
G. Steiner.
Acute effects of insulin in the control of VLDL production in humans.
Diabetes Care
19:
390-393,
1996[Abstract].
21.
Lupien, J. R.,
and
A. A. Young.
No measurable effect of amylin on lipolysis in either white or brown isolated adipocytes from rats.
Diabet. Nutr. Metab.
6:
13-18,
1993.
22.
McGarry, J. D.
What if Minkowski had been ageusic? An alternative angle on diabetes.
Science
258:
766-770,
1992[Medline].
23.
Molina, J. M.,
G. J. S. Cooper,
B. Leighton,
and
J. M. Olefsky.
Induction of insulin resistance in vivo by amylin and calcitonin gene-related peptide.
Diabetes
39:
260-265,
1990[Abstract].
24.
Moore, C. X.,
and
G. J. S. Cooper.
Co-secretion of amylin and insulin from cultured islet -cells: modulation by nutrient secretagogues, islet hormones and hypoglycemic agents.
Biochem. Biophys. Res. Commun.
179:
1-9,
1991[Medline].
25.
Nishizuka, Y.
Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J.
9:
484-496,
1995
26.
Oakes, N. D.,
C. J. Kennedy,
A. B. Jenkins,
D. R. Laybutt,
D. J. Chisholm,
and
E. W. Kraegen.
A new antidiabetic agent, BRL 49653, reduces lipid availability and improves insulin action and glucoregulation in the rat.
Diabetes
43:
1203-1210,
1995[Abstract].
27.
Peiro, E.,
P. Degano,
R. A. Silvestre,
and
J. Marco.
Inhibition of insulin release by amylin is not mediated by changes in somatostatin output.
Life Sci.
49:
761-765,
1991[Medline].
28.
Pieber, T. R.,
J. Roitelman,
Y. Lee,
K. L. Luskey,
and
D. T. Stein.
Direct plasma radioimmunoassay for rat amylin-(137): concentrations with acquired and genetic obesity.
Am. J. Physiol.
267 (Endocrinol. Metab. 30):
E156-E164,
1994
29.
Prentki, M.,
and
B. Corkey.
Are the -cell signaling molecules malonyl-CoA and cytosolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM?
Diabetes
45:
273-283,
1996[Abstract].
30.
Shrago, E.,
G. Woldegiorgis,
A. E. Ruoho,
and
C. C. DiRusso.
Fatty acyl CoA esters as regulators of cell metabolism.
Prostaglandins Leukotrienes Essent. Fatty Acids
52:
163-166,
1995[Medline].
31.
Storlien, L. H.,
A. B. Jenkins,
D. J. Chisholm,
W. S. Pascoe,
S. Khouri,
and
E. W. Kraegen.
Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid.
Diabetes
40:
280-289,
1991[Abstract].
32.
Unger, R. H.
Lipotoxicity in the pathogenesis of obesity-dependent NIDDM - genetic and clinical implications.
Diabetes
44:
863-870,
1995[Abstract].
33.
Wagoner, P. K.,
C. Chen,
J. F. Worley,
I. D. Dukes,
and
G. S. Oxford.
Amylin modulates beta-cell glucose sensing via effects on stimulus secretion coupling.
Proc. Natl. Acad. Sci. USA
90:
9145-9149,
1993[Abstract].
34.
Wang, M. W.,
A. A. Young,
T. J. Rink,
and
G. J. S. Cooper.
(8-37)h-CGRP antagonizes actions of amylin on carbohydrate metabolism in vitro and in vivo.
FEBS Lett.
291:
195-198,
1991[Medline].
35.
Wang, Z.-L.,
W. M. Bennet,
M. A. Ghatei,
P. G. H. Byfield,
D. M. Smith,
and
S. R. Bloom.
Influence of islet amyloid polypeptide and 8-37 fragment of islet amyloid polypeptide on insulin release from perifused rat islets.
Diabetes
42:
330-335,
1993[Abstract].
36.
Weissberger, A. J.,
K. K. Y. Ho,
and
L. Lazarus.
Contrasting effects of oral and transdermal routes of estrogen replacement therapy on 24-h growth hormone GH secretion, insulin-like growth factor I, and GH-binding protein in postmenopausal women.
J. Clin. Endocrinol. Metab.
72:
374-381,
1991[Abstract].
37.
Young, A. A.,
G. J. S. Cooper,
P. Carlo,
T. J. Rink,
and
M. W. Wang.
Response to intravenous injections of amylin and glucagon in fasted, fed, and hypoglycemic rats.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E943-E950,
1993
38.
Young, A. A.,
M. W. Wang,
and
G. J. S. Cooper.
Amylin injection causes elevated plasma lactate and glucose in the rat.
FEBS Lett.
291:
101-104,
1991[Medline].
39.
Young, D. A.,
R. O. Deems,
R. W. Deacon,
R. H. McIntosh,
and
J. E. Foley.
Effects of amylin on glucose metabolism and glycogenolysis in vivo and in vitro.
Am. J. Physiol.
259 (Endocrinol. Metab. 22):
E457-E461,
1990