Amylin and insulin in rat soleus muscle: dose responses for cosecreted noncompetitive ANDREW HOWARD Department
antagonists
A. YOUNG, BRONISLAVA E. GREENE, TIMOTHY
of Physiology,
Amylin
GEDULIN, DEBORAH WOLFE-LOPEZ, J. RINK, AND GARTH J. S. COOPER Pharmaceuticals, San Diego, California 92121
Young, Andrew A., Bronislava Gedulin, Deborah example, the published data do not readily distinguish Wolfe-Lopez, Howard E. Greene, Timothy J. Rink, and between competitive and noncompetitive (or insurGarth J. S. Cooper. Amylin and insulin in rat soleusmuscle: mountable) inhibition of insulin action by amylin. This
dose responsesfor cosecreted noncompetitive antagonists. J. Physiol. 263 (Endocrinol. Metab. 26): E274-E281, 1992.-Increasing concentrations of amylin progressively depressedthe maximal insulin-stimulated radioglucoseincorporation into soleus muscle glycogen, but did not substantively changethe EC& (range 0.78 to 1.52 nM); these findings show noncompetitive, insurmountable antagonism of insulin action by amylin. The resultsfrom 36 combinationsof different insulin and amylin concentrations were used to construct a response surfacethat can be usedto predict the responsefor any combination of insulin and amylin concentration. The predicted responseto a constant ratio of insulin and amylin concentration is a bell-shapedcurve. The experimentally determinedresponse to increasingamountsof an amylin-insulin mixture (molar ratio of 0.14:1, within the range measuredfor pancreatic secretion and plasmalevels) gave a bell-shapedresponserather than the sigmoidal responseseenwith insulin alone. The amylin doseresponserelation in the soleussystemprovides a usefulbioassay for amylin agonists.The doseresponsefor highly purified, synthetic human amylin obtained by measuringamylin concentrations by radioimmunoassayin the incubation medium gave an E& of 456pM (kO.18 log units). Human amylin had a potency greater than or equal to that of human insulin in this highly insulin-sensitive preparation. glycogen synthesis;glycogenolysis;in vitro
is critically important for understanding the physiological and pathophysiological role of amylin. For instance, if amylin and insulin are secreted in a fixed molar ratio, as some have suggested (26), then a competitive antagonism would simply shift the insulin dose-response curve to the right. However, the cosecretion of noncompetitive antagonist would have very different consequences. In the present study, we report the response of rat soleus muscle to insulin at a range of amylin concentrations, and the response to amylin at a range of insulin concentrations, and interpolate the amylin-insulin response surface. We verify the independence of insulin and amylin effects, and we predict and verify the effects of increasing concentrations of a fixed-ratio amylininsulin mixture on one aspect of glycogen metabolism in the isolated soleus muscle of the rat. This soleus muscle preparation can be used as a quantitative bioassay of amylin agonists; we report the potency of a highly purified preparation of synthetic human amylin when the amylin concentration applied to muscle was determined by immunoassay of the incubation medium at the end of the incubation.
AMYLIN is a 37amino
METHODS
Am.
acid peptide (5) cosecreted with insulin from the pancreatic ,&cell in response to nutrient secretagogues such as glucose and arginine (27). It was first sequenced and fully characterized after purification to homogeneity from amyloid typically found in the islets of individuals with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM) (6). The first reported activity of purified amylin was a decrease in the rate of insulin-stimulated incorporation of radioglucose into glycogen in the isolated stripped soleus muscle of the rat (19, 20). Amylin has since been shown to activate muscle glycogen phosphorylase by conversion to the active a-form (9, 32). This action is consistent with observations of increased glucose 6-phosphate concentration (34), and decreased total glycogen content (34) after amylin stimulation. The isolated stripped soleus muscle of the rat and mouse is composed predominantly of type I (oxidative, red) fibers. It has been used extensively (2,3,7,&l& 21, 24) as a model of insulin action; it is highly insulin sensitive in comparison to other muscle groups (17); its sensitivity to amylin in comparison to glycolytic (type II) muscles has not yet been measured. Although amylin has been demonstrated to depress the insulin dose-response curve in soleus muscle (20), the quantitative relationship between insulin and amylin dose responses has not been explored in detail. For E274
0193-1849/92
$2.00
Copyright
Soleus assay, dissection. Male Harlan-Sprague-Dawley rats of -200 g masswereusedto maintain the massof soleusmuscle strips ~40 mg. The animals were fasted for 4 h before being killed by decapitation. The skin was stripped from the lower limb, which was then pinned out on corkboard. The Achilles tendon was cut just above OScalcis and gastrocnemiusmuscle reflected out from the posterior aspect of the tibia. The soleus muscle,a small 15 to 20-mm-long, l-mm-thick flat muscleon the bone surface of gastrocnemiusmuscle was then stripped clear, and loose connective tissue was cleaned off using fine scissorsand forceps.The soleusmusclewasthen split into equal parts using a bladepassedanteroposteriorly through the belly of the muscle to obtain a total of four muscle strips from each animal. After the musclefrom the animal wasdissected,it was kept for a short period in physiological saline.The musclewas not held under tension, as reported in other methodologies(7), since in our hands this had no demonstrableeffect on the insulin sensitivity of radioglucoseincorporation into glycogen(results not shown). Incubation. Muscles were addedto 50-ml Erlenmeyer flasks containing 10 ml of a pregassedKrebs-Ringer bicarbonate buffer containing (in mM) 118.5 NaCl, 5.94 KCl, 2.54 CaCl,, 1.19 MgSO*, 1.19 KH2P04, 25 NaHCO,, 5.5 glucose,and recombinant human insulin (Humulin-R, Eli Lilly) and either synthetic rat (Bachem,lot no. ZG485) or synthetic humanamylin (Bachem,lot no. ZH144) asdetailedbelow. Albumin wasnot added, since early studies indicated that it increasedthe variability of the responseto amylin. Subsequentstudiesindicated
0 1992 the American
Physiological
Society
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AMYLIN
AND
INSULIN
IN
that serum albumin did not significantly prevent the absorption of amylin to glassware (C. Peterson, unpublished observations) or help to maintain immunoreactive amylin in solution. Methods used to determine the nature and purity of rat and human amylin included quantitative amino acid analysis, gas-phase amino acid sequencing, reverse-phase high-performance liquid chromatography (HPLC), and fast-atom-bombardment mass spectrometry. By the latter two methods, purity was 98.4 and 97.9%) respectively. However, application of these methods failed to detect one atom of mercury per molecule of rat amylin (J. Leighton and J. Cobb, Glaxo, Research Triangle Park, NC, personal communication), which was subsequently detected by electrospray ionization mass spectrometry (10) and atomic absorption spectroscopy. Subsequent analysis of the method of synthesis of the rat amylin suggests that this atom is interposed between the sulfur atoms of cysteine residues 2 and 7, and therefore forms part of the ring structure near the aminoterminus. Incorporation of mercury into rat amylin may be a feature of many commercially available batches that have already been used in published studies. Because the mercury adduct has all of the described actions of mercury-free amylin, including stimulation of glycogenolysis in vitro, production of hyperlactemia, hyperglycemia, hypocalcemia, and hypotension in vivo, and is blockable by specific amylin antagonists in vitro and in vivo, we consider that its actions can be used to at least qualitatively predict those of mercury-free amylins. A mercuric method of cyclization was not used in the synthesis of human amylin, which was found by electrospray ionization mass spectrometry and atomic absorption spectroscopy to be mercury free. Muscles were assigned to different flasks so that the four muscle pieces from each animal were evenly distributed among the different assay conditions. The incubation media were gassed by gently blowing carbogen (95% O,-5% CO,) over the surface while they were continuously agitated at 37°C in an oscillating water bath. pH of gassed media at 37°C was verified as being between 7.1 and 7.4. After a one-half-hour preincubation, 0.5 &i of [U-14C]glucose was added to each flask for a further 60 min. Each muscle piece was then rapidly removed, trimmed of tendons, blotted, frozen in liquid N2, weighed, and then stored at -20” C for subsequent determination of [14C]glycogen. The incubation medium was also frozen for subsequent analysis. Addition of 10,3, 1, and 0.3 nmol of amylin to 10 ml of buffer (39.18, 11.75,3.92, and 1.18 pg rat amylin; 39.05, 11.72,3.91, and 1.17 pg human amylin) was intended to result in final concentrations of 1,000,300, 100, and 30 nM, respectively. For both human and rat amylin, the mass of peptide actually present in a volume of diluted amylin solution added to the incubation medium was determined by quantitative amino acid analysis (15). For insulin and human amylin dose-response analyses, the concentrations present in the medium at the end and at a series of time points (0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120 min) after addition of amylin was determined by radioimmunoassay. At these time points, the incubation medium was sampled and added to buffer containing 0.1% Triton X-100, at which stage further adsorption to container walls was presumably inhibited. Amylin and insulin radioimmunoassay. Unextracted samples were diluted as necessary in buffer containing 0.1% Triton X-100. The assayused rabbit antibody raised against human amylin (Peninsula, catalog no. RAS7321). The antibody was 100%cross-reactiveagainst rat and cat amylin and human amylin-acid with a half-maximal inhibitory concentration of 58 pM; it had 0.04% cross-reactivity against rat calcitonin gene-related peptide I (CGRP-I) and 0% against rat CGRP-II. Standards were madefrom synthetic human amylin (Bachem), which was biologically active in the isolated soleusmuscle,asdescribedin the present report. Peptide content of standards was deter-
RAT
SOLEUS
MUSCLE
E275
mined by quantitative amino acid analysis (15) to the limits of sensitivity of the method. Thereafter, the assumptionwasmade that the standard was retained in solution (diluted linearly) with serial dilution in Triton X-loo-containing buffer. Interassay coefficient of variation (CV) was5.12%, intraassay CV was 0.6-3.7%, and the detection limit in buffer was -20 pM. Insulin concentrationswere determinedusinga commercially available kit (code IM78, Amersham) employing guinea pig anti-insulin serum, 12Wabeled purified bovine insulin as a tracer, and human insulin as a standard. Intraassay coefficient was 6.5-12.0%. [14C]gZycogen determination. This method was modified to allow determination in a 7-ml scintillation vial. Each frozen musclespecimenwasplaced in a vial with 1 ml 60% potassium hydroxide (wt/vol) and digestedat 70°C for 45 min under intermittent vigorous agitation. Dissolved glycogen was precipitated onto the walls of the vial by addition of 3 ml absolute ethanol and overnight cooling at -20°C. After centrifugation for 30 min at 2,000g, the supernatant wasgently aspirated, the glycogen was again washed with ethanol and centrifuged, the ethanol was aspirated, and the precipitate was dried under vacuum. It was important to evaporate all ethanol to avoid quenching during scintillation counting. The remaining glycogenwasredissolvedin 1 ml water and 4 ml scintillation fluid and counted for 14C Numerical methods. A rate of glucoseincorporation into glycogen (expressedin prnol. h-l l g wet tissue-l) was obtained from the specific activity of [14C]glucosein the 5.5 mM glucose of the incubation medium, and the total 14Ccounts remaining in the glycogen extracted from each muscle. Dose-responsecurves were fitted to a four-parameter logistic model using a least-squares iterative routine (ALLFIT, ~2.7, National Institutes of Health) to derive half-maximal effective concentration (EC,,) values. Since EC,, is lognormally distributed, it is expressed+SE of the logarithm. Pairwise comparisonswere performed using t test-based routines of SYSTAT (30). Insulin dose response.Insulin dose-responsecurves were obtained by incubation in media containing insulin added to result in final (nominal) concentrations of 0, 0.071, 0.21, 0.71, 2.1, 7.1, and 71 nM. The conversion factor usedwas 1 pU/ml equals7.1 pM. Amylin dose response. Amylin dose-responsecurves were generated using musclesadded to media containing 7.1 nM recombinant human insulin and either synthetic rat or synthetic human amylin (Bachem) addedto result in final (nominal) concentrations of 0, 1, 3, 10, 30, 100, 300, and 1,000 nM. A supraphysiologicalinsulin concentration wasusedfor two reasons:1) the precisionof estimation of E& wasrelated to the magnitude of 14Cincorporation into glycogen,sogreater amylin suppressionof the insulin responsewaspossiblewith near-maximal insulin stimulation; and 2) using an insulin responsenear the plateau (asymptotic part) of the dose-responsecurve in contrast to the 50% responseobtained at the often-usedinsulin concentration of 710pM minimized variation in responsedueto variation in the bathing insulin concentration or individual muscleinsulin sensitivities. The effect of using higher insulin concentrations is seen in the reduced standard error of the amylin dose-response EC&, values detailed in Table 1. Each assayalso contained internal positive controls consisting of a single batch (Bachem, lot no. ZG485) of archived rat amylin, lyophilized and stored at -20°C. Insulin-amylin response matrix. The insulin-amylin doseresponsematrix was obtained by repeating the insulin doseresponseanalyseswith amylin addedto result in nominal final concentrations of 0, 3, 10, 30, 100,and 300 nM, thereby generating a 6 X 6 table with quadruplicatesat each combination of insulin and amylin concentrations.
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E276
AMYLIN
AND INSULIN
Muscleswere incubated in media containing either synthetic rat amylin and recombinant human insulin added in a 0.14:1 molar ratio, or synthetic (mercury-free) human amylin and recombinant human insulin in the samemolar ratio. Insulin-
to-amylin
fixed-ratio
dose response.
IN RAT SOLEUS MUSCLE
.
RESULTS
Insulin and amylin dose responses. Data pooled from seven insulin dose-response assays performed in the absence of amylin are shown in Fig. 1. There was a 3.7-fold increment in the rate of glycogen labeling. When the insulin concentration in the incubation was assumed equal to the nominal concentration, an EC& of the response of 1.02 nM t 0.04 log units was obtained (n = 8-28 muscle strips/point). When the incubation insulin concentration was determined by immunoassay of the thawed media, an E& of 503 pM t 0.03 log units was obtained. Data pooled from four amylin dose-response assays performed using rat amylin in the isolated soleus muscle in the presence of 7.1 nM insulin are shown in Fig. 2. The bars indicate the 4.4.fold increase in the rate of radioglucase incorporation into glycogen evoked by insulin in this series of experiments. Progressive increases in amylin concentration in the incubation media reduced the rate of radioglucose incorporation into muscle glycogen. The E& for the response was 3.1 nM t 0.07 log units (concentration estimated by amount of peptide added, as determined by quantitative amino acid analysis). Combined insulin-amylin dose response. Figure 3A shows a series of insulin dose-response curves obtained in the presence of different amylin concentrations as indicated. Data for the maxima and EC& values of the insulin responses are presented in Table 1. They indicate that the E& of the insulin response was not progressively right-
Zero Fig. 2. Amylin aose response, rat amylin. Rates of radioglucose incorporation into glycogen in isolated rat soleus muscle incubated with 7.1 nM recombinant human insulin and varying concentrations of synthetic rat amylin (mercuric; see text). Symbols are as in Fig. 1; n = 16 for each point. E& = 3.1 nM k 0.07 log units (SE). Bars indicate response with and without insulin in the absence of rat amylin.
shifted with increasing amylin concentrations. Figure 3B shows amylin dose-response curves obtained with different insulin concentrations, as indicated. Amylin ECFjo -values and maximal responses, presented in Table 1, similarly indicate that the E& of the amylin response was independent of the prevailing insulin concentration. In other words, amylin could suppress labeling of glycogen at all insulin concentrations. Figure 4 combines the data from the insulin and amylin dose responses into a single fitted surface. The surface was generated as the product of fractional responses to insulin and amylin, using means of parameters for slope and EC& for the insulin and amylin transects, and plotted on log-log-linear axes. The equation for the surface is response =
I 1 + (inSUlin&JBinS
L
+ Uins 1
A amy - D amy ’ i 1 + (amylin/CamV)BamY
+
Damy 1
xF+K Thus Eq. 1 has the general form response = [insulin 1 Zero
0.1
1 Insulin (nM)
I 10
100
Fig. 1. Insulin dose response. Rates of radioglucose incorporation into glycogen in isolated rat soleus muscle incubated with varying concentrations of recombinant human insulin in the absence of amylin. Symbols represent means t SE of the response; n = 24-28 for all data points except for [insulin] = 71 pM (n = 8). Curve A, with an EC,, of 1.02 nM 2 0.04 log units (SE), was obtained when insulin concentration in incubation medium was assumed to be a simple function of its dilution. Curve B, with an EC,, of 503 pM t 0.03 log units, was fitted to insulin concentrations obtained from immunoassay of the thawed media. * Glucose, [UJ4C]glucose; * glycogen, [ 14C]glycogen.
factor] x [amylin
factor]
x
F +K
where Ains = 1 and Aamy = 1 (maximal responses); Bins = -0.889, Bamy = 0.835 (slope factors); Gins = 1.42 nM, Gamy = 9.0 nM (EC50 values); Dins = 0.18, Damy = 0.15 (basal responses); F (response factor) = 4.0 prnol. h-l l g wet muscle-l; and K (constant) = 0.14 (common baseline). Insulin and amylin concentrations are in nanomoles per liter. Because the baseplane of the graph (Fig. 4) is defined by log [insulin] and log [amylin] axes, combinations of insulin and amylin in any given ratio are represented as straight diagonals on this plane. Lines describing amylinto-insulin ratios of O.Ol:l, 0.14:1, and 1:l are projected up
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AMYLIN
AND INSULIN
E277
IN RAT SOLEUS MUSCLE
Table 1. Dose-related effects of insulin and amylin on rates of radioglucose incorporation into glycogen in isolated rat soleus muscle
1.9 3.4
Maximal Response, pmol - h-l . g wet tissue-l
10
Insulin
[Amylin] , nM 0 3 10 30 100 300
97
I
B
.,,I
I
0.1
Zero
1 Insulin
L_
,,,
10 (nM)
100
I
[Insulin], nM 0
1000
0.21 0.71 2.1 7.1 71
n
71 7.1
EGO,
nM
dose response
4.09 3.45 3.12 2.13 1.15 0.93 Amylin dose response 0.85kO.17 1.26t0.18 2.11k0.18 2.94&O. 18 3.56&O. 19 4.07t0.18
1.16kO.10 0.78-tO.12 1.52t0.15 1.33t0.20
Undetermined Undetermined 12.15t0.94 10.27t0.48 8.24k0.24 10.45-t-O. 16 8.69&O. 13 11.93t0.11
Concentrations are nominal values; maximal response values are given as mean and means t SE; EC& values are means t log SE. Insulin dose response at various constant amylin concentrations and amylin dose response at various constant insulin concentrations. For fitting insulin dose-response curves, maximal values were constrained to those indicated. For two insulin dose-response curves, amylin suppression of the response made the curves too shallow for reliable analysis. Maximal response, calculated quantity of glucosyl units (hexose moieties) transferred onto glycogen per gram muscle (wet weight) per hour.
,”
j
Zero
”
0.1
”
”
1
Amylin
A’
IO (nM)
1
1’
100
1
1000
Fig. 3. Interaction of insulin and amylin actions. A: insulin doseresponse relationships in the presence of different amylin concentrations, as indicated. Symbols representing means of actual responses are connected to relevant curves. Amylin concentrations in media predicted by quantitative amino acid analysis (nM) are indicated for each curve. B: amylin dose-response relations in presence of different insulin concentrations, as indicated. Means of actual responses are connected to relevant curve. Predicted media insulin concentrations (nM) are indicated for each curve.
onto the surface (labeled 0.01, 0.14, and 1, respectively). Insulin- to-amylin fixed-ratio doseresponse. The experimentally determined response to increasing concentrations of a fixed 0.14: 1 ratio of amylin to insulin are plotted as open circles (Hg-rat amylin) and squares (Hg-free human amylin) in Fig. 5. Both data sets clearly fit a bellshaped distribution, predicted for noncompetitive functional antagonism, rather than a shifted sigmoid response curve as would be predicted for competitive antagonism. For comparison, we have plotted both the bellshaped function generated from Eq. 1 using [amylin] = O.l4[insulin] (labeled 0.14 in Fig. 4) and the sigmoid dose response predicted for insulin in the absence of amylin. ECSOof synthetic human amylin. Data pooled from five amylin dose-response assays performed on a single batch of human amylin in the presence of 7.1 nM insulin are shown in Fig. 6. At high amylin concentrations, glycogen labeling was suppressed to below the “basal” rate obtained in the absence of insulin and amylin (0.51 vs. 0.76 pmol . h-l l g wet muscle- ‘; P < 0.001, unpaired t test). The ECSO of the response for human amylin was 456 pM t 0.18 log units when analyzed using amylin concentra-
Fig. 4. Insulin-amylin response surface. Response surface obtained from Eq. 1 (see text) fitting the 6 [insulin] x 6 [amylin] matrix of data used to plot Fig. 3, A and B. Lines labeled 0.01, 0.14, and 1 represent responses predicted for mixtures where amylin is at 1, 14, and 100% of the molar concentration of insulin.
tions obtained by radioimmunoassay of the incubation media. If the data were analyzed using amylin concentrations derived from mass added (from amino acid analysis) or from mass originally weighed out, the ECSO values were, respectively, 4.5 and 22 times greater and the doseresponse curves were, respectively, right-shifted and made steeper as shown in Fig. 6; the Hill coefficients were 0.54, 0.68, and 0.77, respectively. In summary, as the
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AMYLIN
lo-"
'O-10
'O-9
'09
'o-7
IO6
AND INSULIN
1o-5 IO”
Insulin (molar)
Amylin (molar) Fig. 5. Response to increasing concentrations of insulin and amylin in a fixed ratio. Rate of radiolabeling of glycogen in isolated rat soleus muscle as a consequence of increasing concentrations of a mixture of amylin (0, rat amylin, Hg adduct; q , Hg-free human amylin) and insulin where [amylin] is 14% of [insulin]. Symbols have same meaning as in Fig. 1; -n. 4 4 for each point. Bell-shaped curve is same data set that defines line labeled 0.14 in Fig. 4. Sigmoid curve is insulin response component of bell-shaped curve; that is, [amylin factor] in Eq. 1 = 1.
IN RAT SOLEUS
MUSCLE
These reductions in immunoreactive amylin were apparent at the first of several time series samplings of incubation media, and did not change appreciably in any measurement in the subsequent 2 h. When added to incubation media to achieve a nominal concentration of 100 pM, with or without muscle strips, the immunoreactive amylin remained essentially at or below the detection limit of the assay; when added in greater amounts, recoveries were somewhat higher. Regardless of the presence or absence of muscle strips, there was no indication of a time dependence of amylin concentration in the incubation media. An analysis similar to that described above for human amylin was performed using rat amylin. When weighed out and diluted to give predicted concentrations of 1,000 or 300 nM, radioimmunoassay returned similar values (785 and 255 nM). When diluted to give a concentration of 1 nM, however, immunoassay indicated rat amylin concentrations of -105 pM, indicating the loss of -90% of immunoreactivity. Again, from time series analysis, it appeared that the loss of immunoreactivity was a rapid event that had substantially occurred before incubation started. The relationship between observed concentration (by immunoassay) and expected concentration is plotted for both human and rat synthetic amylin in Fig. 7. In contrast to amylin, insulin diluted linearly. That is, the concentration of immunoreactive insulin was related to the degree of dilution over the range 71 pM to 71 nM. With or without muscle present in the media, there was no indication of a change in insulin immunoreactivity over 2-h incubations.
I
Zero
0.01
0.1
1
10
100
1000
Amylin (nM) Fig. 6. Amylin dose response, human amylin. Dose response for chemically synthesized human amylin performed in the presence of 7.1 nM recombinant human insulin in isolated rat soleus muscle. Symbols have same meaning as in Fig. 1; n = 18-20 for each point. Curve A was fitted to data obtained when amylin concentration is derived from mass initially weighed out and diluted. Curve B was fitted to data obtained when amylin concentration is derived from mass of peptide added to assay media as determined by quantitative amino acid analysis. Curve C was fitted to data obtained when amylin concentration in assay media is measured by radioimmunoassay. ECsO for curue C is 456 pM t 0.18 log units (SE).
quantities of amylin added to the assay medium became small, a smaller proportion remained as immunoreactive peptide in solution. For example, with dilutions aimed at producing a final concentration of 1 nM human amylin, the measured concentration of immunoreactive peptide was at or below the detection limit of the assay (-20 PM).
0 Rat amylin o Human amylin
Fig. 7. Relationship between expected amylin concentrations and concentrations measured using radioimmunoassay. Rat (0) and human (0) synthetic amylin concentrations in incubation media measured by specific radioimmunoassay as a function of values expected after addition of dilutions of weighed-out peptide. Observed and expected values converge at higher peptide concentrations. At lower concentrations similar to those reported in vivo, immunoreactive amylin present is much less than the amount nominally added. Line of identity is indicated. Symbols represent geometric mean and SE; n = 4-9 for each point.
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AMYLIN
AND
INSULIN
DISCUSSION
The finding that the E& values for amylin and insulin action on soleus muscle are not altered by increasing concentrations of the other hormone are consistent with biochemical and pharmacological data (11, 33) indicating that they act via separate receptors rather than by competing at a common receptor. Amylin behaves as a noncompetitive, functional antagonist to insulin. That is, amylin reduces the magnitude of the insulin response without affecting insulin potency Criti tally importa nt for analyzing the role of amylin i.s the findi ng that it causes insurmountable inhibition of insulin action. Amylin’s biological activity was originally described as opposing the muscle response to insulin (20). Some researchers have therefore regarded insulin and amylin as acting in opposition to each other, representing selfcanceling signals. By this reasoning, if these hormones were secreted in a constant ratio, the effects of any increase in plasma amylin would be nullified by the proportionate increase in insulin. The present report instead predicts and verifies that, with fixed ratios of insulin and amylin, suppression by amylin of the insulin response will eventually supervene as the concentrations of both hormones increase, resulting in the bell-shaped doseresponse relationship in Fig. 5. In describing this bell-shaped response, we have provided a theoretical construct that could explain how insulin resistance would eventually result, even though hyperinsulinemia occurred pari passu with hyperamylinemia. Even so, the constancy of the amylin-to-insulin ratio is conjectura 1. Although it has been repor ted that in vitro stimulation of P-cell secretio n under a variety of conditions results in amylin and insulin release in an essentially fixed ratio (16, 26, 27), there is mounting evidence for the dissociation of secretion of amylin and insulin; reported amylin-to-insulin ratios have ranged from l-2% (14, 16) to 37% (27), and in experiments in which the isolated perfused rat pancreas was stimulated with a pro longed glucose sti .mul us, the ra tio of amylin to insulin secreted varied between 2 and X00%, with the fasting ratio being around 13% (13). Although in the present report we focused on experiments using a ratio of l4%, the bell-shaped response was also evident at amylin-to-insulin ratios of 1 and 100%. On the other hand, the choice of a ratio of 14% to illustrate the point may have had some physiological validity; amylin-to-insulin ratios of 15-20% have been reported in humans (28), and in our hands, during oral glucose tolerance testing in nondiabetic human subjects (Ha) the molar ratios of plasma amylin to insulin have varied between 0 and 94% (minima 4.3 t 1.5%, maxima 20.3 t 7.6%). The response surface we have delineated in the present study describes the theoretical bounds of the steady-state response of a soleus muscle to all combinations of amylin and insulin. Fixed ratios of amylin to insulin define bellshaped rather than flat responses on this surface, exemplifying the observa tion that it is the absolute concentration of amylin and insulin, rather than their ratio, that determines tissue response. The relevance of the soleus response surface to the response of the intact organism is yet to be determined. Different tissues may be expected to
IN RAT
SOLEUS
MUSCLE
E279
have different sensitivities to insulin and amylin, and hence exhibit different response surfaces; adipocytes, for example, are sensitive to insulin, yet are apparently unaffected by amylin (4) (J. Lupien, unpublished observations). It is also possible, with progressing insulin resistance, that the change in insulin sensitivity of a tissue is not matched by an equal change in amylin sensitivity. The impact of changes in shape of the response surface with progressing insulin resistance is yet to be determined. James et al. (17) measured rates of radio-2-deoxyglucase uptake into soleus and other muscles during euglycemia at different steady-state insulin concentrations. Their data yield a greater potency for insulin in vivo than is typically reported in vitro. Possible reasons for such apparent discrepancies include concentration gradients between plasma and the receptor. Such gradients will occur where there is degradation or uptake of a ligand in the vicinity of the receptor, and where there is any impediment to its diffusion. The observation that lymphatic insulin concentration is
antagonists
A. YOUNG, BRONISLAVA E. GREENE, TIMOTHY
of Physiology,
Amylin
GEDULIN, DEBORAH WOLFE-LOPEZ, J. RINK, AND GARTH J. S. COOPER Pharmaceuticals, San Diego, California 92121
Young, Andrew A., Bronislava Gedulin, Deborah example, the published data do not readily distinguish Wolfe-Lopez, Howard E. Greene, Timothy J. Rink, and between competitive and noncompetitive (or insurGarth J. S. Cooper. Amylin and insulin in rat soleusmuscle: mountable) inhibition of insulin action by amylin. This
dose responsesfor cosecreted noncompetitive antagonists. J. Physiol. 263 (Endocrinol. Metab. 26): E274-E281, 1992.-Increasing concentrations of amylin progressively depressedthe maximal insulin-stimulated radioglucoseincorporation into soleus muscle glycogen, but did not substantively changethe EC& (range 0.78 to 1.52 nM); these findings show noncompetitive, insurmountable antagonism of insulin action by amylin. The resultsfrom 36 combinationsof different insulin and amylin concentrations were used to construct a response surfacethat can be usedto predict the responsefor any combination of insulin and amylin concentration. The predicted responseto a constant ratio of insulin and amylin concentration is a bell-shapedcurve. The experimentally determinedresponse to increasingamountsof an amylin-insulin mixture (molar ratio of 0.14:1, within the range measuredfor pancreatic secretion and plasmalevels) gave a bell-shapedresponserather than the sigmoidal responseseenwith insulin alone. The amylin doseresponserelation in the soleussystemprovides a usefulbioassay for amylin agonists.The doseresponsefor highly purified, synthetic human amylin obtained by measuringamylin concentrations by radioimmunoassayin the incubation medium gave an E& of 456pM (kO.18 log units). Human amylin had a potency greater than or equal to that of human insulin in this highly insulin-sensitive preparation. glycogen synthesis;glycogenolysis;in vitro
is critically important for understanding the physiological and pathophysiological role of amylin. For instance, if amylin and insulin are secreted in a fixed molar ratio, as some have suggested (26), then a competitive antagonism would simply shift the insulin dose-response curve to the right. However, the cosecretion of noncompetitive antagonist would have very different consequences. In the present study, we report the response of rat soleus muscle to insulin at a range of amylin concentrations, and the response to amylin at a range of insulin concentrations, and interpolate the amylin-insulin response surface. We verify the independence of insulin and amylin effects, and we predict and verify the effects of increasing concentrations of a fixed-ratio amylininsulin mixture on one aspect of glycogen metabolism in the isolated soleus muscle of the rat. This soleus muscle preparation can be used as a quantitative bioassay of amylin agonists; we report the potency of a highly purified preparation of synthetic human amylin when the amylin concentration applied to muscle was determined by immunoassay of the incubation medium at the end of the incubation.
AMYLIN is a 37amino
METHODS
Am.
acid peptide (5) cosecreted with insulin from the pancreatic ,&cell in response to nutrient secretagogues such as glucose and arginine (27). It was first sequenced and fully characterized after purification to homogeneity from amyloid typically found in the islets of individuals with type 2 (non-insulin-dependent) diabetes mellitus (NIDDM) (6). The first reported activity of purified amylin was a decrease in the rate of insulin-stimulated incorporation of radioglucose into glycogen in the isolated stripped soleus muscle of the rat (19, 20). Amylin has since been shown to activate muscle glycogen phosphorylase by conversion to the active a-form (9, 32). This action is consistent with observations of increased glucose 6-phosphate concentration (34), and decreased total glycogen content (34) after amylin stimulation. The isolated stripped soleus muscle of the rat and mouse is composed predominantly of type I (oxidative, red) fibers. It has been used extensively (2,3,7,&l& 21, 24) as a model of insulin action; it is highly insulin sensitive in comparison to other muscle groups (17); its sensitivity to amylin in comparison to glycolytic (type II) muscles has not yet been measured. Although amylin has been demonstrated to depress the insulin dose-response curve in soleus muscle (20), the quantitative relationship between insulin and amylin dose responses has not been explored in detail. For E274
0193-1849/92
$2.00
Copyright
Soleus assay, dissection. Male Harlan-Sprague-Dawley rats of -200 g masswereusedto maintain the massof soleusmuscle strips ~40 mg. The animals were fasted for 4 h before being killed by decapitation. The skin was stripped from the lower limb, which was then pinned out on corkboard. The Achilles tendon was cut just above OScalcis and gastrocnemiusmuscle reflected out from the posterior aspect of the tibia. The soleus muscle,a small 15 to 20-mm-long, l-mm-thick flat muscleon the bone surface of gastrocnemiusmuscle was then stripped clear, and loose connective tissue was cleaned off using fine scissorsand forceps.The soleusmusclewasthen split into equal parts using a bladepassedanteroposteriorly through the belly of the muscle to obtain a total of four muscle strips from each animal. After the musclefrom the animal wasdissected,it was kept for a short period in physiological saline.The musclewas not held under tension, as reported in other methodologies(7), since in our hands this had no demonstrableeffect on the insulin sensitivity of radioglucoseincorporation into glycogen(results not shown). Incubation. Muscles were addedto 50-ml Erlenmeyer flasks containing 10 ml of a pregassedKrebs-Ringer bicarbonate buffer containing (in mM) 118.5 NaCl, 5.94 KCl, 2.54 CaCl,, 1.19 MgSO*, 1.19 KH2P04, 25 NaHCO,, 5.5 glucose,and recombinant human insulin (Humulin-R, Eli Lilly) and either synthetic rat (Bachem,lot no. ZG485) or synthetic humanamylin (Bachem,lot no. ZH144) asdetailedbelow. Albumin wasnot added, since early studies indicated that it increasedthe variability of the responseto amylin. Subsequentstudiesindicated
0 1992 the American
Physiological
Society
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AMYLIN
AND
INSULIN
IN
that serum albumin did not significantly prevent the absorption of amylin to glassware (C. Peterson, unpublished observations) or help to maintain immunoreactive amylin in solution. Methods used to determine the nature and purity of rat and human amylin included quantitative amino acid analysis, gas-phase amino acid sequencing, reverse-phase high-performance liquid chromatography (HPLC), and fast-atom-bombardment mass spectrometry. By the latter two methods, purity was 98.4 and 97.9%) respectively. However, application of these methods failed to detect one atom of mercury per molecule of rat amylin (J. Leighton and J. Cobb, Glaxo, Research Triangle Park, NC, personal communication), which was subsequently detected by electrospray ionization mass spectrometry (10) and atomic absorption spectroscopy. Subsequent analysis of the method of synthesis of the rat amylin suggests that this atom is interposed between the sulfur atoms of cysteine residues 2 and 7, and therefore forms part of the ring structure near the aminoterminus. Incorporation of mercury into rat amylin may be a feature of many commercially available batches that have already been used in published studies. Because the mercury adduct has all of the described actions of mercury-free amylin, including stimulation of glycogenolysis in vitro, production of hyperlactemia, hyperglycemia, hypocalcemia, and hypotension in vivo, and is blockable by specific amylin antagonists in vitro and in vivo, we consider that its actions can be used to at least qualitatively predict those of mercury-free amylins. A mercuric method of cyclization was not used in the synthesis of human amylin, which was found by electrospray ionization mass spectrometry and atomic absorption spectroscopy to be mercury free. Muscles were assigned to different flasks so that the four muscle pieces from each animal were evenly distributed among the different assay conditions. The incubation media were gassed by gently blowing carbogen (95% O,-5% CO,) over the surface while they were continuously agitated at 37°C in an oscillating water bath. pH of gassed media at 37°C was verified as being between 7.1 and 7.4. After a one-half-hour preincubation, 0.5 &i of [U-14C]glucose was added to each flask for a further 60 min. Each muscle piece was then rapidly removed, trimmed of tendons, blotted, frozen in liquid N2, weighed, and then stored at -20” C for subsequent determination of [14C]glycogen. The incubation medium was also frozen for subsequent analysis. Addition of 10,3, 1, and 0.3 nmol of amylin to 10 ml of buffer (39.18, 11.75,3.92, and 1.18 pg rat amylin; 39.05, 11.72,3.91, and 1.17 pg human amylin) was intended to result in final concentrations of 1,000,300, 100, and 30 nM, respectively. For both human and rat amylin, the mass of peptide actually present in a volume of diluted amylin solution added to the incubation medium was determined by quantitative amino acid analysis (15). For insulin and human amylin dose-response analyses, the concentrations present in the medium at the end and at a series of time points (0, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120 min) after addition of amylin was determined by radioimmunoassay. At these time points, the incubation medium was sampled and added to buffer containing 0.1% Triton X-100, at which stage further adsorption to container walls was presumably inhibited. Amylin and insulin radioimmunoassay. Unextracted samples were diluted as necessary in buffer containing 0.1% Triton X-100. The assayused rabbit antibody raised against human amylin (Peninsula, catalog no. RAS7321). The antibody was 100%cross-reactiveagainst rat and cat amylin and human amylin-acid with a half-maximal inhibitory concentration of 58 pM; it had 0.04% cross-reactivity against rat calcitonin gene-related peptide I (CGRP-I) and 0% against rat CGRP-II. Standards were madefrom synthetic human amylin (Bachem), which was biologically active in the isolated soleusmuscle,asdescribedin the present report. Peptide content of standards was deter-
RAT
SOLEUS
MUSCLE
E275
mined by quantitative amino acid analysis (15) to the limits of sensitivity of the method. Thereafter, the assumptionwasmade that the standard was retained in solution (diluted linearly) with serial dilution in Triton X-loo-containing buffer. Interassay coefficient of variation (CV) was5.12%, intraassay CV was 0.6-3.7%, and the detection limit in buffer was -20 pM. Insulin concentrationswere determinedusinga commercially available kit (code IM78, Amersham) employing guinea pig anti-insulin serum, 12Wabeled purified bovine insulin as a tracer, and human insulin as a standard. Intraassay coefficient was 6.5-12.0%. [14C]gZycogen determination. This method was modified to allow determination in a 7-ml scintillation vial. Each frozen musclespecimenwasplaced in a vial with 1 ml 60% potassium hydroxide (wt/vol) and digestedat 70°C for 45 min under intermittent vigorous agitation. Dissolved glycogen was precipitated onto the walls of the vial by addition of 3 ml absolute ethanol and overnight cooling at -20°C. After centrifugation for 30 min at 2,000g, the supernatant wasgently aspirated, the glycogen was again washed with ethanol and centrifuged, the ethanol was aspirated, and the precipitate was dried under vacuum. It was important to evaporate all ethanol to avoid quenching during scintillation counting. The remaining glycogenwasredissolvedin 1 ml water and 4 ml scintillation fluid and counted for 14C Numerical methods. A rate of glucoseincorporation into glycogen (expressedin prnol. h-l l g wet tissue-l) was obtained from the specific activity of [14C]glucosein the 5.5 mM glucose of the incubation medium, and the total 14Ccounts remaining in the glycogen extracted from each muscle. Dose-responsecurves were fitted to a four-parameter logistic model using a least-squares iterative routine (ALLFIT, ~2.7, National Institutes of Health) to derive half-maximal effective concentration (EC,,) values. Since EC,, is lognormally distributed, it is expressed+SE of the logarithm. Pairwise comparisonswere performed using t test-based routines of SYSTAT (30). Insulin dose response.Insulin dose-responsecurves were obtained by incubation in media containing insulin added to result in final (nominal) concentrations of 0, 0.071, 0.21, 0.71, 2.1, 7.1, and 71 nM. The conversion factor usedwas 1 pU/ml equals7.1 pM. Amylin dose response. Amylin dose-responsecurves were generated using musclesadded to media containing 7.1 nM recombinant human insulin and either synthetic rat or synthetic human amylin (Bachem) addedto result in final (nominal) concentrations of 0, 1, 3, 10, 30, 100, 300, and 1,000 nM. A supraphysiologicalinsulin concentration wasusedfor two reasons:1) the precisionof estimation of E& wasrelated to the magnitude of 14Cincorporation into glycogen,sogreater amylin suppressionof the insulin responsewaspossiblewith near-maximal insulin stimulation; and 2) using an insulin responsenear the plateau (asymptotic part) of the dose-responsecurve in contrast to the 50% responseobtained at the often-usedinsulin concentration of 710pM minimized variation in responsedueto variation in the bathing insulin concentration or individual muscleinsulin sensitivities. The effect of using higher insulin concentrations is seen in the reduced standard error of the amylin dose-response EC&, values detailed in Table 1. Each assayalso contained internal positive controls consisting of a single batch (Bachem, lot no. ZG485) of archived rat amylin, lyophilized and stored at -20°C. Insulin-amylin response matrix. The insulin-amylin doseresponsematrix was obtained by repeating the insulin doseresponseanalyseswith amylin addedto result in nominal final concentrations of 0, 3, 10, 30, 100,and 300 nM, thereby generating a 6 X 6 table with quadruplicatesat each combination of insulin and amylin concentrations.
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E276
AMYLIN
AND INSULIN
Muscleswere incubated in media containing either synthetic rat amylin and recombinant human insulin added in a 0.14:1 molar ratio, or synthetic (mercury-free) human amylin and recombinant human insulin in the samemolar ratio. Insulin-
to-amylin
fixed-ratio
dose response.
IN RAT SOLEUS MUSCLE
.
RESULTS
Insulin and amylin dose responses. Data pooled from seven insulin dose-response assays performed in the absence of amylin are shown in Fig. 1. There was a 3.7-fold increment in the rate of glycogen labeling. When the insulin concentration in the incubation was assumed equal to the nominal concentration, an EC& of the response of 1.02 nM t 0.04 log units was obtained (n = 8-28 muscle strips/point). When the incubation insulin concentration was determined by immunoassay of the thawed media, an E& of 503 pM t 0.03 log units was obtained. Data pooled from four amylin dose-response assays performed using rat amylin in the isolated soleus muscle in the presence of 7.1 nM insulin are shown in Fig. 2. The bars indicate the 4.4.fold increase in the rate of radioglucase incorporation into glycogen evoked by insulin in this series of experiments. Progressive increases in amylin concentration in the incubation media reduced the rate of radioglucose incorporation into muscle glycogen. The E& for the response was 3.1 nM t 0.07 log units (concentration estimated by amount of peptide added, as determined by quantitative amino acid analysis). Combined insulin-amylin dose response. Figure 3A shows a series of insulin dose-response curves obtained in the presence of different amylin concentrations as indicated. Data for the maxima and EC& values of the insulin responses are presented in Table 1. They indicate that the E& of the insulin response was not progressively right-
Zero Fig. 2. Amylin aose response, rat amylin. Rates of radioglucose incorporation into glycogen in isolated rat soleus muscle incubated with 7.1 nM recombinant human insulin and varying concentrations of synthetic rat amylin (mercuric; see text). Symbols are as in Fig. 1; n = 16 for each point. E& = 3.1 nM k 0.07 log units (SE). Bars indicate response with and without insulin in the absence of rat amylin.
shifted with increasing amylin concentrations. Figure 3B shows amylin dose-response curves obtained with different insulin concentrations, as indicated. Amylin ECFjo -values and maximal responses, presented in Table 1, similarly indicate that the E& of the amylin response was independent of the prevailing insulin concentration. In other words, amylin could suppress labeling of glycogen at all insulin concentrations. Figure 4 combines the data from the insulin and amylin dose responses into a single fitted surface. The surface was generated as the product of fractional responses to insulin and amylin, using means of parameters for slope and EC& for the insulin and amylin transects, and plotted on log-log-linear axes. The equation for the surface is response =
I 1 + (inSUlin&JBinS
L
+ Uins 1
A amy - D amy ’ i 1 + (amylin/CamV)BamY
+
Damy 1
xF+K Thus Eq. 1 has the general form response = [insulin 1 Zero
0.1
1 Insulin (nM)
I 10
100
Fig. 1. Insulin dose response. Rates of radioglucose incorporation into glycogen in isolated rat soleus muscle incubated with varying concentrations of recombinant human insulin in the absence of amylin. Symbols represent means t SE of the response; n = 24-28 for all data points except for [insulin] = 71 pM (n = 8). Curve A, with an EC,, of 1.02 nM 2 0.04 log units (SE), was obtained when insulin concentration in incubation medium was assumed to be a simple function of its dilution. Curve B, with an EC,, of 503 pM t 0.03 log units, was fitted to insulin concentrations obtained from immunoassay of the thawed media. * Glucose, [UJ4C]glucose; * glycogen, [ 14C]glycogen.
factor] x [amylin
factor]
x
F +K
where Ains = 1 and Aamy = 1 (maximal responses); Bins = -0.889, Bamy = 0.835 (slope factors); Gins = 1.42 nM, Gamy = 9.0 nM (EC50 values); Dins = 0.18, Damy = 0.15 (basal responses); F (response factor) = 4.0 prnol. h-l l g wet muscle-l; and K (constant) = 0.14 (common baseline). Insulin and amylin concentrations are in nanomoles per liter. Because the baseplane of the graph (Fig. 4) is defined by log [insulin] and log [amylin] axes, combinations of insulin and amylin in any given ratio are represented as straight diagonals on this plane. Lines describing amylinto-insulin ratios of O.Ol:l, 0.14:1, and 1:l are projected up
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AMYLIN
AND INSULIN
E277
IN RAT SOLEUS MUSCLE
Table 1. Dose-related effects of insulin and amylin on rates of radioglucose incorporation into glycogen in isolated rat soleus muscle
1.9 3.4
Maximal Response, pmol - h-l . g wet tissue-l
10
Insulin
[Amylin] , nM 0 3 10 30 100 300
97
I
B
.,,I
I
0.1
Zero
1 Insulin
L_
,,,
10 (nM)
100
I
[Insulin], nM 0
1000
0.21 0.71 2.1 7.1 71
n
71 7.1
EGO,
nM
dose response
4.09 3.45 3.12 2.13 1.15 0.93 Amylin dose response 0.85kO.17 1.26t0.18 2.11k0.18 2.94&O. 18 3.56&O. 19 4.07t0.18
1.16kO.10 0.78-tO.12 1.52t0.15 1.33t0.20
Undetermined Undetermined 12.15t0.94 10.27t0.48 8.24k0.24 10.45-t-O. 16 8.69&O. 13 11.93t0.11
Concentrations are nominal values; maximal response values are given as mean and means t SE; EC& values are means t log SE. Insulin dose response at various constant amylin concentrations and amylin dose response at various constant insulin concentrations. For fitting insulin dose-response curves, maximal values were constrained to those indicated. For two insulin dose-response curves, amylin suppression of the response made the curves too shallow for reliable analysis. Maximal response, calculated quantity of glucosyl units (hexose moieties) transferred onto glycogen per gram muscle (wet weight) per hour.
,”
j
Zero
”
0.1
”
”
1
Amylin
A’
IO (nM)
1
1’
100
1
1000
Fig. 3. Interaction of insulin and amylin actions. A: insulin doseresponse relationships in the presence of different amylin concentrations, as indicated. Symbols representing means of actual responses are connected to relevant curves. Amylin concentrations in media predicted by quantitative amino acid analysis (nM) are indicated for each curve. B: amylin dose-response relations in presence of different insulin concentrations, as indicated. Means of actual responses are connected to relevant curve. Predicted media insulin concentrations (nM) are indicated for each curve.
onto the surface (labeled 0.01, 0.14, and 1, respectively). Insulin- to-amylin fixed-ratio doseresponse. The experimentally determined response to increasing concentrations of a fixed 0.14: 1 ratio of amylin to insulin are plotted as open circles (Hg-rat amylin) and squares (Hg-free human amylin) in Fig. 5. Both data sets clearly fit a bellshaped distribution, predicted for noncompetitive functional antagonism, rather than a shifted sigmoid response curve as would be predicted for competitive antagonism. For comparison, we have plotted both the bellshaped function generated from Eq. 1 using [amylin] = O.l4[insulin] (labeled 0.14 in Fig. 4) and the sigmoid dose response predicted for insulin in the absence of amylin. ECSOof synthetic human amylin. Data pooled from five amylin dose-response assays performed on a single batch of human amylin in the presence of 7.1 nM insulin are shown in Fig. 6. At high amylin concentrations, glycogen labeling was suppressed to below the “basal” rate obtained in the absence of insulin and amylin (0.51 vs. 0.76 pmol . h-l l g wet muscle- ‘; P < 0.001, unpaired t test). The ECSO of the response for human amylin was 456 pM t 0.18 log units when analyzed using amylin concentra-
Fig. 4. Insulin-amylin response surface. Response surface obtained from Eq. 1 (see text) fitting the 6 [insulin] x 6 [amylin] matrix of data used to plot Fig. 3, A and B. Lines labeled 0.01, 0.14, and 1 represent responses predicted for mixtures where amylin is at 1, 14, and 100% of the molar concentration of insulin.
tions obtained by radioimmunoassay of the incubation media. If the data were analyzed using amylin concentrations derived from mass added (from amino acid analysis) or from mass originally weighed out, the ECSO values were, respectively, 4.5 and 22 times greater and the doseresponse curves were, respectively, right-shifted and made steeper as shown in Fig. 6; the Hill coefficients were 0.54, 0.68, and 0.77, respectively. In summary, as the
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AMYLIN
lo-"
'O-10
'O-9
'09
'o-7
IO6
AND INSULIN
1o-5 IO”
Insulin (molar)
Amylin (molar) Fig. 5. Response to increasing concentrations of insulin and amylin in a fixed ratio. Rate of radiolabeling of glycogen in isolated rat soleus muscle as a consequence of increasing concentrations of a mixture of amylin (0, rat amylin, Hg adduct; q , Hg-free human amylin) and insulin where [amylin] is 14% of [insulin]. Symbols have same meaning as in Fig. 1; -n. 4 4 for each point. Bell-shaped curve is same data set that defines line labeled 0.14 in Fig. 4. Sigmoid curve is insulin response component of bell-shaped curve; that is, [amylin factor] in Eq. 1 = 1.
IN RAT SOLEUS
MUSCLE
These reductions in immunoreactive amylin were apparent at the first of several time series samplings of incubation media, and did not change appreciably in any measurement in the subsequent 2 h. When added to incubation media to achieve a nominal concentration of 100 pM, with or without muscle strips, the immunoreactive amylin remained essentially at or below the detection limit of the assay; when added in greater amounts, recoveries were somewhat higher. Regardless of the presence or absence of muscle strips, there was no indication of a time dependence of amylin concentration in the incubation media. An analysis similar to that described above for human amylin was performed using rat amylin. When weighed out and diluted to give predicted concentrations of 1,000 or 300 nM, radioimmunoassay returned similar values (785 and 255 nM). When diluted to give a concentration of 1 nM, however, immunoassay indicated rat amylin concentrations of -105 pM, indicating the loss of -90% of immunoreactivity. Again, from time series analysis, it appeared that the loss of immunoreactivity was a rapid event that had substantially occurred before incubation started. The relationship between observed concentration (by immunoassay) and expected concentration is plotted for both human and rat synthetic amylin in Fig. 7. In contrast to amylin, insulin diluted linearly. That is, the concentration of immunoreactive insulin was related to the degree of dilution over the range 71 pM to 71 nM. With or without muscle present in the media, there was no indication of a change in insulin immunoreactivity over 2-h incubations.
I
Zero
0.01
0.1
1
10
100
1000
Amylin (nM) Fig. 6. Amylin dose response, human amylin. Dose response for chemically synthesized human amylin performed in the presence of 7.1 nM recombinant human insulin in isolated rat soleus muscle. Symbols have same meaning as in Fig. 1; n = 18-20 for each point. Curve A was fitted to data obtained when amylin concentration is derived from mass initially weighed out and diluted. Curve B was fitted to data obtained when amylin concentration is derived from mass of peptide added to assay media as determined by quantitative amino acid analysis. Curve C was fitted to data obtained when amylin concentration in assay media is measured by radioimmunoassay. ECsO for curue C is 456 pM t 0.18 log units (SE).
quantities of amylin added to the assay medium became small, a smaller proportion remained as immunoreactive peptide in solution. For example, with dilutions aimed at producing a final concentration of 1 nM human amylin, the measured concentration of immunoreactive peptide was at or below the detection limit of the assay (-20 PM).
0 Rat amylin o Human amylin
Fig. 7. Relationship between expected amylin concentrations and concentrations measured using radioimmunoassay. Rat (0) and human (0) synthetic amylin concentrations in incubation media measured by specific radioimmunoassay as a function of values expected after addition of dilutions of weighed-out peptide. Observed and expected values converge at higher peptide concentrations. At lower concentrations similar to those reported in vivo, immunoreactive amylin present is much less than the amount nominally added. Line of identity is indicated. Symbols represent geometric mean and SE; n = 4-9 for each point.
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AMYLIN
AND
INSULIN
DISCUSSION
The finding that the E& values for amylin and insulin action on soleus muscle are not altered by increasing concentrations of the other hormone are consistent with biochemical and pharmacological data (11, 33) indicating that they act via separate receptors rather than by competing at a common receptor. Amylin behaves as a noncompetitive, functional antagonist to insulin. That is, amylin reduces the magnitude of the insulin response without affecting insulin potency Criti tally importa nt for analyzing the role of amylin i.s the findi ng that it causes insurmountable inhibition of insulin action. Amylin’s biological activity was originally described as opposing the muscle response to insulin (20). Some researchers have therefore regarded insulin and amylin as acting in opposition to each other, representing selfcanceling signals. By this reasoning, if these hormones were secreted in a constant ratio, the effects of any increase in plasma amylin would be nullified by the proportionate increase in insulin. The present report instead predicts and verifies that, with fixed ratios of insulin and amylin, suppression by amylin of the insulin response will eventually supervene as the concentrations of both hormones increase, resulting in the bell-shaped doseresponse relationship in Fig. 5. In describing this bell-shaped response, we have provided a theoretical construct that could explain how insulin resistance would eventually result, even though hyperinsulinemia occurred pari passu with hyperamylinemia. Even so, the constancy of the amylin-to-insulin ratio is conjectura 1. Although it has been repor ted that in vitro stimulation of P-cell secretio n under a variety of conditions results in amylin and insulin release in an essentially fixed ratio (16, 26, 27), there is mounting evidence for the dissociation of secretion of amylin and insulin; reported amylin-to-insulin ratios have ranged from l-2% (14, 16) to 37% (27), and in experiments in which the isolated perfused rat pancreas was stimulated with a pro longed glucose sti .mul us, the ra tio of amylin to insulin secreted varied between 2 and X00%, with the fasting ratio being around 13% (13). Although in the present report we focused on experiments using a ratio of l4%, the bell-shaped response was also evident at amylin-to-insulin ratios of 1 and 100%. On the other hand, the choice of a ratio of 14% to illustrate the point may have had some physiological validity; amylin-to-insulin ratios of 15-20% have been reported in humans (28), and in our hands, during oral glucose tolerance testing in nondiabetic human subjects (Ha) the molar ratios of plasma amylin to insulin have varied between 0 and 94% (minima 4.3 t 1.5%, maxima 20.3 t 7.6%). The response surface we have delineated in the present study describes the theoretical bounds of the steady-state response of a soleus muscle to all combinations of amylin and insulin. Fixed ratios of amylin to insulin define bellshaped rather than flat responses on this surface, exemplifying the observa tion that it is the absolute concentration of amylin and insulin, rather than their ratio, that determines tissue response. The relevance of the soleus response surface to the response of the intact organism is yet to be determined. Different tissues may be expected to
IN RAT
SOLEUS
MUSCLE
E279
have different sensitivities to insulin and amylin, and hence exhibit different response surfaces; adipocytes, for example, are sensitive to insulin, yet are apparently unaffected by amylin (4) (J. Lupien, unpublished observations). It is also possible, with progressing insulin resistance, that the change in insulin sensitivity of a tissue is not matched by an equal change in amylin sensitivity. The impact of changes in shape of the response surface with progressing insulin resistance is yet to be determined. James et al. (17) measured rates of radio-2-deoxyglucase uptake into soleus and other muscles during euglycemia at different steady-state insulin concentrations. Their data yield a greater potency for insulin in vivo than is typically reported in vitro. Possible reasons for such apparent discrepancies include concentration gradients between plasma and the receptor. Such gradients will occur where there is degradation or uptake of a ligand in the vicinity of the receptor, and where there is any impediment to its diffusion. The observation that lymphatic insulin concentration is
