03.pdf

JOP. J. Pancreas (Online) 2001; 2(4):140-149. Effect of Treatment with Different Doses of 17-β-Estradiol on Insulin
Receptor Substrate-1.
Celestino González, Ana Alonso, Natalia A Grueso, Fernando Díaz, Manuel M Esteban, Serafina
Fernández, Angeles M Patterson
Department of Functional Biology, Physiology Area, University of Oviedo, Oviedo, Spain.
ABSTRACT
levels in insulin dependent tissues, but in adifferent manner in each tissue. These novel Context Ovarian hormones modulate insulin
sensitivity, but their exact role remains unclear.
knowledge about the possible risk for insulin Objective We tried to determine whether
resistance in women taking oral contraceptives different doses of 17-β-estradiol cause changes or receiving hormone replacement therapy at in the regulation of insulin receptor substrate (IRS-1) levels, and if so, the possible
implications in insulin sensitivity.
Design Ovariectomized rats were treated with
INTRODUCTION
different doses of 17-β-estradiol at 6, 11 and 16days.
Various clinical observations and experimental Main outcome measures Immunoprecipitation
data suggest that estrogen and progesterone can and Western blotting for IRS-1 were performed modulate insulin sensitivity in females [1, 2]. In Results We found that estradiol treatment has
pregnancy, where estrogen and progesterone an influence on the amount of IRS-1 but that it concentrations are markedly elevated, have a acts in different ways depending on the tissue substantial effect on carbohydrate metabolism studied, on the length of treatment, and on the as well as on the alteration of insulin sensitivity [3]. Moreover, in women taking the combined Conclusions Our results suggest that low
oral contraceptive pill, artificially increased concentrations of 17-β-estradiol could be levels of the sex steroid hormones estrogen and responsible for the upregulation of insulin progesterone were found to affect glucose tolerance and insulin sensitivity [4, 5].
sensitivity in muscle and adipose tissue.
However, it remains unclear whether estrogens alone or progestins alone can cause insulin downregulated with high concentrations of 17- resistance, or whether it is only a combination β-estradiol, thus these high hormone plasma levels could favour insulin resistance in Insulin action begins upon its binding to its cell peripheral tissues. The role of 17-β-estradiol surface receptor [6, 7]. The discovery of seems to modulate insulin receptor substrate 1 tyrosine kinase activity in the insulin receptor JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. implies that the mechanism of insulin action of phosphorylation may also play a role in its reduced response to insulin in the insulin- transmitting the insulin signal. Insulin receptor On the other hand, the possible contribution of substrate 1 (IRS-1) is considered the major estradiol to the degradation pathway of IRS-1 is substrate for the insulin receptor, and contains not sufficiently known, but the identification of motifs that, after tyrosine phosphorylation, are well-defined intermediates suggests that there binding sites for proteins containing Src may be a specific degradation pathway. Since homology (SH2) domains [9]. The association the role of IRS-1 is to act as a docking protein of some proteins (phosphatidylinositol 3´- which assembles a signaling complex, IRS-1 kinase: PI3-K, growth factor receptor-bound protein 2: Grb2, and tyrosine phosphatase SHP2) with IRS-1 permits their activation and the transmission of the insulin signal [10].
demonstrate whether different doses of 17-β- Thus, IRS proteins amplify the insulin receptor estradiol could influence the regulation of the amount of IRS-1 and the possible implication constraints encountered by receptors that of this hormone in insulin sensitivity during directly recruit proteins containing SH2 site gestation. Therefore, this study was designed to domains to their autophosphorylation sites.
test whether the amount of IRS-1 in the liver, Molloy et al. [11] and Lee et al. [12] showed skeletal muscle and adipose tissue could be that estrogen induces the expression of the modified by the concentration of 17-β-estradiol downstream signaling molecules, IRS-1 and IRS-2. Estrogen induction of IRS-1 expression was associated with increased tyrosinephosphorylation of IRS-1 and correlated with MATERIAL AND METHODS
protein kinase (MAPK) activation. While the increase in IRS-1 expression generally mirroredthe increase in tyrosine phosphorylation, the Twelve-week-old virgin female Wistar rats authors could not rule out the possibility that (from the Biotery of the University of Oviedo) the increase in tyrosine phosphorylation of IRS- 1 results from a change in stoichiometry or the conditions of temperature (23±3 ºC) and sites of phosphorylation. Furthermore, there humidity (65±1%), with a regular lighting schedule of a 12 h light/dark cycle (08:00 am - 08:00 pm) were used. The animals were fed a decreased phosphorylation of IRS-1 may be standard diet (Panlab A04, Barcelona, Spain) mediated by the antiestrogen-induction of a and had free access to water. All experimental specific tyrosine phosphatase activity [13, 14].
manipulations were performed between 09:30 phosphorylation of IRS-1 on serine/threonineresidue has an inhibitory effect on insulin Experimental Design
signaling [15, 16, 17]. Following the increase inIRS-1 serine/threonine phosphorylation, the Three days before initiating the hormonal ability of insulin to phosphorylate IRS-1 on treatment (day -7), the rats were ovariectomized tyrosine residues is decreased. It can be through a midline incision using light ether concluded, therefore, that a modulated pattern anaesthesia. The ovariectomized rats were JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. randomly separated into four groups: control to the temporal diagram reported in Table 1.
(V), estradiol (E), estradiol x 10 (EX10) and Animals were sacrificed randomly on the 6th, 11th and 16th days (6 animals/subgroup). These individually throughout the experiment.
days were selected as changes were found in After surgery, the ovariectomized rats were allowed 3 days to recover from the stress of surgery and decrease their hormonal levels.
On the day of sacrifice and after 12 h of fasting subcutaneously every twelve hours (09.00 am anesthetized with 3.3 mL/kg body weight of and 09:00 pm) for 20 days with 0.1 ml of a 17- intraperitoneal ekytesin (0.96 g/mL sodium β-estradiol (SIGMA Chemical Co., San Louis, pentobarbital, 4.02 g/mL chloral hydrate, 2.12 USA) suspension in olive oil/ethanol (3:2 v/v).
g/mL magnesium sulphate, 40% propilenglicol, The control group (V) injected with the vehicle (olive oil/ethanol 3:2 v/v) was followed in parallel. In the E group, different doses of 17-β- reflexes, a blood sample (1 mL) was collected estradiol were injected in order to simulate the from the jugular vein in heparinized tubes, centrifuged at 3000 rpm during 20 min at 4 ºC pregnant rats [19, 20]. In the EX10 group, the doses of 17-β-estradiol injected were ten times stored frozen at –20 ºC until assayed. Plasma E and in the EX100 group, the doses of 17-β- 17-β-estradiol was measured by RIA using estradiol injected were one hundred times E.
The hormonal treatment was applied according Biomedicals Inc., Costa Mesa, USA). The assaysensitivity was 10 pg/mL, and the intra-assay Table 1. Temporal diagram of the 17- β-estradiol doses.
Day 17-β-estradiol dose (E) Note
samples were measured on the same day. Each Finally, samples of different tissues (liver; skeletal muscle: flexor digitorum superficialis, extensor digitorum longus, soleus and extensor digitorum lateralis; retroperitoneal adipose tissue) were collected and immediately frozen in liquid nitrogen for future experiments and the animals were killed by bleeding.
Immunoprecipitation and Western Blotting
The samples of liver, skeletal muscle and adipose tissue were washed with ice-cold sterile Barcelona, Spain), 0.05% sodium deoxycholate, JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. speed for 30 sec. The extracts were centrifuged (Western-Light, Chemiluminescent Detection at 12,000 g at 4º C for 10 min in order to System, TROPIX Inc., Bedford, USA) using a remove insoluble material. After centrifugation, 1:2,000 dilution of polyclonal antibody against the IRS-1 as the primary antibody, followed by Bradford dye-binding method [21] using the alkaline-phosphatase-conjugated anti-rabbit Bio-Rad (Hercules, USA) reagents and BSA as standard. The aqueous fraction containing 250 for detection. Finally, the membranes were µg of protein for liver and muscle and 150 µg rinsed several times with blocking buffer without BSA and proteins were detected with immunoprecipitation (IP) with 0.25 µg of polyclonal antibody against the insulin receptor Biotech, Barcelona, Spain) according to the substrate 1 (IRS-1) (sc-559-G, Santa Cruz Biotech, Inc., Santa Cruz, USA). The immune- complexes were precipitated with protein G- agarose beads (Roche Diag., Barcelona, Spain) overnight at 4º C in a rocking platform and Western blots were quantified using a digital were washed several times in wash buffer (50 scanner (AX-110, Nikon, Surrey, UK) and NIH Image 1.57 software (Scion Co., Maryland, 0.1% ortovanadate 1 M). After washing, thepellet was suspended in protein loading buffer (250 mM Tris-HCl pH 6.8, 8% SDS, 8 mMEDTA, 35% glycerol, 2.5% β-mercapto- The experiments were carried out in accordance ethanol, Bromophenol Blue) and heated in a with the rules of laboratory animal care.
boiling water bath for 5 min.
For total extraction, similar size aliquots were STATISTICS
subjected to SDS-PAGE (7% Tris-Acri-Bis) ina miniature slab gel apparatus (Bio-Rad, Data are expressed as mean ± SEM. We used Hercules, USA). The prestained molecular- analysis of variance followed by a Tukey test in mass standards used were myosin (218 kDa), β- the statistical analysis of 17-β-estradiol plasma galactosidase (126 kDa), bovine serum albumin levels. Since the distribution of the IRS-1 levels in different tissues was skewed, the Mann- soybean trypsin inhibitor (33.9 kDa), lysozyme (17.4 kDa) and aprotinin (7.6 kDa) (Bio-Rad, used. A P value less than 0.05 was considered Hercules, USA). Electrotransfer of proteins significant. Statistical analysis was performed (Hybond-ECL, Amersham Pharmacia Biotech,Barcelona, Spain) was performed for 60 min at 50 V (constant) in a miniature transferapparatus (Mini-Protean, Bio-Rad) as described Plasma Levels of 17-β-Estradiol
by Towbin et al. [22].
Non-specific protein binding to the The plasma levels of 17-β-estradiol are shown in Figure 1. The results obtained in the E group preincubating the filter for two hours at room were similar to normal pregnant rats at 5, 10 temperature in blocking buffer (TNT, 7% BSA) and 15 days of pregnancy [19, 20]. In this group, we found similar values at 6 and 11 days JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. In the liver, at day 6 of experimentation weobserved a significant increase in the IRS-1level in the E and the EX10 groups ascompared to the V and the EX100 groups. Thesame result was observed at day 11 but thedifference between the EX10 and the V groupsdid not reach the level of significance. At day16, we found a significant increase in the Egroup as compared to the other groups and asignificant decrease in the EX100 group ascompared to the EX10 group; no significantdifferences were found between the V and the Figure 1. 17-β-estradiol plasma levels in ovariectomized
EX10 groups. The length of treatment does not rats (V) and rats treated with different doses of 17-β- significantly change the amount of IRS-1, but estradiol (E, EX10, EX100). Data are expressed as mean we observed a remarkable progressive increase ± SEM. Only significant differences are shown.
Significant interday comparison: * day 16 vs. days 6 and in the V group from day 6 to day 16 and a remarkable decrease in the EX10 and theEX100 groups in the same period.
of treatment and a significant increase was The level of IRS-1 in skeletal muscle was observed at day 16 vs. days 6 and 11. The significantly increased in the E group as results obtained in the EX10 and the EX100 compared to the other groups at day 6.
groups were significantly higher than E.
However, at day 11 we found a significant increase in the V group as compared to the parallel with the E group. The 17-β-estradiol other groups; we also showed that the EX100 plasma levels were dependent on the solution group had significantly higher IRS-1 levels than injected. Thus we observed that the plasma the E and the EX10 groups and these levels concentration of 17-β-estradiol increases: at were significantly higher in the E group as day 6 (238% in E vs. V, 509% in EX10 vs. V compared to the EX10 group. Only significant and 787% in EX100 vs. V), at day 11 (188% in differences were found between V and EX10 E vs. V, 488% in EX10 vs. V and 1,145% in groups at day 16. As far as the comparisons EX100 vs. V) and at day 16 (401% in E vs. V, between the different days were concerned, we 803% in EX10 vs. V and 1,463% in EX100 vs. observed a significant increase of IRS-1 levels in the V group from day 6 to day 11 and asignificant decrease from day 11 to day 16. In Protein Content of IRS-1
the R group, the IRS-1 levels were significantlydecreased at days 11 and 16 vs. day 6, while the Figure 2 shows a representative experiment in length of treatment significantly increased these which the solubilized liver, skeletal muscle and levels in the EX10 (day 16 vs. days 6 and 11) and the EX100 (days 11 and 16 vs. day 6) antibody. After SDS-PAGE and electrotransfer, In adipose tissue, the level of IRS-1 was found nitrocellulose membranes were incubated in the to be significantly higher in the E and the EX10 groups as compared to the V group at day 6 of immunoprecipitated materials, ECL detection treatment. However, this tendency changes at days 11 and 16 of treatment: the level of IRS-1 JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. Figure 2. IRS-1 levels in liver, skeletal muscle and
adipose tissue in ovariectomized rats treated with
different doses of 17-β-estradiol. The proteins were
isolated, immunoprecipitated with anti-IRS-1 antibody
and immunoblotted with anti-IRS-1 antibody. Scanning
densitometry was performed for five independent
experiments. Data are expressed as mean ± SEM. Only
significant differences are shown.
Significant interday comparison: * day 6 vs. days 11 and
16; ** day 11 vs. day 16; *** day 6 vs. day 16
A.S.U.: Arbitrary scanning units
between days 6 and 16, whereas in the EX10and the EX100 groups, the opposite occurred:this level decreased at day 16 vs. days 6 and 11in the EX10 group, as well as progressivelydecreasing during the treatment period in theEX100 group. However, in the E group wefound a significant decrease between days 6and 11 and a significant increase between days11 and 16.
compared to the other groups at days 11 and 16.
We also observed that the tendency of the IRS- DISCUSSION
1 levels throughout treatment was differentdepending on the timing and the 17-β-estradiol We have recently showed that 17-β-estradiol dose. In the V group, the level of IRS-1 rose could be responsible for the increase in insulin JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. sensitivity during early pregnancy when the These findings confirm a previous study which plasma concentrations of 17-β-estradiol and demonstrated that gestation could be divided into two periods, namely, the period of early pregnancy when the plasma concentrations of gestation, which is characterized by an increase both hormones are high, the role of 17-β- in sensitivity to insulin action in the maternal estradiol could be that of antagonizing the tissues and the period of late gestation, effect of progesterone by diminishing insulin characterized by a decrease in this sensitivity sensitivity. We therefore propose that some of [19]. In the light of the present results our hypothesis is that, during early pregnancy, the metabolism during pregnancy can be focused peripheral "insulin sensitivity" (larger amount of IRS-1) together with high insulin plasma The novel finding of this study is that 17-β- levels (data not shown) could favour the storage estradiol seems to be responsible for controlling of energetic reserve in the adipose tissue, insulin sensitivity in females throughout gestation. In this sense, the present results adaptative changes are reversed. The decrease confirm those of a previous study [23], namely, in insulin plasma levels (data not shown) together with less insulin sensitivity in adipose estradiol is similar to that found in early tissue facilitate the lipolysis and the increase in pregnancy, IRS-1 is upregulated and the insulin sensitivity increases in the peripheral tissues observed at the end of pregnancy [20].
(skeletal muscle and adipose). However, IRS-1 In relation to the EX10 and the EX100 groups, we can note that the higher doses of 17-β- concentration of this hormone is similar to that estradiol (EX100 group) significantly decreased found in late pregnancy, diminishing the insulin the amount of IRS-1 with respect to the E group sensitivity in peripheral tissues. We suggest, in in the liver and adipose tissue. However, in accordance with Lee et al. [12], that a low skeletal muscle, this fact only occurs at day 6 of concentration of 17-β-estradiol increases IRS-1 expression by a transcriptional mechanism hormone action seems to amplify the decrease because the IRS-1 promoter does have four in IRS-1 levels in the EX100 group, also with consensus half- estrogen response elements the exception of skeletal muscle. Similar results [24]. However, when the concentration of 17-β- findings seem to demonstrate that liver and complexes of estradiol receptor-estradiol to the adipose tissue are the tissues most affected by IRS-1 promoter could induce a decrease in IRS- 17-β-estradiol action with respect to the amount 1 expression, probably by displacement of other of IRS-1, and that the effect of this hormone transcription factors linked to the IRS-1 was dose and timing dependent in agreement promoter. Among these factors, we could point with other authors postulating that the effect of out progesterone, lactogenic hormones, growth hormone, etc, [1, 2, 3, 23, 25, 26].
metabolism depend on the type of estrogen, In the present study, in the E group, the tissues dose, length of treatment, etc. [4, 27, 28]. The studied were found to perform differently. In roles of liver and adipose tissue are very this way, there are no significant changes in the important during pregnancy, because the liver treatment, but, in skeletal muscle and adipose clearance [29] and adipose tissue is the most tissue, a significant decrease was observed from important energetic reserve. In this sense, our day 6 to day 11 and from day 6 to day 16.
hypothesis, in the light of the present results, is JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. that during early pregnancy, the liver could bediscouraging insulin clearance, so the high Received March 2nd, 2001 – Accepted June insulin plasma levels (data not shown) together with high sensitivity in adipose tissue couldfavour the storage of energetic reserve.
Key words Insulin; Insulin Resistance; Rats
However, in late pregnancy, the role of the livercould be to increase insulin clearance, so the Abbreviations Grb2: growth factor receptor-
decrease in insulin plasma levels (data not bound protein 2; IP: immunoprecipitation; IRS- shown) together with less insulin sensitivity in adipose tissue facilitate the lypolisis and the increase in triglycerides plasma levels which can be observed at the end of pregnancy [20].
We consider that these novel findings are veryimportant for a better understanding of the role Acknowledgements This study was supported
of estrogen during pregnancy. Moreover, these by grants from the University of Oviedo (NP- results also illustrate the great importance of estrogen dosage and concentration as regards Universidades e Investigación del Principado glucose metabolism in hormonal replacement de Asturias as part of the II Plan Regional de therapy in women at menopause and in women taking oral contraceptives.
In summary, our present findings suggest that Correspondence
low concentrations of 17-β-estradiol similar to early pregnancy levels could be responsible for the upregulation of IRS-1, increasing insulin sensitivity in peripheral tissues (muscle and downregulated with high concentrations of 17- β-estradiol similar to late pregnancy, thus these insulin resistance in the peripheral tissues.
Consequently, the role of 17-β-estradiol seems to be to modulate the amount of IRS-1 ininsulin dependent tissues, but in a differentmanner in each tissue. In spite of this, the role References
of this hormone could appear to be slightlyaltered in the presence of high plasma concentrations of progesterone, lactogenic H, Bakker A, Heine RJ. Induction of insulin hormones and growth hormone, just as occurs resistance by androgens and estrogens. J Clin during normal pregnancy. Moreover, we think that these novel findings are very important in order to improve knowledge about the possible risk for insulin resistance in women taking oral Adipocyte insulin action during the normal contraceptives or hormone replacement therapy menstrual cycle. Hum Reprod 1996; 11:968-74.
JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. 3 Saad MJA, Maeda L, Brenelli SL, Carvalho signaling in human breast cancer: estrogen CRO, Paiva RS, Velloso LA. Defects in insulin regulation of insulin receptor substrate-1 signal transduction in liver and muscle of expression in vitro and in vivo. Mol Endocrinol pregnant rats. Diabetologia 1997; 40:179-86.
13 Freiss G, Vignon F. Antiestrogens increase 4 Godsland IF, Walton C, Felton C, Proudler protein tyrosine phosphatase activity in human breast cancer cells. Mol Endocrinol 1994; secretion, and metabolism in users of oral contraceptives. J Clin Endocrinol Metab 1992; 14 Freiss G, Puech C, Vignon F. Extinction of associated protein tyrosine phosphatase-1 in metabolism in oral contraceptive users without human breast cancer cells. Mol Endocrinol Endocrinol Metabolism 1994; 79:1277-83.
15 Tanti JF, Grémeaux T, Van Obberghen E, phosphorylation of insulin receptor substrate 1 pathological conditions. Annu Rev Med 1990; modulates insulin receptor signaling. J Biol 16 De Fea K, Roth RA. Modulation of insulin JM, Araki E, Wilden PA, et al. Structure of the receptor substrate 1 tyrosine phosphorylation insulin receptor substrate IRS-1 defines a and function by mitogen-activated protein unique signal transduction protein. Nature kinase. J Biol Chem 1997; 272:31400-6.
8 White MF, Kahn CR. The insulin signaling modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612.
Biochemistry 1997; 36:12939-47. [97477343] 9 Sun XJ, Miralpeix M, Myers MG, GlasheenEM, Backer JM, Kahn CR, White MF.
Expression and function of IRS-1 in insulin mechanism of insulin action in normal and insulin-resistant states. Exp Clin Endocrinol 19 González C, Díaz F, Fernández S, Patterson signaling system. Trends Biochem Sci 1994; AM. Role of 17-β-estradiol and progesterone 11 Molloy CA, May FEB, Westley BR. Insulin restriction (50%) in pregnant and non-pregnant receptor substrate-1 expression is regulated by rats. J Endocrinol Invest 1997; 20:397-403.
estrogen in the MCF-7 human breast cancer cell line. J Biol Chem 2000; 275:12565-71.
20 González C, Díaz F, Fernández S, Patterson AM. Pregnancy in rats and food restriction 12 Lee AV, Jackson JG, Gooch JL, Hilsenbeck (50%): insulin response in relation to serum lipids and lipoprotein levels. Nutr Research D. Enhancement of insulin-like growth factor JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001 JOP. J. Pancreas (Online) 2001; 2(4):140-149. transduction in rat tissues. Mol Cell Endocrinol quantities utilizing the principle of protein dye binding. Anal Biochem 1976; 72:248-54.
26 Kawai M, Kishi K. Adaptation of pancreatic islet B-cells during the last third of pregnancy: Electrophoretic transfer of proteins from regulation of B-cell function and proliferation polyacrylamide gels to nitrocellulose sheets.
Procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350-4. [80056736] 27 Kojima T, Lindheim SR, Duffy DM, Vijod 23 González C, Alonso A, Alvarez N, Díaz F, MA, Stanczyk FZ, Lobo RA. Insulin sensitivity Martínez M, Fernández S, Patterson AM. Role of 17-β-estradiol and/or progesterone on insulin ethinyl estradiol used in oral contraceptives.
sensitivity in the rat: implications during Am J Obstet Gynecol 1993; 169:1540-4.
pregnancy. J Endocrinol 2000; 166:283-91.
28 Lindheim SR, Duffy DM, Kojima T, Vijod 24 Kato S, Tora l, Yamauchi J, Masushige S, administration influences the effect of estrogen estrogen response element of the ovalbumin gene contains several half- palindromic 5´- women. Fertil Steril 1994; 62:1176-80.
TGACC-3´ motifs acting synergistically. Cell 25 Thirone ACP, Carvalho CRO, Brenelli SL, mechanism, products, and significance. Endocr Velloso LA, Saad MJ. Effect of chronic growth JOP. Journal of the Pancreas – http://www.joplink.net – Vol.2, No.4 – July 2001

Source: http://www.omicsonline.com/open-access/effect-of-treatment-with-different-doses-of-bestradiol-on-insulin-receptor-substrate.pdf