Kevin Beaumont

Preclinical pharmacology of pramlintide in the rat: Comparisons with human and rat amylin

The pancreatic β‐cell hormone, amylin, is absent or reduced in individuals with type I diabetes mellitus and in many insulin‐treated patients with type II diabetes. Amylin replacement therapy may be beneficial in these individuals, but the pharmaceutically inconvenient physicochemical properties of native human amylin led to the development instead of the amylin agonist, [Pro25,28,29]human amylin, or pramlintide (formerly designated AC137). Here we compare for rat amylin, human amylin and pramlintide, receptor binding and biological actions in rats in vivo and in rat soleus muscle. In the rat, the spectrum of actions and pharmacokinetic and pharmacodynamic properties of pramlintide are either very similar to, or indistinguishable from, those of rat or human amylin.

Molecular cloning of two receptors from rat brain with high affinity for salmon calcitonin

Two receptors with high affinity for salmon calcitonin were cloned from the nucleus accumbens region of rat brain. The deduced 479 amino acid sequence of cDNA clone L2175‐D20 (designated Cla receptor) is 78% and 66% identical with those reported for human and porcine calcitonin receptors, respectively. Clone U3237‐A2 codes for a receptor (designated Clb) that is identical to Cla except for a 37 amino acid insert in the second extracellular domain. COS‐7 cells transfected with either transcript bound [125I]salmon calcitonin with high affinity (K d = 8 pM for Cla; K d = 48 pM for Clb) and responded to salmon calcitonin with increases in cAMP. Tissue distribution studies revealed Cla transcript in rat brain, skeletal muscle, kidney and lung, whereas Clb was predominantly found in brain.

Structure and biology of amylin

Amylin is a recently discovered 37 amino acid peptide secreted into the bloodstream, along with insulin, from pancreatic beta-cells. It is about 50% identical to calcitonin gene-related peptides (CGRP alpha and CGRP beta) and structurally related to the calcitonins. Amylin can elicit the vasodilator effects of CGRP and the hypocalcaemic actions of calcitonin, while these peptides can mimic newly discovered actions of amylin on carbohydrate metabolism. The different relative potencies of these peptides suggest that they act with different selectivities at a family of receptors. Amylin is deficient in insulin-dependent diabetes mellitus, while plasma levels are elevated in insulin-resistant conditions such as obesity and impaired glucose tolerance. In this Viewpoint article, Tim Rink and colleagues propose that amylin is an endocrine partner to insulin and glucagon; deficiency or excess of amylin may therefore contribute to important metabolic diseases.

SPECIAL REPORT Regulation of muscle glycogen metabolism by CGRP and amylin: CGRP receptors not involved

The aim of the present study was to determine whether amylin and calcitonin gene‐related peptide (CGRP) act through shared or distinct receptors to inhibit insulin‐stimulated incorporation of [14C]‐glucose into glycogen. Rat amylin was 3 fold more potent than either rat αCGRP or rat βCGRP at reducing glycogen synthesis from [14C]‐glucose in insulin‐treated rat soleus muscle. This action was blocked by peptide antagonists, with the rank order of potency being AC187> salmon calcitonin 8–32 (sCT8.32) > h‐αCGRP8–37 for antagonism of either amylin or CGRP. The antagonist potency order correlated with affinity for amylin receptors measured in rat nucleus accumbens but not CGRP receptors measured in rat L6 muscle cells. Inhibition of glucose incorporation into glycogen by amylin and CGRP appears to be mediated by shared receptors that have the pharmacological characteristics of amylin receptors, and are distinct from previously described CGRP receptors.

Selective amylin antagonist suppresses rise in plasma lactate after intravenous glucose in the rat. Evidence for a metabolic role of endogenous amylin.

Data presented here provide the first demonstration that circulating amylin regulates metabolism in vivo, and support an endocrine hormonal role that is distinct from its autocrine action at pancreatic islets. When rats were pre‐treated with the potent amylin antagonist AC187 (n = 18), and then administered a 2 mmol glucose load, the rise in plasma lactate was less than in rats administered glucose only (n = 27; P < 0.02). When rats were treated so that plasma glucose and insulin profiles were similar (n = 8), the increase in plasma lactate in the presence of AC187 was only 50.3% as high as the increase when AC187 was absent (P < 0.001). These experimental results fit with the view that some of the lactate appearing in plasma after a glucose load comes from insulin‐sensitive tissues. The experiments also support the view that an important fraction of the increase in lactate depends on processes inhibited by a selective amylin antagonist, most likely amylin action in muscle.

Diabetogenic Effects of Salmon Calcitonin Are Attributable to Amylin-Like Activity

During the development of synthetic calcitonins for therapeutic use in bone disease, a diabetogenic (hyperglycemic) effect was observed, particularly with salmon calcitonin. The effect was attributed by some to inhibition of insulin secretion. We have recently reported high-affinity (28 pmol/L) amylin-binding sites in certain areas of rat brain, and found that these sites also bind salmon but not rat calcitonin with comparable high affinity. Rat amylin and salmon calcitonin have been determined to have significant structural homology. In vitro and in vivo studies indicate that rat amylin can exert calcitonin-like effects on osteoclasts and on plasma calcium. Here we report that salmon calcitonin mimics the actions of rat amylin on skeletal muscle glycogen metabolism in vitro; it stimulates glycogenolysis and inhibits incorporation of radiolabeled glucose into glycogen (50% effective concentration [EC50], 0.4 +/- 0.11 nmol/L log and 8.4 +/- 0.05 nmol/L log, respectively). In anesthetized rats, salmon calcitonin, like rat amylin, rapidly increases plasma lactate concentration, followed by a slower increase in glucose concentration. Like amylin, salmon calcitonin also inhibits the insulin response to 2 mmol infused glucose (insulin increments suppressed by 52% and 57% at 10 minutes for salmon calcitonin and amylin). Other shared actions, such as suppression of appetite, stimulation of renin secretion, inhibition of gastric acid secretion, and inhibition of gastric emptying, further affirm our proposal that the exogenous peptide, salmon calcitonin, is a mimic of endogenous amylin in the rat.

Dose-dependent elevation of cyclic AMP, activation of glycogen phosphorylase, and release of lactate by amylin in rat skeletal muscle

We report here our investigation of the role of cyclic AMP (cAMP) in amylin signal transduction in isolated strips of soleus muscle. Rat amylin, at 100 nM, increased cAMP levels, from 0.431 +/- 0.047 to a peak of 1.24 +/- 0.01 pmol cAMP/mg wet wt. after 5 min, in the absence of added phosphodiesterase inhibitor. The EC50 of the response was 0.48 nM (+/- 0.12 log units) in the absence of insulin and 0.3 nM (+/- 0.18 log units) in the presence of 7.1 nM insulin. The response seen with a maximally effective concentration of amylin (10 nM) was similar to that seen with a maximally effective concentration of epinephrine (1 microM) under the same conditions. Consistent with the observed rise in cAMP there was an increase in glycogen phosphorylase a (EC50 2.2 nM +/- 0.25 log units), decreased glycogen content (EC50 0.9 nM +/- 0.22 log units) and enhanced production of lactate (EC50 1.5 nM +/- 0.33 log units). These data support the concept that amylin promotes glycogenolysis in skeletal muscle and enhances production of lactate through glycolysis as a result of activation of Gs coupled receptors, stimulation of adenylate cyclase, elevation of cAMP levels and activation of glycogen phosphorylase.

Comparison of the in vitro and in vivo pharmacology of adrenomedullin, calcitonin gene-related peptide and amylin in rats

Adrenomedullin has been reported to be structurally similar to a group of peptides that includes amylin, calcitonin and calcitonin gene-related peptide (CGRP). Human and rat adrenomedullin displaced [125I]CGRP from membranes of SK-N-MC cells (CGRP receptors) with affinities intermediate between those of rat amylin and rat CGRP alpha (Ki values 0.12 +/- 0.06, 0.017 +/- 0.007, 3.83 +/- 1.14 and 0.007 +/- 0.001 nM, respectively). In contrast Ki values for displacement of [125I]rat amylin from accumbens membranes (amylin receptors), and [125I]salmon calcitonin from T47D cells (calcitonin receptors) were lower than with rat amylin or rat CGRP alpha in these preparations (51 +/- 5, 34 +/- 2, 0.024 +/- 0.002, 0.31 +/- 0.07 nM, respectively, at amylin receptors; 33 +/- 5, 69 +/- 29, 2.7 +/- 1.5 and 13 +/- 3 nM, respectively, at calcitonin receptors). In anesthetized rats, the hypotensive potency of adrenomedullin was between that of amylin and CGRP alpha. In contrast, for amylin or calcitonin agonist actions (inhibition of [14C]glycogen formation in soleus muscle, hyperlactemia, hypocalcemia and inhibition of gastric emptying), human adrenomedullin was without measurable effect. Thus, in its binding behaviour and in its biological actions, adrenomedullin appeared to behave as a potent CGRP agonist, but as a poor amylin or calcitonin agonist.

Different pharmacological characteristics in L6 and C2C12 muscle cells and intact rat skeletal muscle for amylin, CGRP and calcitonin

1 We compared the ability of rat amylin, rat calcitonin gene‐related peptide (CGRP) and rat and salmon calcitonins to elevate cyclic AMP levels and to inhibit [U‐14C]‐glucose incorporation into glycogen in insulin‐stimulated intact rat soleus muscle and in two cell lines derived from rodent skeletal muscle, L6 and C2C12 2 In intact soleus muscle, both amylin (EC50s of 0.7‐6.1 nM) and salmon calcitonin (EC50s of 0.5‐1.4 nM) were more potent than CGRP (EC50s of 5.6–15.8 nM) and were much more potent than rat calcitonin (EC50s of 50–137 nM) at stimulating cyclic AMP production, activating glycogen phosphorylase and inhibiting insulin‐stimulated [14C]‐glycogen formation. 3 In contrast, in both L6 and C2C12 cells, CGRP (EC50s of 0.042‐0.12 nM) stimulated cyclic AMP formation and inhibited insulin‐stimulated [U‐14C]‐glucose incorporation into glycogen approximately 1000 times more potently than amylin (EC50s 34–240 nM), while salmon calcitonin was without measurable effect. 4 There was a correlation between elevation of cyclic AMP and inhibition of insulin‐stimulated [U‐14C]‐glucose incorporation into glycogen evoked by these peptides in both intact muscle (r2 = 0.69, P <0.0004) and muslce cell lines (r2 = 0.96, P <0.0001). 5 In conclusion, the effects of amylin, CGRP, and calcitonin on soleus muscle glycogen metabolism appear to be mediated by adenylyl cyclase‐coupled receptors which show a pharmacological profile similar to high affinity amylin binding sites that have been previously reported in rat brain. In contrast, the effects of amylin and CGRP in L6 and C2C12 rodent muscle cell lines appear to be mediated by adenylyl cyclase‐coupled receptors that behave like CGRP receptors.