Defining the pathway of worm-like amyloid fibril formation by the mouse prion protein by delineation of the productive and unproductive oligomerization reactions.

Aggregation reactions of proteins leading to amyloid fibril formation are often characterized by early transient accumulation of a heterogeneous population of soluble oligomers differing in size and structure. Delineating the kinetic roles of the different oligomeric forms in fibril formation has been a major challenge. The aggregation of the mouse prion protein to form worm-like amyloid fibrils at low pH is known to proceed via a ß-rich oligomer ensemble, which is shown here to be comprised of two subpopulations of oligomers that differ in size and internal structure. The relative populations of the two oligomers can be tuned by varying the concentration of NaCl present. By demonstrating that the apparent rate constant for the formation of fibrils is dependent linearly on the concentration of the larger oligomer and is independent of the concentration of the smaller oligomer, we show that the larger oligomer is a productive intermediate that accumulates on the direct pathway of aggregation from monomer to worm-like fibrils. The smaller oligomer is shown to be populated off the pathway of the larger oligomer and, hence, is not directly productive for fibril formation. The relative populations of the two oligomers can also be tuned by single-amino acid residue changes in the sequence of the protein. The different protein variants yield worm-like fibrils of different lengths, and the apparent rate of formation of the fibrils by the mutant variants is also shown to be dependent on the concentration of the larger but not of the smaller oligomer formed.

Food protein amyloid fibrils: Origin, structure, formation, characterization, applications and health implications.

Amyloid fibrils have traditionally been considered only as pathological aggregates in human neurodegenerative diseases, but it is increasingly becoming clear that the propensity to form amyloid fibrils is a generic property for all proteins, including food proteins. Differently from the pathological amyloid fibrils, those derived from food proteins can be used as advanced materials in biomedicine, tissue engineering, environmental science, nanotechnology, material science as well as in food science, owing to a combination of highly desirable feature such as extreme aspect ratios, outstanding stiffness and a broad availability of functional groups on their surfaces. In food science, protein fibrillization is progressively recognized as an appealing strategy to broaden and improve food protein functionality. This review article discusses the various classes of reported food protein amyloid fibrils and their formation conditions. It furthermore considers amyloid fibrils in a broad context, from their structural characterization to their forming mechanisms and ensued physical properties, emphasizing their applications in food-related fields. Finally, the biological fate and the potential toxicity mechanisms of food amyloid fibrils are discussed, and an experimental protocol for their health safety validation is proposed in the concluding part of the review.

Pramlintide acetate injection for the treatment of type 1 and type 2 diabetes mellitus.

BACKGROUND: Amylin is a hormone cosecreted with insulin by the beta cells of the pancreas. It suppresses postprandial glucagon secretion and slows gastric emptying. Pramlintide acetate is an amylin analogue that was approved by the US Food and Drug Administration in March 2005. OBJECTIVE: This article reviews the current primary literature on the clinical efficacy and tolerability of pramlintide injection in the treatment of type 1 and type 2 diabetes mellitus (DM). Among other topics covered are the pharmacokinetics, pharmacodynamics, and dosing and administration of pramlintide. METHODS: Pertinent English-language articles were identified through a search of MEDLINE (1966-January 2007), International Pharmaceutical Abstracts (1970-present), Database of Abstracts of Reviews of Effectiveness (1995-January 2007), Cochrane Database of Systematic Reviews (1995-January 2007), and EMBASE Drugs & Pharmacology (1991-1st quarter 2007). The search terms included pramlintide, amylin, gastric emptying, pharmacokinetic, pharmacoeconomic, postprandial hyperglycemia, and glucagon. Articles were selected for review if they described studies having a randomized, double-blind, controlled design and included glycosylated hemoglobin (HbA(1c)) as an end point. RESULTS: Pramlintide is administered subcutaneously in the abdominal area or thigh immediately before each main meal to achieve maximal reductions in post-prandial glucose excursions. Its C(max) is reached within 20 minutes, and its t(1/2) is 48 minutes. Metabolism is primarily via the kidneys. Pramlintide therapy was associated with inhibition of postprandial glucagon secretion in 24 patients with type 2 DM; prolonged gastric emptying in 11 patients with type 1 DM; a 23% reduction in total energy intake in 11 patients with type 2 DM; and a reduction in markers of oxidative stress in 18 patients with type 1 DM (all, P <- 0.05 vs placebo). In two 52-week studies in patients with type 1 DM, the groups that received pramlintide 30 to 60 microg QID (n = 243), 60 microg TID (n = 164), and 60 microg QID (n = 161) had respective 0.39%, 0.29%, and 0.34% reductions in HbA(1c) and 0.5-, 0.3-, and 0.6-kg reductions in body weight, respectively (all, P < 0.05 vs placebo). In two 52-week studies in patients with type 2 DM, the groups that received pramlintide 120 microg BID (n = 166) and 150 microg TID (n = 144) had respective 0.62% and 0.6% reductions in HbA(1c) and 1.4- and 1.3-kg reductions in body weight (all, P < 0.05 vs placebo). Hypoglycemia, nausea, vomiting, and anorexia were the most frequently reported (>/=10% occurrence) adverse events in patients receiving pramlintide compared with placebo. These events were mild to moderate and occurred more frequently during the first month of therapy. CONCLUSIONS: Pramlintide therapy was associated with reductions in HbA(1c) and body weight in four 52-week studies in patients with type 1 DM and type 2 DM. Hypoglycemia, nausea, vomiting, and anorexia were the most frequently occurring adverse events, particularly during the first month of therapy. Pramlintide was associated with reductions in measures of oxidative stress, but studies are needed to evaluate the effects of this agent on DM-related complications.

A beta deposition inhibitor screen using synthetic amyloid.

The formation, growth, and maturation of brain amyloid "senile" plaques are essential pathological processes in Alzheimer's disease (AD) and key targets for therapeutic intervention. The process of in vitro deposition of A beta at physiological concentrations onto plaques in AD brain preparations has been well characterized, but is cumbersome for drug discovery. We describe here a high-through put screen for inhibitors of A beta deposition onto a synthetic template (synthaloid) of fibrillar A beta immobilized in a polymer matrix. Synthaloid is indistinguishable from plaques in AD brain (the natural template) in deposition kinetics, pH profile, and structure-activity relationships for both A beta analogs and inhibitors. Synthaloid, in contrast to current A beta aggregation screens, accurately predicted inhibitor potency for A beta deposition onto AD cortex preparations, validating its use in searching for agents that can slow the progression of AD and exposing a previously inaccessible target for drug discovery.

Amyloid fibril systems reduce, stabilize and deliver bioavailable nanosized iron.

Iron-deficiency anaemia (IDA) is a major global public health problem. A sustainable and cost-effective strategy to reduce IDA is iron fortification of foods, but the most bioavailable fortificants cause adverse organoleptic changes in foods. Iron nanoparticles are a promising solution in food matrices, although their tendency to oxidize and rapidly aggregate in solution severely limits their use in fortification. Amyloid fibrils are protein aggregates initially known for their association with neurodegenerative disorders, but recently described in the context of biological functions in living organisms and emerging as unique biomaterial building blocks. Here, we show an original application for these protein fibrils as efficient carriers for iron fortification. We use biodegradable amyloid fibrils from ß-lactoglobulin, an inexpensive milk protein with natural reducing effects, as anti-oxidizing nanocarriers and colloidal stabilizers for iron nanoparticles. The resulting hybrid material forms a stable protein-iron colloidal dispersion that undergoes rapid dissolution and releases iron ions during acidic and enzymatic in vitro digestion. Importantly, this hybrid shows high in vivo iron bioavailability, equivalent to ferrous sulfate in haemoglobin-repletion and stable-isotope studies in rats, but with reduced organoleptic changes in foods. Feeding the rats with these hybrid materials did not result in abnormal iron accumulation in any organs, or changes in whole blood glutathione concentrations, inferring their primary safety. Therefore, these iron-amyloid fibril hybrids emerge as novel, highly effective delivery systems for iron in both solid and liquid matrices.

Synthesis, Structure Characterization, and Evaluation in Microglia Cultures of Neuromelanin Analogues Suitable for Modeling Parkinson's Disease.

In the substantia nigra of human brain, neuromelanin (NM) released by degenerating neurons can activate microglia with consequent neurodegeneration, typical of Parkinson's disease (PD). Synthetic analogues of NM were prepared to develop a PD model reproducing the neuropathological conditions of the disease. Soluble melanin-protein conjugates were obtained by melanization of fibrillated ß-lactoglobulin (fLG). The melanic portion of the conjugates contains either eumelanic (EufLG) or mixed eumelanic/pheomelanic composition (PheofLG), the latter better simulating natural NMs. In addition, the conjugates can be loaded with controlled amounts of iron. Upon melanization, PheofLG-Fe conjugates maintain the amyloid cross-ß protein core as the only structurally organized element, similarly to human NMs. The similarity in composition and structural organization with the natural pigment is reflected by the ability of synthetic NMs to activate microglia, showing potential of the novel conjugates to model NM induced neuroinflammation. Thus, synthetic NM/microglia constitute a new model to develop anti-Parkinson drugs.

Adjunctive therapy with pramlintide in patients with type 1 or type 2 diabetes mellitus.

Uncontrolled diabetes mellitus is associated with both microvascular and macrovascular complications. Despite an array of treatment options available, achievement of euglycemia in most patients with diabetes is still lacking. Pramlintide acetate, a synthetic analog of the human hormone amylin and belonging to a new class of agents, was approved in March 2005 as adjunctive treatment in patients with type 1 or 2 diabetes mellitus. To evaluate the data available on the efficacy and safety of pramlintide, we conducted a search of MEDLINE (January 1966-May 2006) and International Pharmaceutical Abstracts (January 1970-May 2006). Bibliographies of clinical trials were reviewed for additional references. The literature reviewed demonstrated that pramlintide is effective in reducing levels of glycosylated hemoglobin and potentially preventing weight gain. The most commonly reported adverse effects associated with pramlintide were nausea, anorexia, and hypoglycemia. These adverse effects occurred more often during the initiation of therapy and were usually mild to moderate in nature. Whether this therapy is a cost-effective option for patients with type 1 or type 2 diabetes mellitus is yet to be determined.

Update in the pharmacologic treatment of diabetes mellitus: focus on pramlintide and exenatide.

There are now more than 20 million people in America with diabetes mellitus (DM), and the prevalence of this illness continues to increase especially in those with type 2 DM. Over the past decade, research in the area of DM treatment has focused on pharmacologic approaches to modifying glucose metabolism as well as on lifestyle interventions to prevent and manage DM. Pharmacologic research has been guided by an improved understanding of the human physiology of glucose metabolism, allowing for development of new hormonal drug therapies and improved insulin formulations. As a result, there are several new pharmacologic treatments now available or on the horizon for DM. In this article, the authors review the first of the new hormonal therapies for DM, with a focus on information that will be useful for diabetes educators including the medication actions, side effects, patient counseling points, monitoring, and place in therapy in comparison to existing DM treatments. This series on new therapies has been divided into 3 parts, with this first part devoted to an update on the new incretin mimetic and amylin analog agents recently approved for use in DM. Subsequent parts in this series will focus on the new insulin products and oral therapies available or soon to be available. Cases will be used to assist with understanding the type of patient who will benefit from each of these new therapies.

Enhancement of AA-amyloid formation in mice by transthyretin amyloid fragments and polyethylene glycol.

The mechanism behind amyloid formation is unknown in all types of amyloidosis. Several substances can enhance amyloid formation in animal experiments. To induce secondary systemic amyloid (AA-type amyloid) formation, we injected silver nitrate into mice together with either amyloid fibrils obtained from patients with familial polyneuropathy (FAP) type I or polyethylene glycol (PEG). Mice injected with silver nitrate only served as controls. Amyloid deposits were detectable at day 3 in animals injected with amyloid fibrils and in those injected with PEG, whereas in control mice, deposits were not noted before day 12. Our results indicate that amyloid fibrils from FAP patients and even a non-sulfate containing polysaccharide (PEG) have the potential to act as amyloid-enhancing factors.

An insulin-degrading enzyme inhibitor decreases amylin degradation, increases amylin-induced cytotoxicity, and increases amyloid formation in insulinoma cell cultures.

Amylin (islet amyloid polypeptide) is the chief component of the islet amyloid found in type 2 diabetes, and amylin fibril precursors may be cytotoxic to pancreatic beta-cells. Little is known about the prevention of amylin aggregation. We investigated the role of insulin-degrading enzyme (IDE) in amylin degradation, amyloid deposition, and cytotoxicity in RIN-m5F insulinoma cells. Human (125)I-labeled amylin degradation was inhibited by 46 and 65% with the addition of 100 nmol/l human amylin or insulin, respectively. (125)I-labeled insulin degradation was inhibited with 100 nmol/l human amylin, rat amylin, and insulin (by 50, 50, and 73%, respectively). The IDE inhibitor bacitracin inhibited amylin degradation by 78% and insulin degradation by 100%. Amyloid staining by Congo red fluorescence was detectable at 100 nmol/l amylin and was pronounced at 1,000 nmol/l amylin treatment for 48 h. Bacitracin treatment markedly increased staining at all amylin concentrations. Bacitracin with amylin caused a dramatic decrease in cell viability compared with amylin alone (68 and 25%, respectively, at 10 nmol/l amylin). In summary, RIN-m5F cells degraded both amylin and insulin through a common proteolytic pathway. IDE inhibition by bacitracin impaired amylin degradation, increased amyloid formation, and increased amylin-induced cytotoxicity, suggesting a role for IDE in amylin clearance and the prevention of amylin aggregation.

Properties of pramlintide and insulin upon mixing.

PURPOSE: The pharmacokinetics, pharmacodynamics, and safety of pramlintide and various insulin formulations in patients with type 1 diabetes mellitus (DM) when given as separate injections or mixed in the same syringe before injection were studied. METHODS: In two randomized, open-label, placebo-controlled, five-period-crossover studies, patients with type 1 DM received preprandial injections of pramlintide, short-acting insulin, and long-acting insulin administered either by separate injections or after mixing in various combinations. Serum free insulin and plasma glucose concentrations were measured for 10 hours and plasma pramlintide concentrations for 5 hours after injection. RESULTS: Blood samples were collected from a total of 51 patients. All treatments involving mixtures were comparable to separate injections with respect to the area under the concentration-versus-time curve (AUC) and the maximum concentration (Cmax) of serum free insulin. There were some minor differences in the AUC and Cmax of pramlintide. No injection-site reactions or other unexpected adverse events were observed. CONCLUSION: Mixing pramlintide with short- or long-acting insulin in the same syringe before subcutaneous injection did not affect the pharmacodynamics of glucose or the pharmacokinetics of insulin or pramlintide in a clinically significant manner.

Pramlintide acetate.

PURPOSE: The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage and administration of pramlintide are reviewed. SUMMARY: Pramlintide, a synthetic analogue of the human hormone amylin, is the first of a new class of amylinomimetic compounds. It was approved in March 2005 as a subcutaneous injection for the adjunctive treatment of patients who have type 1 or 2 diabetes mellitus and have failed to achieve glycemic control despite optimal therapy with insulin. Pramlintide complements the effects of insulin in postprandial glucose regulation by decreasing glucagon secretion. Pramlintide exhibits linear pharmacokinetics, and peak serum levels are reached within 30 minutes of administration. The drug is predominantly renally eliminated, with a mean elimination half-life of 30-50 minutes. Clinical trials have shown that pramlintide suppresses postmeal glucagon secretion, slows gastric emptying, reduces postprandial glucose levels, and improves glycemic control while managing weight loss. Pramlintide has also been shown to decrease hemoglobin A(1c), serum fructosamine, and total cholesterol levels. Pramlintide has been associated with an increased risk of insulin-induced severe hypoglycemia; other adverse events include nausea, anorexia, fatigue, and vomiting. The dosage varies with the type of diabetes. Because of the cost associated with the complications of uncontrolled diabetes, pramlintide may have a beneficial effect on total costs associated with this chronic disease. CONCLUSION: Pramlintide in combination with insulin is a potential therapeutic option for improving glycemic control in patients with diabetes, but the increased risk of hypoglycemia must be aggressively monitored.

Clinical studies.

Recognizing that type 1 diabetes was characterized not only by insulin deficiency, but also by amylin deficiency, Cooper (Cooper, 1991) predicted that certain features of the disease could be related thereto, and he proposed amylin/insulin co-replacement therapy. Although the early physiological rationale was flawed, the idea that glucose control could be improved over that attainable with insulin alone without invoking the ravages of worsening insulin-induced hypoglycemia was vindicated. The proposal spawned a first-in-class drug development program that ultimately led to marketing approval by the U.S. Food and Drug Administration of the amylinomimetic pramlintide acetate in March 2005. The prescribers' package insert (Amylin Pharmaceuticals Inc., 2005), which includes a synopsis of safety and efficacy of pramlintide, is included as Appendix 1. Pramlintide exhibited a terminal t1/2, in humans of 25-49 min and, like amylin, was cleared mainly by the kidney. The dose-limiting side effect was nausea and, at some doses, vomiting. These side effects usually subsided within the first days to weeks of administration. The principal risk of pramlintide co-therapy was an increased probability of insulin-induced hypoglycemia, especially at the initiation of therapy. This risk could be mitigated by pre-emptive reduction in insulin dose. Pramlintide dosed at 30-60 microg three to four times daily in patients with type 1 diabetes, and at doses of 120 microg twice daily in patients with type 2 diabetes, invoked a glycemic improvement, typically a decrease in HbA1c of 0.4-0.5% relative to placebo, that was sustained for at least 1 year. This change relative to control subjects treated with insulin alone typically was associated with a reduction in body weight and insulin use, and was not associated with an increase in rate of severe hypoglycemia other than at the initiation of therapy. Effects observed in animals, such as slowing of gastric emptying, inhibition of nutrient-stimulated glucagon secretion, and inhibition of food intake, generally have been replicated in humans. A notable exception appears to be induction of muscle glycogenolysis and increase in plasma lactate.

Acute effects of the human amylin analog AC137 on basal and insulin-stimulated euglycemic and hypoglycemic fuel metabolism in patients with insulin-dependent diabetes mellitus.

Amylin has been reported to decrease glycogen storage in rodent skeletal muscles and produce insulin resistance in intact rats. To test the acute effect of a human amylin analog (AC137) on glucose metabolism in man, seven IDDM patients were infused in a randomized, double blind, cross-over study with AC137 (100 micrograms/h, n = 1; 50 micrograms/h, n = 6) or placebo for 330 min during a two-step euglycemic clamp (insulin infusion rates, 0.2 and 0.6 mU/kg.min; basal and hyperinsulinemic period, respectively) followed by a hyperinsulinemic hypoglycemic clamp (insulin infusion rate, 1.5 mU/kg.min; hypoglycemic period). During euglycemia, no differences were found in glucose disposal (step 1, 2.43 +/- 0.20 vs. 2.03 +/- 0.26; step 2, 4.28 +/- 0.54 vs. 4.11 +/- 0.45 mg/kg.min; AC137 vs. placebo, mean +/- SEM), arteriovenous substrate balances across the forearm, or hepatic glucose production. During hypoglycemia, glucose fluxes were also similar. However, lactate release from the forearm was more pronounced (P < 0.05) with the analog than with placebo (area under the curve, -11.2 +/- 4.6 vs. -1.4 +/- 2.2 mmol/min.L). Despite similar plasma glucose nadirs (2.7 +/- 0.0 vs. 2.6 +/- 0.1 mmol/L; AC137 vs. placebo), circulating cortisol and GH rose to significantly higher levels during hypoglycemia with the amylin analog (P < 0.05). In conclusion, acute administration of the amylin analog AC137 did not influence insulin-stimulated glucose metabolism during euglycemic conditions. During imposed hypoglycemia, lactate release from skeletal muscle was, however, enhanced, and the rise in cortisol and GH was augmented.

Amylin replacement with pramlintide as an adjunct to insulin therapy in type 1 and type 2 diabetes mellitus: a physiological approach toward improved metabolic control.

Destruction and dysfunction of pancreatic beta-cells, resulting in absolute and relative insulin deficiency, represent key abnormalities in the pathogenesis of type 1 and type 2 diabetes, respectively. Following the discovery of amylin, a second beta-cell hormone that is co-secreted with insulin in response to nutrient stimuli, it was realized that diabetes represents a state of bihormonal beta cell deficiency and that lack of amylin action may contribute to abnormal glucose homeostasis. Experimental studies show that amylin acts as a neuroendocrine hormone that complements the effects of insulin in postprandial glucose regulation through several centrally mediated effects. These include a suppression of postprandial glucagon secretion and a vagus-mediated regulation of gastric emptying, thereby helping to control the influx of endogenous and exogenous glucose, respectively. In animal studies, amylin has also been shown to reduce food intake and body weight, consistent with an additional satiety effect. Pramlintide is a soluble, non-aggregating, injectable, synthetic analog of human amylin currently under development for the treatment of type 1 and insulin-using type 2 diabetes. Long-term clinical studies have consistently demonstrated that pre-prandial s.c. injections of pramlintide, in addition to the current insulin regimen, reduce HbA(1c) and body weight in type 1 and type 2 diabetic patients, without an increase in insulin use or in the event rate of severe hypoglycemia. The most commonly observed side effects were gastrointestinal-related, mainly mild nausea, which typically occurred upon initiation of treatment and resolved within days or weeks. Amylin replacement with pramlintide as an adjunct to insulin therapy is a novel physiological approach toward improved long-term glycemic and weight control in patients with type 1 and type 2 diabetes.

Pharmacokinetics and pharmacodynamics of AC137 (25,28,29 tripro-amylin, human) after intravenous bolus and infusion doses in patients with insulin-dependent diabetes.

A study was conducted to evaluate the effect of 30-mug, 100-mug, and 300-mug 2-minute bolus doses and 2-hour infusion doses of AC137 (25,28,29 tripro-amylin, human) on plasma AC137 concentrations and plasma glucose and lactate responses in patients with insulin-dependent diabetes mellitus (IDDM). The study design was an imbedded two-way cross-over wherein patients received placebo and active boluses in one period and placebo and active infusions in the other period. Two patients in each dose group received placebo throughout the two periods. Pharmacokinetics and pharmacodynamics (PK/PD) were determined during the 6-hour period after initiation of dosing. Data were fitted with a linked PK/PD model. Pharmacokinetics were linear over the dose range studied, and attenuation of glucose and lactate responses to a mixed meal was dose and concentration dependent. The results of the PK/PD model indicate that the attenuation of glucose and lactate responses was greater after AC137 infusion doses than after the same doses given as a bolus. Glucose and lactate responses to a mixed meal were essentially negated by the 300-mug infusion dose.

Differential permeability of the blood-brain barrier to two pancreatic peptides: insulin and amylin.

Insulin and amylin are cosecreted by pancreatic B cells and have receptors within the central nervous system (CNS), where they exert multiple effects. Although these peptides are not produced in the CNS, their ability to cross the blood-brain barrier (BBB) explains their presence there. We used multiple-time regression analysis to measure, in mice, the unidirectional influx constant (Ki) of each of these peptides to compare their rates of transport with each other and in different regions of the brain. The uptake of amylin by whole brain and by the cerebellum, midbrain, frontal cortex, parietal cortex, and occipital cortex was greater than that for insulin. For amylin, the areas of highest uptake were the pons-medulla and the cerebellum, and the areas of lowest uptake were the thalamus and midbrain. For insulin, the areas of highest uptake were the pons-medulla and the hypothalamus, whereas three regions (midbrain, thalamus, and occipital cortex) did not have a measurable Ki. The peak percent of injected dose taken up by whole brain was 0.12% for amylin and 0.046% for insulin. These results show that the permeabilities of these two peptides across the BBB differed from each other and among brain regions, suggesting that differential permeability of the BBB for blood-borne peptides could provide a mechanism by which their effects on the CNS are regulated.

Permeability of the blood-brain barrier to amylin.

Amylin is co-secreted with insulin from the pancreas of patients with non-insulin dependent diabetes mellitus, and its deposition may contribute to the central nervous system (CNS) manifestations of this disease. Amylin, but not its mRNA, is found in brain, suggesting that CNS amylin is derived from the circulation. This would require amylin to cross the blood-brain barrier (BBB). We used multiple-time regression analysis to determine the unidirectional influx constant (Ki) of blood-borne, radioactively labeled amylin (I-Amy) into the brain of mice. The Ki was 8.99(10(-4)) ml/g-min and was not inhibited with doses up to 100 micrograms/kg, but it was inhibited by aluminum (Al). About 0.11 to 0.13 percent of the injected dose of I-Amy entered each gram of brain. Radioactivity recovered from brain and analyzed by HPLC showed that the majority of radioactivity taken up by the brain represented intact I-Amy. Capillary depletion confirmed that blood-borne I-Amy completely crossed the BBB to enter the parenchymal/interstitial fluid space of the cerebral cortex. Taken together, these results show that blood-borne amylin has access to brain tissue and may be involved in some of the CNS manifestations of diabetes mellitus.

Dose response characteristics for the hyperglycemic, hyperlactemic, hypotensive and hypocalcemic actions of amylin and calcitonin gene-related peptide-I (CGRP alpha) in the fasted, anaesthetized rat.

Amylin, a 37 amino-acid peptide secreted from the pancreatic beta-cells, exerts marked effects on carbohydrate metabolism in intact rats. It has approximately 50% amino-acid identity with the calcitonin gene-related peptides (CGRP) as well as certain shared biological actions. In vivo potencies were determined for four responses (increases in plasma glucose, increases in plasma lactate, decreases in plasma calcium, and depression of arterial pressure). These responses were measured in fasted, lightly anaesthetized rats given single intravenous bolus injections of rat amylin or rat CGRP alpha at doses of 0.01, 0.1, 1, 10, 100 and 1000 micrograms (about 7 pmol/kg-700 nmol/kg). Control animals received an equal volume of saline. The order of potency for the different responses was as follows: (i) increase in plasma glucose concentration, amylin approximately 2 times more potent than CGRP (by ED50) with detectable responses occurring at doses 100-fold less; (ii) decrease in plasma total calcium concentration, CGRP of equal or greater potency than amylin; and (iii) decrease in arterial pressure, CGRP 44-fold more potent than amylin. An increase in plasma lactate occurred with amylin doses 1000-fold lower than the CGRP doses producing such effects. Saturation of the dose-dependent increase in lactate was not observed, so median effective doses (ED50) were not obtained. These results are consistent with the existence of separate receptor systems for amylin and CGRP. The effects of amylin on plasma glucose and lactate concentrations were demonstrable at doses of 0.1-1.0 micrograms (70-700 pmol/kg). These doses produced plasma levels that were within the concentration range previously reported for insulin-resistant rats, supporting the proposal that amylin is a physiologic endocrine regulator of carbohydrate metabolism in vivo.