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.

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.

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.

Enhanced weight loss with pramlintide/metreleptin: an integrated neurohormonal approach to obesity pharmacotherapy.

The neurohormonal control of body weight involves a complex interplay between long-term adiposity signals (e.g., leptin), and short-term satiation signals (e.g., amylin). In diet-induced obese (DIO) rodents, amylin/leptin combination treatment led to marked, synergistic, fat-specific weight loss. To evaluate the weight-lowering effect of combined amylin/leptin agonism (with pramlintide/metreleptin) in human obesity, a 24-week, randomized, double-blind, active-drug-controlled, proof-of-concept study was conducted in obese or overweight subjects (N = 177; 63% female; 39 +/- 8 years; BMI 32.0 +/- 2.1 kg/m(2); 93.3 +/- 13.2 kg; mean +/- s.d.). After a 4-week lead-in period with pramlintide (180 microg b.i.d. for 2 weeks, 360 microg b.i.d. thereafter) and diet (40% calorie deficit), subjects achieving 2-8% weight loss were randomized 1:2:2 to 20 weeks of treatment with metreleptin (5 mg b.i.d.), pramlintide (360 microg b.i.d.), or pramlintide/metreleptin (360 microg/5 mg b.i.d.). Combination treatment with pramlintide/metreleptin led to significantly greater weight loss from enrollment to week 20 (-12.7 +/- 0.9%; least squares mean +/- s.e.) than treatment with pramlintide (-8.4 +/- 0.9%; P < 0.001) or metreleptin (-8.2 +/- 1.3%; P < 0.01) alone (evaluable, N = 93). The greater reduction in body weight was significant as early as week 4, and weight loss continued throughout the study, without evidence of a plateau. The most common adverse events with pramlintide/metreleptin were injection site events and nausea, which were mostly mild to moderate and decreased over time. These results support further development of pramlintide/metreleptin as a novel, integrated neurohormonal approach to obesity pharmacotherapy.

Continuous subcutaneous pramlintide infusion therapy in patients with type 1 diabetes: observations from a pilot study.

OBJECTIVE: To investigate the efficacy and safety of continuous (basal-bolus) subcutaneous pramlintide infusion (CSPI) in patients with type 1 diabetes mellitus. METHODS: A 16-week, open-label, single-arm pilot study enrolled 11 patients (mean +/- SD values: age, 39.9 +/- 4.0 years; hemoglobin A1c, 8.20% +/- 0.60%; weight, 92.3 +/- 18.4 kg; body mass index, 29.7 +/- 5.1 kg/m2) with longterm type 1 diabetes mellitus (20.7 +/- 1.3 years; duration of pump therapy, 9.5 +/- 6.0 years). Pramlintide basal infusion was begun with continuous subcutaneous infusion at 9 microg/h. After 3 days, premeal bolus doses of pramlintide were initiated at 15 microg and titrated to 60 microg per meal. Basal and bolus insulin doses were reduced 10% on initiation of CSPI and adjusted thereafter as needed to prevent hypoglycemia. RESULTS: After 16 weeks of pramlintide therapy, mean +/- SD hemoglobin A1c decreased to 7.85% +/- 0.74% (-0.35%). The fasting glucose level declined from 198.2 +/- 66.9 mg/dL to 135.8 +/- 63.9 mg/dL. The mean weight decreased to 91.8 +/- 20.1 kg (-0.5 kg) at week 12. The daily bolus insulin requirement decreased 20%; daily basal insulin was unchanged (27.7 +/- 11.7 U). All patients experienced mild postprandial hypoglycemia, but no severe hypoglycemia was reported. Three of the 11 study participants experienced mild initial nausea, but all patients successfully titrated bolus doses to 60 microg within 3 weeks. CONCLUSION: In this pilot study of 11 patients with type 1 diabetes using insulin pumps, CSPI seemed safe and well tolerated, did not alter pramlintide pharmacokinetic variables, and reduced fasting glucose levels. Larger studies of this method for pramlintide administration seem warranted.

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.

Mechanism of islet amyloid polypeptide fibrillation at lipid interfaces studied by infrared reflection absorption spectroscopy.

Islet amyloid polypeptide (IAPP) is a pancreatic hormone and one of a number of proteins that are involved in the formation of amyloid deposits in the islets of Langerhans of type II diabetes mellitus patients. Though IAPP-membrane interactions are known to play a major role in the fibrillation process, the mechanism and the peptide's conformational changes involved are still largely unknown. To obtain new insights into the conformational dynamics of IAPP upon its aggregation at membrane interfaces and to relate these structures to its fibril formation, we studied the association of IAPP at various interfaces including neutral as well as charged phospholipids using infrared reflection absorption spectroscopy. The results obtained reveal that the interaction of human IAPP with the lipid interface is driven by the N-terminal part of the peptide and is largely driven by electrostatic interactions, as the protein is able to associate strongly with negatively charged lipids only. A two-step process is observed upon peptide binding, involving a conformational transition from a largely alpha-helical to a beta-sheet conformation, finally forming ordered fibrillar structures. As revealed by simulations of the infrared reflection absorption spectra and complementary atomic force microscopy studies, the fibrillar structures formed consist of parallel intermolecular beta-sheets lying parallel to the lipid interface but still contain a significant number of turn structures. We may assume that these dynamical conformational changes observed for negatively charged lipid interfaces play an important role as the first steps of IAPP-induced membrane damage in type II diabetes.

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.

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.

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.

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.

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.

Pramlintide for the treatment of diabetes mellitus.

OBJECTIVE: To provide an overview of the role of amylin, as well as that of pramlintide, a synthetic analog of amylin, in maintaining glucose homeostasis; and discuss the pharmacology, pharmacokinetics, efficacy, adverse effects, and role of pramlintide in the control of postprandial hyperglycemia. DATA SOURCES: The data presented in this review were obtained from published literature, abstracts presented at scientific meetings, and information on file with the manufacturer. MEDLINE searches (1986-March 2003) using the search terms pramlintide and amylin were conducted to identify clinical trials and review articles. Additionally, the bibliographies of the identified articles were reviewed. DATA SYNTHESIS: Clinical trials have demonstrated that amylin in combination with insulin controls postprandial glucose levels by decreasing food intake, slowing gastric emptying, and suppressing glucagon secretion. Clinical trials also showed significant decreases in mean plasma glucose levels and glycosylated hemoglobin, as well as the added benefits of weight loss and reduction in insulin doses. The most commonly reported adverse effects associated with pramlintide in clinical trials were gastrointestinal complaints and hypoglycemia, which occurred most frequently during initiation of therapy. CONCLUSIONS: The administration of insulin alone often does not result in optimal metabolic control. The treatment of amylin deficiency, in addition to insulin deficiency, may be warranted in order to obtain glucose homeostasis. The role of pramlintide, an amylin analog, will become clearer as more clinical data become available.

Ex vivo human placental transfer of the peptides pramlintide and exenatide (synthetic exendin-4).

Two peptides, pramlintide (37 amino acids), an analog of human amylin, and exenatide, synthetic exendin-4 (39 amino acids), are both in late-stage clinical development as potential new treatments for people with diabetes. Both are potential long-term treatments, and there is the likelihood that some women will become pregnant while using one of these peptide therapies. Therefore, it was important to evaluate the potential for each peptide to cross the placental barrier and thereby result in exposure to the fetus. This was examined using ex vivo perfusions of human placentas. The fetal and maternal side of a cotyledon were cannulated and perfused first with buffer, and then with radioactive antipyrine in order to establish the integrity of the system and the perfusion constants. Either pramlintide or exenatide was then added to each acceptable cotyledon perfusate on the maternal side. Each peptide was evaluated at an initial concentration near the therapeutic plasma concentration and at approximately 10-50 times that concentration in each of the three cotyledons. Maternal and fetal perfusate samples were assayed for peptide concentrations using an immunoassay. The ratio of fetal-to-maternal peptide concentrations during equilibrium perfusion were extremely low (pramlintide < or = 0.006, exenatide < or = 0.017). These data demonstrate negligible passage of either peptide across the placental barrier. It is, therefore, likely that maternal use of either peptide during gestation will result in negligible exposure to the fetus.

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.

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.