Diabetic retinopathy (DR) is the leading etiology of blindness in the working population of the USA. Its long-term management relies on effective glycemic control. Seven anti-diabetic classes have been introduced for patients with type 2 diabetes (T2D) in the past two decades, with different glucose-lowering and cardiovascular benefits. Yet, their effects specifically on DR have not been studied in detail. A systematic review of the literature was conducted to investigate this topic, focusing on the available clinical data for T2D. Published studies were evaluated based on their level of statistical evidence, as long as they incorporated at least one endpoint or adverse event pertaining to retinal health. Fifty nine articles met our inclusion criteria and were grouped per anti-diabetic class as follows: alpha-glucosidase inhibitors (1), peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists (8), amylin analogs (1), glucagon-like peptide-1 (GLP-1) receptor agonists (28), dipeptidyl peptidase 4 (DPP-4) inhibitors (9), and sodium glucose co-transporter-2 (SGLT-2) inhibitors (9), plus one retrospective study and two meta-analyses evaluating more than one of the aforementioned anti-diabetic categories. We also reviewed publicly-announced results of trials for the recently-introduced class of twincretins. The available data indicates that most drugs in the newer anti-diabetic classes are neutral to DR progression; however, there are subclasses differences in specific drugs and T2D populations. In particular, there is evidence suggesting there may be worse diabetic macular edema with PPAR-gamma agonists, potential slight DR worsening with semaglutide (GLP-1 receptor agonist), and potential slight increase in the incidence of retinal vein occlusion in elderly and patients with advanced kidney disease receiving SGLT-2 inhibitors. All these warrant further investigation. Longer follow-up and systematic assessment of at least one DR-related endpoint are highly recommended for all future trials in the T2D field, to ultimately address this topic.

Misfolding of human islet amyloid polypeptide (hIAPP) into insoluble aggregates is associated with Type 2 diabetes. It has been suggested that hIAPP toxicity may be due to its accumulation in pancreatic islets, causing membrane disruption and cell permeabilization, however the molecular basis underlying its lipid association are still unclear. Here, we combine solid-state NMR, fluorescence and bright field microscopy to investigate hIAPP - lipid membrane interactions. Real-time microscopy highlights a time-dependent penetration of hIAPP oligomers toward the most buried layers of the lipid vesicles until the membrane disrupts. Deuterium NMR was conducted on liposomes at different hIAPP concentration to probe lipid internal order and thermotropism. The gel-to-fluid phase transition of the lipids is decreased by the presence of hIAPP, and site-specific analysis of the order parameter showed a significant increase of lipid order for the first eight positions of the acyl chain, suggesting a partial insertion of the peptide inside the bilayer. These results offer experimental insight into the membrane destabilization of hIAPP on model membrane vesicles.

BACE2 (β-site APP-cleaving enzyme 2) is a protease expressed in the brain, but also in the pancreas, where it seems to play a physiological role. Amyloidogenic diseases, including Alzheimer's disease and type 2 diabetes (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. In T2D, islet amyloid polypeptide (IAPP) deposits have been shown to be a pathogenic key feature of the disease. The aim of the present study was to investigate the effect of BACE2 modulation on β-cell alterations in a mouse model of T2D induced by IAPP overexpression. Heterozygous mice carrying the human transcript of IAPP (hIAPP-Tg) were used as a model to study the deleterious effects of IAPP upon β-cell function. These animals showed glucose intolerance and impaired insulin secretion. When crossed with BACE2-deficient mice, the animals presented a significant improvement in glucose tolerance accompanied with an enhanced insulin secretion, as compared to hIAPP-Tg mice. BACE2 deficiency also partially reverted gene expression changes observed in islets from hIAPP-Tg mice, including a set of genes related to inflammation. Moreover, homozygous hIAPP mice presented a severe hyperglycemia and a high lethality rate from 8 weeks onwards due to a massive destruction of β-cell mass. This process was significantly reduced when crossed with the BACE2-KO model, improving the survival rate of the animals. Altogether, the absence of BACE2 ameliorates glucose tolerance defects induced by IAPP overexpression in the β-cell and promotes β-cell survival. Thus, targeting BACE2 may represent a promising therapeutic strategy to improve β-cell function in T2D.

The neuroendocrine hormone amylin, also known as islet amyloid polypeptide, is co-localized, co-packaged and co-secreted with insulin from adult pancreatic islet β cells to maintain glucose homeostasis. Specifically, amylin reduces secretion of nutrient-stimulated glucagon, regulates blood pressure with an effect on renin-angiotensin system, and delays gastric emptying. The physiological actions of human amylin attribute to the conformational α-helix monomers whereas the misfolding instable oligomers may be detrimental to the islet β cells and further transform to β-sheet fibrils as amyloid deposits. No direct evidence proves that the amylin fibrils in amyloid deposits cause diabetes. Here we also have performed a systematic review of human amylin gene changes and reported the S20G mutation is minor in the development of diabetes. In addition to the metabolic effects, human amylin may modulate autoimmunity and innate inflammation through regulatory T cells to impact on both human type 1 and type 2 diabetes.

Cyanidin-3-rutinoside (C3R), a naturally occurring anthocyanin, is present in various fruits and vegetables as a colorant. C3R has been well characterized and demonstrated a number of biological activities attributed to its antioxidant properties. The present study compared the effectiveness of C3R against monosaccharide-induced protein glycation and oxidation in vitro using bovine serum albumin (BSA).The results demonstrated that C3R (0.125-1.00 mM) inhibited the formation of fluorescent AGEs in ribose-glycated BSA (2-52%), fructose-glycated BSA (81-93%), glucose-glycated BSA (30-74%) and galactose-glycated BSA (6-79%).Correspondingly, C3R (1.00 mM) decreased the level of N(ɛ)-(carboxymethyl) lysine (56-86%) in monosaccharide-induced glycation in BSA. C3R also reduced the level of fructosamine, β-amyloid cross structure, protein carbonyl content as well as the depletion of thiol in BSA/monosaccharide system. In summary, C3R might offer a new promising antiglycation agent for the prevention of diabetic complications by inhibiting AGE formation and oxidation-dependent protein damage.

Amyloid fibrils associated with many neurodegenerative diseases are the most intriguing targets of modern structural biology. Significant knowledge has been accumulated about the morphology and fibril-core structure recently. However, no conventional methods could probe the fibril surface despite its significant role in the biological activity. Tip-enhanced Raman spectroscopy (TERS) offers a unique opportunity to characterize the surface structure of an individual fibril due to a high depth and lateral spatial resolution of the method in the nanometer range. Herein, TERS is utilized for characterizing the secondary structure and amino acid residue composition of the surface of insulin fibrils. It was found that the surface is strongly heterogeneous and consists of clusters with various protein conformations. More than 30% of the fibril surface is dominated by β-sheet secondary structure, further developing Dobson's model of amyloid fibrils (Jimenez et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9196-9201). The propensity of various amino acids to be on the fibril surface and specific surface secondary structure elements were evaluated. β-sheet areas are rich in cysteine and aromatic amino acids, such as phenylalanine and tyrosine, whereas proline was found only in α-helical and unordered protein clusters. In addition, we showed that carboxyl, amino, and imino groups are nearly equally distributed over β-sheet and α-helix/unordered regions. Overall, this study provides valuable new information about the structure and composition of the insulin fibril surface and demonstrates the power of TERS for fibril characterization.

Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly, therefore, is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were nontoxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of nontoxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Of 10 variation sites between sequences of amyloid-resistant porcine islet amyloid polypeptide (pIAPP) and amyloid-prone human IAPP (hIAPP), seven locate within residues 17-29, the most amyloidogenic fragment within hIAPP. To investigate how these variations affect amyloidogenicity, 26 IAPP(17-29) or IAPP(20-29) variants were synthesized and their secondary structures, amyloidogenicity, oligomerization and cytotoxicity were studied. Our results indicated that pIAPP fragments are refractory to amyloid formation and significantly less cytotoxic compared with hIAPP fragments. A novel stable dimer was observed in pIAPP(20-29) solution, whereas hIAPP(20-29) exists mostly as monomers and trimers. Among all human to porcine substitutions, S20R caused the most prolonged lag time and significantly attenuated cytotoxicity. The different oligomerization and amyloidogenic properties of hIAPP and pIAPP fragments are discussed.

Amylin is the major constituent of pancreatic islet amyloid whose accumulation characterizes patients with type 2 diabetes mellitus (T2DM). Although amylin is tightly linked with T2DM, in many cases, proamylin may be the more toxic species. As the precursor of amylin, however, the pathophysiological role of proamylin remains unknown. In this study, we investigate whether serum levels of proamylin or amylin or the proamylin/amylin ratios are different among normal subjects and patients with impaired glucose regulation (IGR) and T2DM. Totally 79 subjects were divided into three groups according to the results of oral glucose tolerance test (OGTT); they were T2DM group (32 cases), IGR group (23cases), and normal glucose tolerance (NGT) group (24cases). Serum levels of amylin and proamylin were measured with an enzyme-linked immunosorbent assay (ELISA). The relationships between serum levels of proamylin, amylin, their ratios and anthropometric and metabolic parameters were also analyzed. The serum levels of proamylin were significantly higher in patients with IGR and T2DM than in control subjects. The serum levels of proamylin were significantly associated with IGR and T2DM, with the odds ratios of 1.589 (95%CI, 1.228-2.055, P < 0.01) and 1.860 (95%CI, 1.342-2.587, P < 0.01), respectively. Both fasting serum levels of proamylin and proamylin/amylin ratios were found to correlate negatively with HOMA-B and DeltaI30/DeltaG30. Serum levels of proamylin, amylin, and their ratios were positively correlated with HOMA-IR. BMI and HOMA-B were independent related factors with serum levels of proamylin. Our results suggest that proamylin may play an important role in amyloid deposit in patients with IGR and T2DM.

Hydrophobic interactions play an important role in assembly processes in aqueous environments. In case of peptide amphiphiles, hydrophobicity is combined with hydrogen bonding to yield well-defined peptide-based aggregates. Here, we report a systematic study after the role of hydrophobic interactions on both stabilization and morphology of a peptide fibrillar assembly. For this purpose, alkyl tails were connected to a known beta-sheet forming peptide with the sequence KTVIIE. The introduction of n-alkyl groups induced thermal stability to the assemblies without affecting the morphology of the peptide aggregates.

Amyloid fibril formation is a nucleation dependent process characterized by a lag-phase prior to the appearance of detectable amyloid fibrils. While the three-dimensional structure of amyloid fibrils at atomic resolution is just beginning to be elucidated, the early process of monomers assembly into oligomers is less understood. Understanding the dynamic processes that lead to the formation of these intermediates is highly important as these assemblies might be the most pathological ones. Here, we investigated the biophysical and structural features characterizing the early stage assemblies formed by the human hormone calcitonin. We calculated the initial nucleus size by experimentally determining the dependence between the lag-time length and the hCT concentrations. We used size exclusion chromatography and dynamic light scattering in order to characterize the dynamic growth process of preliminary intermediates transformed into larger structures. The early structures were visualized using high-resolution transmission electron microscopy. Annular pore-like structures were observed along with protofibrilar structures. This observed morphology is similar to structures revealed during the fibrillization processes of beta-amyloid, alpha-synuclein, and islet amyloid polypeptide, suggesting that these intermediates represent a generic early structure conformation. The results introduced here imply that a variety of intermediate assemblies are formed during the early stages of amyloid fibril formation. The characterizing of their structural features and assembly kinetics will contribute to the rational design of inhibitors directed towards early structure assemblies.

The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2-terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into beta-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in beta-TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding beta-cells.

Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth.

Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation.

Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest. 81, 493-499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.

Molecular mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene expression remain unclear. In the present study, we investigated the effects of glucose and forskolin on IAPP gene regulation in the INS-1 islet beta-cell line. Both glucose and forskolin increased the level of expression of this gene, as measured by Northern blot analysis, and increased IAPP gene transcription in a time- and concentration-dependent manner, as demonstrated in a reporter gene assay. Although inhibition of protein kinase A activity with H-89 eliminated the effect of forskolin on this gene, the glucose effect was unaffected. This supported the predominant use of a protein kinase A-independent signaling pathway for glucose regulation of the IAPP gene. Electrophoretic mobility shift assay further indicated that glucose and forskolin regulated expression of this gene by targeting different elements of the promoter. Mutation of the cAMP regulatory element flanking the IAPP coding region resulted in the loss of most of the forskolin-stimulated IAPP gene promoter activity, whereas glucose-enhanced IAPP gene transcription was unaffected. These results demonstrate parallel and distinct regulatory pathways involved in glucose- and forskolin-induced IAPP gene expression in this model beta-cell system.

Human amylin, a major constituent of pancreatic amyloid deposits, may be a pathogenetic factor for noninsulin-dependent diabetes mellitus (NIDDM). We demonstrated that the human amylin S20G gene mutation (S20G) was associated with a history of early onset, more severe type of NIDDM, linking the amylin gene to this disease. Also, we demonstrated that expression of human wild-type (WT) amylin in COS-1 cells leads to intracellular amyloidogenesis and induction of apoptosis, suggesting a possible mechanism for disease induction. Therefore we compared the abilities of S20G and WT amylin to induce apoptosis in transfected COS-1 cells and form amyloid in vitro. We transfected the rat (RAT), mutated human (MUT), WT, and S20G amylin genes into COS-1 cells and measured apoptosis using fluorescent-activated cell sorting analysis at 48, 72, and 96 hours. At 96 hours apoptosis increased significantly (P < 0.01) in cells transfected with WT and S20G over RAT or MUT (WT, 19%; S20G, 25%; RAT, 13%; and MUT, 12%) and the difference between WT and S20G was significant (P < 0.05). Synthetic WT and S20G monomeric peptides were used to generate amyloid fibrils in vitro as measured by the thioflavin T binding assay. The S20G amylin formed approximately twofold more amyloid at a rate approximately threefold higher than WT. Electron micrography indicated that the in vitro amyloid generated by WT and S20G amylins were morphologically indistinguishable. The results suggest that increased cytotoxicity by S20G is because of increased amyloidogenicity, which may be a causative factor in the early development of NIDDM, possibly through loss of ss cell mass.

Recent progress has improved our knowledge of how proteins form amyloid fibrils. Both 'natively unfolded' and globular proteins have been shown to initiate fibrillization by adopting a partially structured conformation. Oligomeric prefibrillar intermediates have been extensively characterized with respect to their morphology and temporal evolution. Three-dimensional models obtained using biophysical and computational methods have provided information about fibril structure. All of these advances suggest common features of self-assembly pathways, with subtle variations accounting for differences among distinct amyloid fibrils.

Central obesity is increasingly recognized as a risk factor for atherosclerosis and type 2 diabetes mellitus. Here we present a hypothesis that may explain the excess atherosclerosis, endothelial dysfunction and progressive beta-cell failure. Central obesity is associated with increased cytosolic triglyceride stores in non-adipose tissues such as muscles, liver and pancreatic beta-cells. A high cytosolic triglyceride content is accompanied by elevated concentrations of cytosolic long-chain acyl-CoA esters, the metabolically active form of fatty acids. These esters inhibit mitochondrial adenine nucleotide translocators, resulting in an intramitochondrial ADP deficiency. In vitro, such ADP deficiency is a potent stimulator of mitochondrial oxygen free radical production, and we assume that this mechanism is also active in vivo. The decline of organ function with normal ageing is thought to be due, at least partly, to a continuous low-grade mitochondrial oxygen free radical production. In tissues containing increased cytosolic triglyceride stores this process will be accelerated. Tissues with a high-energy demand or poor free radical scavenging capacity, such as pancreatic beta-cells, are likely to be more susceptible to this process. This is how we explain their gradual dysfunctioning in central obesity. Likewise we propose that the enhanced production of oxygen free radicals in endothelial cells, or vascular smooth muscle cells, leads to the increased subendothelial oxidation of LDL and atherosclerosis, as well as to the endothelial dysfunction and microalbuminuria.

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, "neurogenic inflammation," which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic beta-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.

Human islet amyloid polypeptide (hIAPP) is co-secreted with insulin from pancreatic islet beta cells. This peptide spontaneously aggregates in the form of fibrils, and amyloid deposits are associated with dead or degenerating beta cells, a hallmark of noninsulin-dependent diabetes mellitus. We demonstrated that COS-1 cells transfected with vectors expressing hIAPP exhibited intracellular amyloid deposits that were associated with cell death (O'Brien, Butler, Kreutter, Kane, Eberhardt, Am J Pathol 1995, 147:609-616). To establish the mechanism of cell death, we transfected COS-1 cells with vectors expressing amyloidogenic hIAPP or nonamyloidogenic rat IAPP and mutant hIAPP constructs and assayed them for markers characteristic of apoptosis and necrosis by fluorescence-activated cell sorting analysis. Amyloidogenic hIAPP-transfected COS cells contained up to threefold more apoptotic cells present at 96 hours after transfection compared with the nonamyloidogenic vector controls. The hIAPP-induced apoptosis was negligible at 24 and 48 hours after transfection and was maximal at 96 hours which parallels the time course of amyloidogenesis. Immunohistochemical staining and confocal microscopy showed that hIAPP is localized with distinct clustering in the endoplasmic reticulum and Golgi apparatus with no discernable extracellular staining. These experiments provide direct evidence that intracellular hIAPP amyloid causes cell death by triggering apoptotic pathways.

We studied whether fibrils spontaneously formed by islet amyloid polypeptide (IAPP, also designated amylin) are cytotoxic to insulin producing cells by examining two different insulin producing cell lines, HIT-T15 and RINm5F. IAPP fibrils (</=10microM) added to HIT-T15 cells for one week did not diminish cell viability (tetrazolium bioreduction) or DNA synthesis (3H-thymidine incorporation) nor did it increase cell death (trypan blue staining) or degree of apoptosis (TUNEL assay), and glucose-stimulated insulin secretion and the cytosolic concentration of Ca2+ were unaffected. Similarly, control fibrils (Alzheimer's peptide, Abeta1-42, fibrils) did not reduce cellular function. In contrast, IAPP fibrils decreased cell viability (tetrazolium bioreduction) and increased number of apoptotic cells in RINm5F cells. Furthermore, hydrogen peroxide markedly impaired tetrazolium bioreduction in RINm5F cells but not in HIT-T15 cells. Glutathione reductase activity was increased by IAPP fibrils in RINm5F cells but not in HIT-T15 cells. Our data suggest a different sensitivity for the cytotoxic action of IAPP fibrils between RINm5F and HIT-T15 cells, which may be ascribed to different sensitivity to formation and action of oxygen intermediates.

After unilateral adjuvant-induced inflammation, expression of neuropeptides believed to be involved in the inflammatory response, e.g. substance P and calcitonin gene-related peptide (CGRP), is upregulated in innervating sensory neurons. Islet amyloid polypeptide (IAPP) is structurally related to CGRP and constitutively expressed in sensory CGRP-containing neurons; the role of IAPP in sensory neurons is unknown. To examine whether IAPP could play a role in inflammation, IAPP expression in L5 dorsal root ganglion (DRG) and its distribution in the dorsal horn were investigated after unilateral adjuvant-induced inflammation in the rat paw and compared with CGRP, using in situ hybridization and immunocytochemistry. At 12 h and day 3, but not day 21, the percentage of nerve cell profiles expressing IAPP and CGRP mRNA was greater in the ipsilateral L5 DRG; these changes paralleled the occurrence of edema around the tarsotibial joint and a slight limp. IAPP expression in individual nerve cell profiles was higher in the ipsilateral L5 DRG at 12 h, but not at days 3 and 21; the corresponding CGRP mRNA level was higher at days 3 and 21. At day 3, the higher expression of IAPP and CGRP on the ipsilateral side was accompanied by increased numbers of immunoreactive DRG neurons and fibers in the spinal cord dorsal horn. Largely, expression of IAPP and CGRP seems to be co-ordinately regulated by localized inflammation, although the rapid, but transient, upregulation in DRG neurons of IAPP mRNA expression and the slower, but sustained, upregulation of CGRP mRNA expression may indicate dissociated regulation of the peptides. Thus, IAPP could play a role in the initial phase of localized inflammation.

Islet amyloid polypeptide (IAPP) is structurally related to calcitonin gene-related peptide (CGRP) and has been implicated in glucose homeostasis and diabetes pathogenesis because it is expressed in insulin cells and forms amyloid in pancreatic islets from type II diabetic patients. IAPP is also constitutively co-expressed with CGRP in rat sensory neurons. Whether expression of IAPP is altered by nerve injury with or without regeneration was investigated in adult rats subjected to unilateral sciatic axotomy; IAPP and CGRP expression were determined by quantitative in situ hybridization and immunocytochemistry at days 3, 10 and 30 after axotomy. In ipsilateral L4-L5 dorsal root ganglia (DRG), the percentages of nerve cell profiles labelled for IAPP and CGRP mRNA were reduced at all time points studied. IAPP and CGRP mRNA expression were lower in nerve cell profiles in ipsilateral DRGs compared to the contralateral side after axotomy alone whereas epineurial nerve suture maintained or restored IAPP and CGRP expression. The numbers of IAPP- and CGRP-immunoreactive DRG nerve cell profiles and dorsal horn fibers were reduced on the ipsilateral side at all time points. Thus, IAPP and CGRP expression are down-regulated upon axotomy. Nerve repair maintains or restores IAPP and CGRP expression in individual neurons but does not prevent the loss of CGRP/IAPP phenotype of some of these neurons in response to axotomy.