Human IAPP is a contributor to painful diabetic peripheral neuropathy.

Peripheral neuropathy is a frequent complication of type 2 diabetes mellitus (T2DM). We investigated whether human islet amyloid polypeptide (hIAPP), which forms pathogenic aggregates that damage pancreatic islet ß cells in T2DM, is involved in T2DM-associated peripheral neuropathy. In vitro, hIAPP incubation with sensory neurons reduced neurite outgrowth and increased levels of mitochondrial reactive oxygen species. hIAPP-transgenic mice, which have elevated plasma hIAPP levels without hyperglycemia, developed peripheral neuropathy as evidenced by pain-associated behavior and reduced intraepidermal nerve fiber (IENF) density. Similarly, hIAPP Ob/Ob mice, which have hyperglycemia in combination with elevated plasma hIAPP levels, had signs of neuropathy, although more aggravated. In wild-type mice, intraplantar and intravenous hIAPP injections induced long-lasting allodynia and decreased IENF density. Non-aggregating murine IAPP, mutated hIAPP (pramlintide), or hIAPP with pharmacologically inhibited aggregation did not induce these effects. T2DM patients had reduced IENF density and more hIAPP oligomers in the skin compared with non-T2DM controls. Thus, we provide evidence that hIAPP aggregation is neurotoxic and mediates peripheral neuropathy in mice. The increased abundance of hIAPP aggregates in the skin of T2DM patients supports the notion that hIAPP is a potential contributor to T2DM neuropathy in humans.

Amyloid Proteins and Peripheral Neuropathy.

Painful peripheral neuropathy affects millions of people worldwide. Peripheral neuropathy develops in patients with various diseases, including rare familial or acquired amyloid polyneuropathies, as well as some common diseases, including type 2 diabetes mellitus and several chronic inflammatory diseases. Intriguingly, these diseases share a histopathological feature-deposits of amyloid-forming proteins in tissues. Amyloid-forming proteins may cause tissue dysregulation and damage, including damage to nerves, and may be a common cause of neuropathy in these, and potentially other, diseases. Here, we will discuss how amyloid proteins contribute to peripheral neuropathy by reviewing the current understanding of pathogenic mechanisms in known inherited and acquired (usually rare) amyloid neuropathies. In addition, we will discuss the potential role of amyloid proteins in peripheral neuropathy in some common diseases, which are not (yet) considered as amyloid neuropathies. We conclude that there are many similarities in the molecular and cell biological defects caused by aggregation of the various amyloid proteins in these different diseases and propose a common pathogenic pathway for "peripheral amyloid neuropathies".

LRIG1 negatively regulates RET mutants and is downregulated in thyroid cancer.

Papillary thyroid carcinoma (PTC) and medullary thyroid carcinoma (MTC) are characterized by genomic rearrangements and point mutations in the proto-oncogene RET. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a suppressor of various receptor tyrosine kinases, including RET. LRIG1 expression levels are associated with patient survival in many cancer types. In the present study, we investigated whether the oncogenic RET mutants RET2A (C634R) and RET2B (M918T) were regulated by LRIG1, and the possible effects of LRIG1 expression in thyroid cancer were investigated in three different clinical cohorts and in a RET2B-driven mouse model of MTC. LRIG1 was shown to physically interact with both RET2A and RET2B and to restrict their ligand-independent activation. LRIG1 mRNA levels were downregulated in PTC and MTC compared to normal thyroid gland tissue. There was no apparent association between LRIG1 RNA or protein expression levels and patient survival in the studied cohorts. The transgenic RET2B mice developed pre-cancerous medullary thyroid lesions at a high frequency (36%); however, no overt cancers were observed. There was no significant difference in the incidence of pre-cancerous lesions between Lrig1 wild-type and Lrig1-deficient RET2B mice. In conclusion, the findings that LRIG1 is a negative regulator of RET2A and RET2B and is also downregulated in PTC and MTC may suggest that LRIG1 functions as a thyroid tumor suppressor.

Variable clinical expression in patients with a germline MEN1 disease gene mutation: clues to a genotype-phenotype correlation.

Multiple endocrine neoplasia type 1 is an inherited endocrine tumor syndrome, predominantly characterized by tumors of the parathyroid glands, gastroenteropancreatic tumors, pituitary adenomas, adrenal adenomas, and neuroendocrine tumors of the thymus, lungs or stomach. Multiple endocrine neoplasia type 1 is caused by germline mutations of the multiple endocrine neoplasia type 1 tumor suppressor gene. The initial germline mutation, loss of the wild-type allele, and modifying genetic and possibly epigenetic and environmental events eventually result in multiple endocrine neoplasia type 1 tumors. Our understanding of the function of the multiple endocrine neoplasia type 1 gene product, menin, has increased significantly over the years. However, to date, no clear genotype-phenotype correlation has been established. In this review we discuss reports on exceptional clinical presentations of multiple endocrine neoplasia type 1, which may provide more insight into the pathogenesis of this disorder and offer clues for a possible genotype-phenotype correlation.

Recent results of basic and clinical research in MEN1: opportunities to improve early detection and treatment.

Due to the variable expression of multiple endocrine neoplasia type 1 (MEN1), it is difficult to predict the course of the disease. However, knowledge about the normal function of the MEN1 gene product, together with the effects of cellular derangement by subsequent genetic events, has increased considerably. At first, the possible existence of a genotype-phenotype correlation is discussed. Thus, mild- and late-onset phenotypes may be distinguished from more malignant phenotypes depending on the character of the primary MEN1 disease gene mutation. Subsequently, tumor-promoting factors such as gender, additional genetic mutations and ecogenetic factors may contribute to the course of the disease. New developments in management are based on the knowledge and experience of the multidisciplinary teams involved. Finally, the metabolic effects of MEN1 mutations in aged patients are discussed. Early identification of predisposition to the disease, together with knowledge about the natural history of specific mutations, risks of additional mutations and periodic clinical monitoring, allow early treatment and may improve life expectancy and quality of life.

Variable clinical expression in patients with a germline MEN1 disease gene mutation: clues to a genotype-phenotype correlation

Multiple endocrine neoplasia type 1 is an inherited endocrine tumor syndrome, predominantly characterized by tumors of the parathyroid glands, gastroenteropancreatic tumors, pituitary adenomas, adrenal adenomas, and neuroendocrine tumors of the thymus, lungs or stomach. Multiple endocrine neoplasia type 1 is caused by germline mutations of the multiple endocrine neoplasia type 1 tumor suppressor gene. The initial germline mutation, loss of the wild-type allele, and modifying genetic and possibly epigenetic and environmental events eventually result in multiple endocrine neoplasia type 1 tumors. Our understanding of the function of the multiple endocrine neoplasia type 1 gene product, menin, has increased significantly over the years. However, to date, no clear genotype-phenotype correlation has been established. In this review we discuss reports on exceptional clinical presentations of multiple endocrine neoplasia type 1, which may provide more insight into the pathogenesis of this disorder and offer clues for a possible genotype-phenotype correlation.

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet beta-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP(1-19), plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP(1-19) in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP(1-19) is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP(1-19) is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.

The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and beta-cell death. Here, we used variants of the hIAPP(1-19) fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, (2)H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP-membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone.

Impaired processing of human pro-islet amyloid polypeptide is not a causative factor for fibril formation or membrane damage in vitro.

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). hIAPP is synthesized by islet beta-cells initially as a preprohormone, processing of which occurs in several steps. It has been suggested that in DM2 this processing is defective and that aggregation of the processing intermediates prohIAPP and prohIAPP(1-48) may represent the initial step in formation of islet amyloid. Here we investigate this possibility by analyzing the aggregation, the structure, and the membrane interaction of mature hIAPP and its precursors, prohIAPP and prohIAPP(1-48), in vitro. Our data reveal that both precursors form amyloid fibrils in solution but not in the presence of membranes. This inhibition is in contrast to the catalyzing effect of membranes on fibril formation of mature hIAPP. Importantly, in the presence of membranes, both precursors are able to inhibit fibrillogenesis of mature hIAPP. These differences in behavior between mature hIAPP and its precursors are most likely related to differences in their mode of membrane insertion. Both precursors insert efficiently and adopt an alpha-helical structure even with a high lipid/peptide ratio, while mature hIAPP rapidly adopts a beta-sheet conformation. Furthermore, while mature hIAPP affects the barrier properties of lipid vesicles, neither of the precursors is able to induce membrane leakage. Our study suggests that the hIAPP precursors prohIAPP and prohIAPP(1-48) do not serve as amyloid initiators but rather prevent aggregation and membrane damage of mature hIAPP in early stages of its biosynthesis and intracellular transport.

A combinatorial approach for the design of complementarity-determining region-derived peptidomimetics with in vitro anti-tumoral activity.

The great success of therapeutic monoclonal antibodies has fueled research toward mimicry of their binding sites and the development of new strategies for peptide-based mimetics production. Here, we describe a new combinatorial approach for the production of peptidomimetics using the complementarity-determining regions (CDRs) from gastrin17 (pyroEGPWLEEEEEAYGWMDF-NH(2)) antibodies as starting material for cyclic peptide synthesis in a microarray format. Gastrin17 is a trophic factor in gastrointestinal tumors, including pancreatic cancer, which makes it an interesting target for development of therapeutic antibodies. Screening of microarrays containing bicyclic peptidomimetics identified a high number of gastrin binders. A strong correlation was observed between gastrin binding and overall charge of the peptidomimetic. Most of the best gastrin binders proceeded from CDRs containing charged residues. In contrast, CDRs from high affinity antibodies containing mostly neutral residues failed to yield good binders. Our experiments revealed essential differences in the mode of antigen binding between CDR-derived peptidomimetics (K(d) values in micromolar range) and the parental monoclonal antibodies (K(d) values in nanomolar range). However, chemically derived peptidomimetics from gastrin binders were very effective in gastrin neutralization studies using cell-based assays, yielding a neutralizing activity in pancreatic tumoral cell lines comparable with that of gastrin-specific monoclonal antibodies. These data support the use of combinatorial CDR-peptide microarrays as a tool for the development of a new generation of chemically synthesized cyclic peptidomimetics with functional activity.

Tissue selectivity in multiple endocrine neoplasia type 1-associated tumorigenesis.

The phenotype of the multiple endocrine neoplasia type 1 (MEN1) syndrome cannot be explained solely by the expression pattern of the predisposing gene MEN1 and its encoded protein, menin. This review addresses putative factors determining MEN1-associated tissue-selective tumorigenesis. Menin's interaction with mixed-lineage leukemia protein-containing histone methyl transferase (MLL-HMT) complex mediates tissue-selective tumor-suppressing and tumor-promoting effects of menin, and as such could be decisive for the predisposition of individual tissues to MEN1-associated tumorigenesis. In tissues in which menin acts as a tumor suppressor, tumorigenesis could depend on the inability of such tissues to adequately compensate for MEN1 gene loss, whereas the variable clinical presentation of MEN1 in individual patients could be a reflection of additional epigenetic factors and/or modifier genes. Further research on this topic may facilitate development of novel therapeutic strategies that could prevent or delay the onset of MEN1-associated tumorigenesis.

The multiple endocrine neoplasia type 1 (MEN1) tumor suppressor regulates peroxisome proliferator-activated receptor gamma-dependent adipocyte differentiation.

Menin, the product of the MEN1 (multiple endocrine neoplasia type 1) tumor suppressor gene, is involved in activation of gene transcription as part of an MLL1 (mixed-lineage leukemia 1)/MLL2 (KMT2A/B)-containing protein complex which harbors methyltransferase activity for lysine 4 of histone H3 (H3K4). As MEN1 patients frequently develop lipomas and peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in several MEN1-related tumor types, we investigated regulation of PPARgamma activity by menin. We found that menin is required for adipocyte differentiation of murine 3T3-L1 cells and PPARgamma-expressing mouse embryonic fibroblasts. Menin augments PPARgamma target gene expression through recruitment of H3K4 methyltransferase activity. Menin interacts directly with the activation function 2 transcription activation domain of PPARgamma in a ligand-independent fashion. Ligand-dependent coactivation, however, is dependent on the LXXLL motif of menin and the intact helix 12 of PPARgamma. We propose that menin is an important factor in PPARgamma-mediated adipogenesis and that loss of PPARgamma function may contribute to lipoma development in MEN1 patients.

P18 is a tumor suppressor gene involved in human medullary thyroid carcinoma and pheochromocytoma development.

In multiple endocrine neoplasia syndrome Type 2 (MEN2), medullary thyroid carcinoma (MTC) and pheochromocytoma (PC) are associated with hereditary activating germ-line mutations in the RET proto-oncogene. Also in a large percentage of sporadic MTCs and PCs, somatic RET mutations appear to be involved in tumor formation. In one single MEN2 family an extensive variety in disease expression may be observed, indicating that additional genetic events are responsible for progression of the disease towards a more aggressive phenotype. However, these additional mutations in both hereditary and sporadic MTC and PC development are largely unknown. Here, we show for the first time the presence of somatic mutations in the cell cycle regulator P18 in human RET-associated MTCs and PCs. Each of these mutations causes an amino acid substitution in the cyclin dependent kinase-interacting region of P18(INK4C). Since these mutations partly inhibited P18(INK4C) function and reduced its stability, our findings implicate P18 as a tumor suppressor gene involved in human MTC and PC development.

Multiple endocrine neoplasia type 2.

Multiple endocrine neoplasia type 2 (MEN 2) is an autosomal dominantly inherited tumor syndrome subclassified into three distinct syndromes: MEN 2A, MEN 2B and familial medullary thyroid carcinoma. In MEN 2 families, medullary thyroid carcinoma, pheochromocytomas and parathyroid adenomas occur with a variable frequency, also depending on the specific genetic defect involved. In 1993, the responsible MEN2 gene was identified. The genetic defect in these disorders involves the RET proto-oncogene on chromosome 10. The germline RET mutations result in a gain-of-function of the RET protein. Extensive studies on large families revealed that there is a strong genotype-phenotype correlation. In this review, guidelines for early diagnosis, including MEN2 gene mutation analysis, and treatment, including preventive surgery, periodic and clinical monitoring, have been formulated, enabling improvement of life expectancy and quality of life. Identification of the RET protein has also provided new insights into its function, and the specific pathways it effects involved in cell proliferation, migration, differentiation and survival. In the near future, identification of biological tumor markers will enable target-directed intervention and may prevent and/or delay progression of both primary and residual tumor growth.

Multiple endocrine neoplasia type 1.

Multiple endocrine neoplasia type 1 (MEN 1) is an autosomal-dominant inherited tumor syndrome characterized by hyperplasia and/or tumors in the parathyroid glands, the pancreatic islets, the anterior pituitary and adrenal glands, as well as neuroendocrine tumors in the thymus, lungs and stomach, and tumors in nonendocrine tissues. In 1997, the responsible MEN1 gene was identified as a tumor-suppressor gene and its product was named menin. In this review, guidelines for early diagnosis, including MEN1 gene mutation analysis, and treatment, including periodic clinical monitoring, have been formulated, enabling improvement of life expectancy and quality of life. Identification of menin-interacting proteins has provided new insights into the function of menin, notably involving regulation of gene transcription related to proliferation and apoptosis, genome stability and DNA repair, and endocrine/metabolic homeostasis. In the near future, target-directed intervention may prevent or delay the onset of MEN 1-related tumors.

Recent insights in islet amyloid polypeptide-induced membrane disruption and its role in beta-cell death in type 2 diabetes mellitus.

The presence of fibrillar protein deposits (amyloid) of human islet amyloid polypeptide (hIAPP) in the pancreatic islets of Langerhans is thought to be related to death of the insulin-producing islet beta-cells in type 2 diabetes mellitus (DM2). The mechanism of hIAPP-induced beta-cell death is not understood. However, there is growing evidence that hIAPP-induced disruption of beta-cell membranes is the cause of hIAPP cytotoxicity. Amyloid cytotoxicity by membrane damage has not only been suggested for hIAPP, but also for peptides and proteins related to other misfolding diseases, like Alzheimer's disease, Parkinson's disease, and prion diseases. Here we review the interaction of hIAPP with membranes, and discuss recent progress in the field, with a focus on hIAPP structure and on the proposed mechanisms of hIAPP-induced membrane damage in relation to beta-cell death in DM2.

Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane.

Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet beta cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this beta cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins.

Synergistic effect of oncogenic RET and loss of p18 on medullary thyroid carcinoma development.

Activating mutations in the RET proto-oncogene are associated with both familial and sporadic medullary thyroid carcinoma (MTC) development; however, the genetic mechanisms underlying MTC tumorigenesis remain largely unknown. Recently, we have identified somatic inactivating mutations in the cell cycle inhibitor gene P18 in human MTC, which coincided with activating RET mutations, suggesting a role for loss of P18 in combination with oncogenic RET in the multistep process of MTC development. Therefore, we crossed transgenic mice expressing oncogenic RET (RET2B) with mice lacking p18 (and p27, another cell cycle inhibitor) and monitored MTC development. RET2B;p18(+/-) mice and RET2B;p18(-/-) mice developed MTC with a highly increased incidence compared with their corresponding single mutant littermates. In addition, expression of oncogenic RET causes an earlier age of onset and larger MTCs in p18(-/-);p27(+/-) mice. In a subset of MTCs of RET2B;p18(+/-)(;p27(+/-)) mice, p18(Ink4c) expression was completely lost. This loss of p18(Ink4c) expression correlated with higher proliferation rates as well as with larger MTCs, indicating that loss of p18 in combination with oncogenic RET not only increases the risk for MTC development but also enhances MTC progression. Our data strongly indicate that oncogenic RET and loss of p18 cooperate in the multistep tumorigenesis of MTC.

A novel RET kinase-beta-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma.

The RET receptor tyrosine kinase has essential roles in cell survival, differentiation, and proliferation. Oncogenic activation of RET causes the cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) and is a frequent event in sporadic thyroid carcinomas. However, the molecular mechanisms underlying RET's potent transforming and mitogenic signals are still not clear. Here, we show that nuclear localization of beta-catenin is frequent in both thyroid tumors and their metastases from MEN 2 patients, suggesting a novel mechanism of RET-mediated function through the beta-catenin signaling pathway. We show that RET binds to, and tyrosine phosphorylates, beta-catenin and show that the interaction between RET and beta-catenin can be direct and independent of cytoplasmic kinases, such as SRC. As a result of RET-mediated tyrosine phosphorylation, beta-catenin escapes cytosolic down-regulation by the adenomatous polyposis coli/Axin/glycogen synthase kinase-3 complex and accumulates in the nucleus, where it can stimulate beta-catenin-specific transcriptional programs in a RET-dependent fashion. We show that down-regulation of beta-catenin activity decreases RET-mediated cell proliferation, colony formation, and tumor growth in nude mice. Together, our data show that a beta-catenin-RET kinase pathway is a critical contributor to the development and metastasis of human thyroid carcinoma.