Nanodroplet Oligomers (NanDOs) of Aß40.

Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.

A versatile method for producing labeled or unlabeled Aß55, Aß40, and other ß-amyloid family peptides.

We present a straightforward, versatile method for expressing and purifying ß-amyloid (Aß40) and transmembrane peptides derived from ß-amyloid precursor protein (Aß55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aß peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aß40 and Aß55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aß55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aß55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option.

ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

ß-Amyloid aggregation and heterogeneous nucleation.

In this article, we consider the role of heterogeneous nucleation in ß-amyloid aggregation. Heterogeneous nucleation is more common and occurs at lower levels of supersaturation than homogeneous nucleation. The nucleation period is also the stage at which most of the polymorphism of amyloids arises, this being one of the defining features of amyloids. We focus on several well-known heterogeneous nucleators of ß-amyloid, including lipid surfaces, especially those enriched in gangliosides and cholesterol, and divalent metal ions. These two broad classes of nucleators affect ß-amyloid particularly in light of the amphiphilicity of these peptides: the N-terminal region, which is largely polar and charged, contains the metal binding site, whereas the C-terminal region is aliphatic and is important in lipid binding. Notably, these two classes of nucleators can interact cooperatively, aggregation begetting greater aggregation.

Pancreatic ß-Cell Adaptive Plasticity in Obesity Increases Insulin Production but Adversely Affects Secretory Function.

Pancreatic ß-cells normally produce adequate insulin to control glucose homeostasis, but in obesity-related diabetes, there is a presumed deficit in insulin production and secretory capacity. In this study, insulin production was assessed directly in obese diabetic mouse models, and proinsulin biosynthesis was found to be contrastingly increased, coupled with a significant expansion of the rough endoplasmic reticulum (without endoplasmic reticulum stress) and Golgi apparatus, increased vesicular trafficking, and a depletion of mature ß-granules. As such, ß-cells have a remarkable capacity to produce substantial quantities of insulin in obesity, which are then made available for immediate secretion to meet increased metabolic demand, but this comes at the price of insulin secretory dysfunction. Notwithstanding, it can be restored. Upon exposing isolated pancreatic islets of obese mice to normal glucose concentrations, ß-cells revert back to their typical morphology with restoration of regulated insulin secretion. These data demonstrate an unrealized dynamic adaptive plasticity of pancreatic ß-cells and underscore the rationale for transient ß-cell rest as a treatment strategy for obesity-linked diabetes.

Evidence against the involvement of oxidative stress in fatty acid inhibition of insulin secretion.

Prolonged exposure to elevated levels of fatty acids adversely affects pancreatic beta-cell function. Here we investigated 1) whether ceramide synthesis, which we reported to mediate fatty acid inhibition of insulin gene expression, also inhibits insulin secretion and 2) whether fatty acid inhibition of insulin secretion involves the generation of reactive oxygen species (ROS), nitric oxide (NO), or prostaglandin E(2) (PGE(2)). A 72-h culture of islets in the presence of palmitate or oleate resulted in a marked decrease in glucose-induced insulin release assessed in 1-h static incubations. This effect was reproduced by exogenous diacylglycerol, but not by a cell-permeable analog of ceramide. Culture in the presence of fatty acids was not associated with an increase in intracellular peroxide or NO levels, neither was insulin secretion restored by antioxidants or an inhibitor of NO production. Exposure to fatty acids led to an increase in PGE(2) release, but an inhibitor of cyclooxygenase 2 was unable to prevent fatty acid inhibition of insulin secretion. These results indicate that fatty acid inhibition of insulin secretion 1) is not mediated by de novo ceramide synthesis, ROS, NO, or PGE(2), and 2) is likely to be caused by the generation of signals or metabolites downstream of diacylglycerol.

Formation of a human ß-cell population within pancreatic islets is set early in life.

CONTEXT: Insulin resistance can be compensated by increased functional pancreatic ß-cell mass; otherwise, diabetes ensues. Such compensation depends not only on environmental and genetic factors but also on the baseline ß-cell mass from which the expansion originates. OBJECTIVE: Little is known about assembly of a baseline ß-cell mass in humans. Here, we examined formation of ß-cell populations relative to other pancreatic islet cell types and associated neurons throughout the normal human lifespan. DESIGN AND METHODS: Human pancreatic sections derived from normal cadavers aged 24 wk premature to 72 yr were examined by immunofluorescence. Insulin, glucagon, and somatostatin were used as markers for ß-, α-, and δ-cells, respectively. Cytokeratin-19 marked ductal cells, Ki67 cell proliferation, and Tuj1 (neuronal class III ß-tubulin) marked neurons. RESULTS: Most ß-cell neogenesis was observed preterm with a burst of ß-cell proliferation peaking within the first 2 yr of life. Thereafter, little indication of ß-cell growth was observed. Postnatal proliferation of α- and δ-cells was rarely seen, but a wave of ductal cell proliferation was found mostly associated with exocrine cell expansion. The ß-cell to α-cell ratio doubled neonatally, reflecting increased growth of ß-cells, but during childhood, there was a 7-fold change in the ß-cell to δ-cell ratio, reflecting an additional loss of δ-cells. A close association of neurons to pancreatic islets was noted developmentally and retained throughout adulthood. Negligible neuronal association to exocrine pancreas was observed. CONCLUSION: Human baseline ß-cell population and appropriate association with other islet cell types is established before 5 yr of age.

Palmitate inhibition of insulin gene expression is mediated at the transcriptional level via ceramide synthesis.

Chronic exposure to elevated levels of fatty acids impairs pancreatic beta cell function, a phenomenon thought to contribute to the progressive deterioration of insulin secretion in type 2 diabetes. We have previously demonstrated that prolonged exposure of isolated islets to elevated levels of palmitate inhibits preproinsulin mRNA levels in the presence of high glucose concentrations. However, whether this occurs via transcriptional or post-transcriptional mechanisms has not been determined. In addition, the nature of the lipid metabolites involved in palmitate inhibition of insulin gene expression is unknown. In this study, we show that palmitate decreases glucose-stimulated preproinsulin mRNA levels in isolated rat islets, an effect that is not mediated by changes in preproinsulin mRNA stability, but is associated with inhibition of glucose-stimulated insulin promoter activity. Prolonged culture of isolated islets with palmitate is associated with increased levels of intracellular ceramide. Palmitate-induced ceramide generation is prevented by inhibitors of de novo ceramide synthesis. Further, exogenous ceramide inhibits insulin mRNA levels, whereas blockade of de novo ceramide synthesis prevents palmitate inhibition of insulin gene expression. We conclude that prolonged exposure to elevated levels of palmitate affects glucose-stimulated insulin gene expression via transcriptional mechanisms and ceramide synthesis.