Publications by authors named "Johan F Paulsson"

20 Publications

  • Page 1 of 1

The Design of a GLP-1/PYY Dual Acting Agonist.

Angew Chem Int Ed Engl 2021 04 1;60(15):8268-8275. Epub 2021 Mar 1.

Global Drug Discovery, Novo Nordisk Research Park, 2760, Maaloev, Denmark.

The two gut hormones GLP-1 and PYY , which are both secreted from the L-cells upon food stimuli, have a stronger inhibitory effect on food intake when they are combined, compared to their individual effects as single agonists. Although they are not homologous and share no sequence similarity, we show that a GLP-1 analogue can be designed to exhibit potent activity on both the Y and GLP-1 receptors. Dual acting hybrid analogues were realized by designing truncated and potent Y receptor PYY analogues, followed by integrating the critical residues into GLP-1. In this study, we show that one of these dual acting agonists acutely reduces food intake significantly more than the respective mono-agonist counterparts.
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http://dx.doi.org/10.1002/anie.202016464DOI Listing
April 2021

Kisspeptin-52 partially rescues the activity of the hypothalamus-pituitary-gonadal axis in underweight male rats dosed with an anti-obesity compound.

Toxicol Appl Pharmacol 2020 10 26;404:115152. Epub 2020 Jul 26.

Novo Nordisk A/S, Måløv, Denmark. Electronic address:

Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.
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http://dx.doi.org/10.1016/j.taap.2020.115152DOI Listing
October 2020

UCP1-independent glucose-lowering effect of leptin in type 1 diabetes: only in conditions of hypoleptinemia.

Am J Physiol Endocrinol Metab 2020 01 19;318(1):E72-E86. Epub 2019 Nov 19.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.
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http://dx.doi.org/10.1152/ajpendo.00253.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985793PMC
January 2020

Design of Y Receptor Selective and Proteolytically Stable PYY Analogues.

J Med Chem 2018 12 20;61(23):10519-10530. Epub 2018 Nov 20.

In recent years peptide YY (PYY) has attracted attention within the area of diabetes and obesity due to its involvement in food intake regulation and glucose homeostasis. It is well-known that PYY is rapidly cleaved by dipeptidyl peptidase-4 to the more Y receptor selective analogue PYY, which is further cleaved to the inactive analogue PYY. In order to improve the selectivity and proteolytic stability of the C-terminus, we synthesized several analogues incorporating N-methyl amino acids or β-homo amino acids and other non-natural amino acids. These were tested against all four NPY receptors, and highly potent and Y receptor selective analogues were identified by combining a tryptophan residue in position 30 with either N-methyl or β-homo arginine in position 35. We also identified an analogue with a MeGln substitution that surprisingly displayed high affinity toward all four receptors. In addition, these analogues displayed improved stability toward C-terminal proteolysis compared to native PYY.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01046DOI Listing
December 2018

Cellular clearance of circulating transthyretin decreases cell-nonautonomous proteotoxicity in .

Proc Natl Acad Sci U S A 2018 08 30;115(33):E7710-E7719. Epub 2018 Jul 30.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037;

Cell-autonomous and cell-nonautonomous mechanisms of neurodegeneration appear to occur in the proteinopathies, including Alzheimer's and Parkinson's diseases. However, how neuronal toxicity is generated from misfolding-prone proteins secreted by nonneuronal tissues and whether modulating protein aggregate levels at distal locales affects the degeneration of postmitotic neurons remains unknown. We generated and characterized animal models of the transthyretin (TTR) amyloidoses that faithfully recapitulate cell-nonautonomous neuronal proteotoxicity by expressing human TTR in the muscle. We identified sensory neurons with affected morphological and behavioral nociception-sensing impairments. Nonnative TTR oligomer load and neurotoxicity increased following inhibition of TTR degradation in distal macrophage-like nonaffected cells. Moreover, reducing TTR levels by RNAi or by kinetically stabilizing natively folded TTR pharmacologically decreased TTR aggregate load and attenuated neuronal dysfunction. These findings reveal a critical role for modulation of aggregation-prone degradation that directly affects postmitotic tissue degeneration observed in the proteinopathies.
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http://dx.doi.org/10.1073/pnas.1801117115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6099907PMC
August 2018

Elucidation of the Binding Mode of the Carboxyterminal Region of Peptide YY to the Human Y Receptor.

Mol Pharmacol 2018 04 24;93(4):323-334. Epub 2018 Jan 24.

Departments of Neuroscience (B.X., J.P., D.L.) and Cell and Molecular Biology (S.V., J.Å., H.G.-T.), Biomedical Centre, Uppsala University, Uppsala, Sweden; and Protein and Peptide Chemistry 2 (S.Ø.) and Obesity Research (J.F.P., B.S.W.), Novo Nordisk A/S, Måløv, Denmark

Understanding the agonist-receptor interactions in the neuropeptide Y (NPY)/peptide YY (PYY) signaling system is fundamental for the design of novel modulators of appetite regulation. We report here the results of a multidisciplinary approach to elucidate the binding mode of the native peptide agonist PYY to the human Y receptor, based on computational modeling, peptide chemistry and in vitro pharmacological analyses. The preserved binding orientation proposed for full-length PYY and five analogs, truncated at the amino terminus, explains our pharmacological results where truncations of the N-terminal proline helix showed little effect on peptide affinity. This was followed by receptor mutagenesis to investigate the roles of several receptor positions suggested by the modeling. As a complement, PYY-(3-36) analogs were synthesized with modifications at different positions in the common PYY/NPY C-terminal fragment (TRQRY-amide). The results were assessed and interpreted by molecular dynamics and Free Energy Perturbation (FEP) simulations of selected mutants, providing a detailed map of the interactions of the PYY/NPY C-terminal fragment with the transmembrane cavity of the Y receptor. The amidated C-terminus would be stabilized by polar interactions with Gln288 and Tyr219, while Gln130 contributes to interactions with Q in the peptide and T is close to the tip of TM7 in the receptor. This leaves the core, -helix of the peptide exposed to make potential interactions with the extracellular loops. This model agrees with most experimental data available for the Y system and can be used as a basis for optimization of Y receptor agonists.
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http://dx.doi.org/10.1124/mol.117.110627DOI Listing
April 2018

In vivo seeding and cross-seeding of localized amyloidosis: a molecular link between type 2 diabetes and Alzheimer disease.

Am J Pathol 2015 Mar 17;185(3):834-46. Epub 2015 Feb 17.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden. Electronic address:

Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aβ) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aβ in islet amyloid in type 2 diabetic patients and Aβ deposits in brains of patients with Alzheimer disease. Aβ reactivity was not detected in islet amyloid although islet β cells express AβPP and convertases necessary for Aβ production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aβ deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aβ shown here may represent a molecular link between type 2 diabetes and Alzheimer disease.
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http://dx.doi.org/10.1016/j.ajpath.2014.11.016DOI Listing
March 2015

Design and Synthesis of Peptide YY Analogues with C-terminal Backbone Amide-to-Ester Modifications.

ACS Med Chem Lett 2013 Dec 21;4(12):1228-32. Epub 2013 Oct 21.

Department of Drug Design and Pharmacology, University of Copenhagen , Universitetsparken 2, DK-2100 Copenhagen, Denmark.

Peptide YY (PYY) is a gut hormone that activates the G protein-coupled neuropeptide Y (NPY) receptors, and because of its appetite reducing actions, it is evaluated as an antiobesity drug candidate. The C-terminal tail of PYY is crucial for activation of the NPY receptors. Here, we describe the design and preparation of a series of PYY(3-36) depsipeptide analogues, in which backbone amide-to-ester modifications were systematically introduced in the C-terminal. Functional NPY receptor assays and circular dichroism revealed that the ψ(CONH) bonds at positions 30-31 and 33-34 are particularly important for receptor interaction and that the latter is implicated in Y2 receptor selectivity.
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http://dx.doi.org/10.1021/ml400335gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027376PMC
December 2013

High plasma levels of islet amyloid polypeptide in young with new-onset of type 1 diabetes mellitus.

PLoS One 2014 26;9(3):e93053. Epub 2014 Mar 26.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Aims/hypothesis: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes.

Results: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels.

Conclusions/interpretation: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3966843PMC
June 2015

A parallel semisynthetic approach for structure-activity relationship studies of peptide YY.

ChemMedChem 2013 Sep 31;8(9):1505-13, 1422. Epub 2013 Jul 31.

Department of Drug Design & Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

The gut hormone peptide YY (PYY) is postprandially secreted from enteroendocrine L cells and is involved in the regulation of energy homeostasis. The N-terminal truncated version PYY(3-36) decreases food intake and has potential as an anti-obesity agent. The anorectic effect of PYY(3-36) is mediated through Y₂ receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C-terminal tetrapeptide sequence of PYY(3-36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C-terminally modified PYY(3-36) analogues. By using an intein-based expression system, PYY(3-29) was generated as a C-terminal peptide α-thioester. Heptapeptides bearing an N-terminal cysteine and modifications at one of the four C-terminal positions were synthesized in a 96-well plate by parallel solid-phase synthesis. In the plate format, an array of [Ala30]PYY(3-36) analogues were generated by ligation, desulfurization, and subsequent solid-phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non-proteinogenic amino acids, were tested in a functional Y₂ receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y₂ receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.
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http://dx.doi.org/10.1002/cmdc.201300290DOI Listing
September 2013

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2-helix variants of PYY3-36.

J Pept Sci 2012 Sep 1;18(9):579-87. Epub 2012 Aug 1.

Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions.
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http://dx.doi.org/10.1002/psc.2436DOI Listing
September 2012

Ferret islet amyloid polypeptide (IAPP): characterization of in vitro and in vivo amyloidogenicity.

Amyloid 2011 Dec 2;18(4):222-8. Epub 2011 Nov 2.

Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.

Diabetes in the domestic ferret (Mustela putorius furo) has previously been described and the purpose of this study was to evaluate if the ferret could serve as a model for the study of β-cell degeneration associated with formation of islet amyloid. The nucleotide and amino acid sequence of ferret islet amyloid polypeptide (IAPP) 1-37 was identified and the synthesized peptide was studied with regards to in vitro amyloidogenicity and potential cellular toxicity in a comparative approach to human, cat and the nonamyloidogenic rat IAPP. Ferret IAPP forms amyloid-like fibrils, but with a longer lag phase than human and cat IAPP and the aggregation process was shown to reduce cell viability of cultured β-cells, but with less potency than these two amyloidogenic counterparts. Immunohistochemistry of ferret pancreas confirmed IAPP expression in the islets of Langerhans, but no islet amyloid was found in a very limited sample size of one diabetic and five healthy ferrets. Islet amyloid has never been described in ferrets, and it is not possible to determine if it is due to lack of studies/material or to the fact that the ferret's life span is too short to present with such pathology.
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http://dx.doi.org/10.3109/13506129.2011.627956DOI Listing
December 2011

Extraneural manifestations of prion infection in GPI-anchorless transgenic mice.

Virology 2011 Mar 11;411(1):1-8. Epub 2011 Jan 11.

Viral Immunobiology Laboratory, Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.

Earlier studies indicated that transgenic (tg) mice engineered to express prion protein (PrP) lacking the glycophosphatidylinositol (GPI⁻/⁻) membrane anchor formed abnormal proteinase-resistant prion (PrPsc) amyloid deposits in their brains and hearts when infected with the RML strain of murine scrapie. In contrast, RML scrapie infection of normal mice with a GPI-anchored PrP did not deposit amyloid with PrPsc in the brain or the heart. Here we report that scrapie-infected GPI⁻/⁻ PrP tg mice also deposit PrP and transmissible infectious material in the gut, kidneys, and islets of Langerhans. Similar to previously reported amyloid deposits in the brain and heart, amyloid deposits were found in the gut; however, no amyloid deposited in the islets. By high-resolution electron microscopy, we show PrP is located primarily in α cells and also β cells. Islets contain abundant insulin and there is no abnormality in glucose metabolism in infected GPI⁻/⁻ PrP tg mice.
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http://dx.doi.org/10.1016/j.virol.2010.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039103PMC
March 2011

Reduced IGF-1 signaling delays age-associated proteotoxicity in mice.

Cell 2009 Dec;139(6):1157-69

Howard Hughes Medical Institute, Glenn Center for Aging Research, Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

The insulin/insulin growth factor (IGF) signaling (IIS) pathway is a key regulator of aging of worms, flies, mice, and likely humans. Delayed aging by IIS reduction protects the nematode C. elegans from toxicity associated with the aggregation of the Alzheimer's disease-linked human peptide, Abeta. We reduced IGF signaling in Alzheimer's model mice and discovered that these animals are protected from Alzheimer's-like disease symptoms, including reduced behavioral impairment, neuroinflammation, and neuronal loss. This protection is correlated with the hyperaggregation of Abeta leading to tightly packed, ordered plaques, suggesting that one aspect of the protection conferred by reduced IGF signaling is the sequestration of soluble Abeta oligomers into dense aggregates of lower toxicity. These findings indicate that the IGF signaling-regulated mechanism that protects from Abeta toxicity is conserved from worms to mammals and point to the modulation of this signaling pathway as a promising strategy for the development of Alzheimer's disease therapy.
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http://dx.doi.org/10.1016/j.cell.2009.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017511PMC
December 2009

Lovastatin ameliorates alpha-synuclein accumulation and oxidation in transgenic mouse models of alpha-synucleinopathies.

Exp Neurol 2010 Feb 26;221(2):267-74. Epub 2009 Nov 26.

Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0624, USA.

Alpha-synuclein (alpha-syn) aggregation is a neuropathological hallmark of many diseases including Dementia with Lewy Bodies (DLB) and Parkinson's Disease (PD), collectively termed the alpha-synucleinopathies. The mechanisms underlying alpha-syn aggregation remain elusive though emerging science has hypothesized that the interaction between cholesterol and alpha-syn may play a role. Cholesterol has been linked to alpha-synucleinopathies by recent work suggesting cholesterol metabolites appear to accelerate alpha-syn fibrillization. Consistent with these findings, cholesterol-lowering agents have been demonstrated to reduce alpha-syn accumulation and the associated neuronal pathology in vitro. In this context, this study sought to investigate the in vivo effects of the cholesterol synthesis inhibitor lovastatin on alpha-syn aggregation in two different transgenic (Tg) mouse models that neuronally overexpress human alpha-syn. Lovastatin-treated mice displayed significantly reduced plasma cholesterol levels and levels of oxidized cholesterol metabolites in the brain in comparison to saline-treated controls. Immunohistochemical analysis demonstrated a significant reduction of neuronal alpha-syn aggregates and alpha-syn immunoreactive neuropil in the temporal cortex of lovastatin-treated Tg mice in comparison to saline-treated alpha-syn Tg controls. Consistently, immunoblot analysis of mouse brain homogenates showed a reduction in levels of total and oxidized alpha-syn in lovastatin-treated alpha-syn Tg mice in comparison to saline-treated alpha-syn Tg controls. The reduced alpha-syn accumulation in lovastatin-treated mice was associated with abrogation of neuronal pathology. The results from this study demonstrate that lovastatin administration can reduce alpha-syn aggregation and associated neuropathology and support the possibility that treatment with cholesterol-lowering agents may be beneficial for patients with PD and/or DLB.
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http://dx.doi.org/10.1016/j.expneurol.2009.11.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812599PMC
February 2010

Site-specific modification of Alzheimer's peptides by cholesterol oxidation products enhances aggregation energetics and neurotoxicity.

Proc Natl Acad Sci U S A 2009 Nov 19;106(44):18563-8. Epub 2009 Oct 19.

Departments of Chemistry and Molecular and Experimental Medicine, Scripps Research Institute and Skaggs Institute for Chemical Biology, La Jolla, CA 92037, USA.

Accumulation of amyloid beta-peptide (Abeta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between Abeta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase Abeta amyloidogenicity. Here, we synthesized Abeta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified Abeta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from Abeta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified Abeta under identical conditions and at the same concentration. Our results show that Abeta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Abeta.
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http://dx.doi.org/10.1073/pnas.0804758106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2774025PMC
November 2009

Differential lipid profile and hormonal response in type 2 diabetes by exogenous insulin aspart versus the insulin secretagogue repaglinide, at the same glycemic control.

Acta Diabetol 2009 Mar 6;46(1):35-42. Epub 2008 Sep 6.

Department of Clinical and Experimental Medicine, Division of Cell Biology, Faculty of Health Science, 581 85 Linköping, Sweden.

Our aim was to study, at the same glycemic control, how treatment with either the insulin secretagogue repaglinide or exogenous insulin aspart affects endogenous insulin secretion, plasma insulin and IAPP (islet amyloid polypeptide) levels, GH-IGF (growth hormone-insulin-like growth factor) axis and plasma lipoprotein concentrations in patients with type 2 diabetes. Five patients, age 65.0+/-4.1 years (mean+/-SE), body weight 82.5+/-5.0 kg, BMI (body mass index) 27.7+/-1.5 kg/m(2) were treated for 10 weeks with repaglinide or insulin aspart in a randomized, cross-over study. At the end of each treatment a 24-h metabolic profile was performed. Blood glucose, C-peptide, free human insulin, free total (human and analogue) insulin, proinsulin, IAPP, IGF-I, IGFBP-1 (IGF binding protein-1), GHBP (growth hormone binding protein) and plasma lipoprotein concentrations were measured. Similar 24-h blood glucose profiles were obtained with repaglinide and insulin aspart treatment. During the repaglinide treatment, the meal related peaks of C-peptide and free human insulin were about twofold higher than during treatment with insulin aspart. Proinsulin, GHBP were higher and IAPP levels tended to be higher during repaglinide compared to insulin aspart. Postprandial plasma total cholesterol, triglycerides and apolipoprotein B concentrations were higher on repaglinide than on insulin aspart treatment. Our results show that, at the same glycemic control, treatment with exogenous insulin aspart in comparison with the insulin secretagogue repaglinide result in a lower endogenous insulin secretion, and a tendency towards a less atherogenic postprandial lipid profile.
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http://dx.doi.org/10.1007/s00592-008-0055-6DOI Listing
March 2009

Real-time monitoring of apoptosis by caspase-3-like protease induced FRET reduction triggered by amyloid aggregation.

Exp Diabetes Res 2008 ;2008:865850

Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 microM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.
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http://dx.doi.org/10.1155/2008/865850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426809PMC
July 2008

Amyloid deposition in transplanted human pancreatic islets: a conceivable cause of their long-term failure.

Exp Diabetes Res 2008 5;2008:562985. Epub 2009 Mar 5.

Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the beta-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.
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http://dx.doi.org/10.1155/2008/562985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652583PMC
March 2009

Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation.

Diabetes 2005 Jul;54(7):2117-25

Deparment of Biomedicine and Surgery, Division of Cell Biology, Linköping University, SE 581 85 Linköping, Sweden.

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.
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http://dx.doi.org/10.2337/diabetes.54.7.2117DOI Listing
July 2005