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      Expression of wild‐type and mutant S20G hIAPP in physiologic knock‐in mouse models fails to induce islet amyloid formation, but induces mild glucose intolerance

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          Abstract

          Aims/Introduction:  Human islet polypeptide S20G mutation (hIAPP S20G) is associated with earlier onset type 2 diabetes and increased amyloidogenicity and cytotoxicity in vitro vs wild‐type hIAPP (hIAPP WT), suggesting that amyloidogenesis may be pathogenic for type 2 diabetes. We compared the contributions of hIAPP S20G and hIAPP WT toward intra islet amyloid formation and development of type 2 diabetes in a unique physiologic knock‐in mouse model.

          Materials and Methods:  We replaced the mouse IAPP gene (M allele) with hIAPP WT (W allele) and hIAPP S20G (G allele) via homologous recombination and backbred transgenic mice against C57Bl/6 strain 5 generations to minimize genetic variation. Mice (3 month old) were maintained on control (CD) or high fat diet (HFD) for 15 months and studied at 3 month intervals by oral glucose tolerance testing (OGTT) and pancreas histology to assess glucose homeostastis, amyloidogeneisis, islet mass, β cell replication, and apoptosis.

          Results:  IAPP blood levels were indistinguishable in all mice. WW and GW mice maintained on both diets lacked intraislet amyloid at all ages. On both diets relative to MM controls WW and GW mice exhibit glucose intolerance ( P < 0.008) with no differences in insulin secretion. However, GW mice secreted significantly more insulin ( P < 0.03 that WW mice on both diets throughout the study. By 12 months on the high fat diet all mice increased their β cell mass about 3‐fold and were indistinguishable.

          Conclusions:  Physiologic expression of hIAPP WT and hIAPP S20G in C57Bl/6 mice produces mild glucose intolerance with inappropriately normal insulin secretion that is independent of intraislet amyloid formation. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2011.00166.x, 2011)

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          Most cited references32

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          Nuclear translocation of caspase-3 is dependent on its proteolytic activation and recognition of a substrate-like protein(s).

          Caspase-3 is thought to play an important role(s) in the nuclear morphological changes that occur in apoptotic cells and many nuclear substrates for caspase-3 have been identified despite the cytoplasmic localization of procaspase-3. Therefore, whether activated caspase-3 is localized in the nuclei and how active caspase-3 has access to its nuclear targets are important and unresolved questions. Here we confirmed nuclear localizations for both caspase-3-p17 and caspase-3-p12 subunits of active caspase in apoptotic cells using subcellular fractionation analysis. We also prepared polyclonal and monoclonal antibodies specific for active caspase-3 to define the subcellular localization of active caspase-3. Immunocytochemical observations using anti-active caspase-3 antibodies showed nuclear accumulation of active caspase-3 during apoptosis. In addition, caspase-3, but not caspase-7, translocated from the cytoplasm into the nucleus after induction of apoptosis. Mutations at the cleavage site between the p17 and p12 subunits and the substrate recognition site for the P3 amino acid of the DXXD substrate cleavage motif inhibited nuclear translocation of caspase-3, indicating that nuclear transport of active caspase-3 required proteolytic activation and substrate recognition. These results suggest that active caspase-3 is translocated in association with a substrate-like protein(s) from the cytoplasm into the nucleus during progression through apoptosis.
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            Increased beta-cell apoptosis prevents adaptive increase in beta-cell mass in mouse model of type 2 diabetes: evidence for role of islet amyloid formation rather than direct action of amyloid.

            Nondiabetic obese humans adapt to insulin resistance by increasing beta-cell mass. In contrast, obese humans with type 2 diabetes have an approximately 60% deficit in beta-cell mass. Recent studies in rodents reveal that beta-cell mass is regulated, increasing in response to insulin resistance through increased beta-cell supply (islet neogenesis and beta-cell replication) and/or decreased beta-cell loss (beta-cell apoptosis). Prospective studies of islet turnover are not possible in humans. In an attempt to establish the mechanism for the deficit in beta-cell mass in type 2 diabetes, we used an obese versus lean murine transgenic model for human islet amyloid polypeptide (IAPP) that develops islet pathology comparable to that in humans with type 2 diabetes. By 40 weeks of age, obese nontransgenic mice did not develop diabetes and adapted to insulin resistance by a 9-fold increase (P < 0.001) in beta-cell mass accomplished by a 1.7-fold increase in islet neogenesis (P < 0.05) and a 5-fold increase in beta-cell replication per islet (P < 0.001). Obese transgenic mice developed midlife diabetes with islet amyloid and an 80% (P < 0.001) deficit in beta-cell mass that was due to failure to adaptively increase beta-cell mass. The mechanism subserving this failed expansion was a 10-fold increase in beta-cell apoptosis (P < 0.001). There was no relationship between the extent of islet amyloid or the blood glucose concentration and the frequency of beta-cell apoptosis. However, the frequency of beta-cell apoptosis was related to the rate of increase of islet amyloid. These prospective studies suggest that the formation of islet amyloid rather than the islet amyloid per se is related to increased beta-cell apoptosis in this murine model of type 2 diabetes. This finding is consistent with the hypothesis that soluble IAPP oligomers but not islet amyloid are responsible for increased beta-cell apoptosis. The current studies also support the concept that replicating beta-cells are more vulnerable to apoptosis, possibly accounting for the failure of beta-cell mass to expand appropriately in response to obesity in type 2 diabetes.
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              Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation.

              Islet amyloid polypeptide (IAPP), a putative polypeptide hormone, is a product of pancreatic beta-cells and the major constituent of the amyloid deposits seen mainly in islets of type 2 diabetic humans and diabetic cats. The connection between IAPP amyloid formation and diabetes is unknown, but a limited segment of the IAPP molecule, positions 20-29, seems responsible for the aggregation to fibrils. Differences in the amino acid sequence of this region probably determine whether or not islet amyloid can develop in a particular species. Amyloid fibril formation can be mimicked in vitro with the aid of synthetic peptides. With this technique we show that peptides corresponding to IAPP positions 20-29 of human and cat, species that develop IAPP-derived islet amyloid, form amyloid-like fibrils in vitro. The corresponding IAPP segment from three rodent species that do not develop IAPP-derived amyloid did not give rise to fibrils. Substitution of the human IAPP-(20-29) decapeptide with one or two amino acid residues from species without islet amyloid generally reduced the capacity to form fibrils. We conclude that the sequence Ala-Ile-Leu-Ser-Ser, corresponding to positions 25-29 of human IAPP, is strongly amyloidogenic and that a proline-for-serine substitution in position 28, as in several rodents, almost completely inhibits formation of amyloid fibrils.
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                Author and article information

                Journal
                J Diabetes Investig
                J Diabetes Investig
                10.1111/(ISSN)2040-1124
                JDI
                ST
                Journal of Diabetes Investigation
                Blackwell Publishing Ltd (Oxford, UK )
                2040-1116
                2040-1124
                11 October 2011
                28 March 2012
                : 3
                : 2 ( doiID: 10.1111/jdi.2012.3.issue-2 )
                : 138-147
                Affiliations
                [ 1 ]Department of Medicine/Divisions of Endocrinology
                [ 2 ]Pediatrics
                [ 3 ]Biochemistry/Molecular Biology
                [ 4 ]Human Cellular Therapy Laboratory, Mayo Clinic Rochester, Rochester, MN
                [ 5 ]Department of Biochemistry/Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, AZ, USA
                [ 6 ]The First Department of Medicine, Wakayama University of Medical Science
                [ 7 ]Department of Clinical Laboratory Medicine, Wakayama Medical University, Wakayama, Japan
                Author notes
                [*] [* ] Corresponding Author. Norman L Eberhardt Tel.: +1‐507‐255‐6554 Fax: +1‐507‐293‐1249 E‐mail address: eberhardt@ 123456mayo.edu
                Article
                JDI166
                10.1111/j.2040-1124.2011.00166.x
                4020731
                66b0de24-a17b-49e1-bf55-a48290b79939
                © 2011 Asian Association for the Study of Diabetes and Blackwell Publishing Asia Pty Ltd
                History
                Page count
                Figures: 6, Tables: 0, Pages: 10
                Categories
                Articles
                Basic Science and Research
                Original Article
                Custom metadata
                2.0
                April 2012
                Converter:WILEY_ML3GV2_TO_NLM version:3.9.3 mode:remove_FC converted:04.02.2014

                islet amyloid polypeptide,transgenic mice,type 2 diabetes

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