Identification of the Amyloid-Degrading Enzyme Neprilysin in Mouse Islets and Potential Role in Islet Amyloidogenesis

Abstract

Islet amyloid contributes to loss of ␤-cell mass and function in type 2 diabetes. It is poorly understood how the building block of amyloid, islet amyloid polypeptide (IAPP), misfolds and accumulates within the islet to contribute to cellular dysfunction. We sought to determine whether neprilysin, an amyloid-degrading enzyme, is present in islets and plays a role in the accumulation of amyloid fibrils. Human IAPP (hIAPP) transgenic mice, a model of islet amyloid in which primarily male mice develop amyloid by 12 months of age, were studied at 10 weeks and 6 months of age, enabling investigation of islet changes before and during early amyloidogenesis. Neprilysin was present in islets, including ␤-cells, and islet neprilysin mRNA and activity were found to decline with age in nontransgenic mice as well as in hIAPP transgenic female mice. In contrast, neprilysin mRNA and activity did not decrease in amyloid-prone hIAPP transgenic male mice at 6 months compared with nontransgenic mice and female hIAPP transgenic mice. Islet amyloid was detected in 43% of the 6-month-old hIAPP transgenic male mice only, suggesting the sustained elevation of islet neprilysin in these mice was a compensatory mechanism aimed at preventing amyloid accumulation. In keeping with amyloid formation, the proportion of insulin-positive area to islet area was significantly reduced in 6-month-old hIAPP transgenic male mice, which also displayed mild fasting hyperglycemia compared with age-matched transgenic female and nontransgenic mice. Together, these findings demonstrate that neprilysin is a factor associated with islet amyloid accumulation and subsequent deterioration of ␤-cell function in hIAPP transgenic male mice. Diabetes 56:304 -310, 2007 I slet amyloid is a pathological characteristic of the endocrine pancreas in type 2 diabetes thought to contribute to reduced ␤-cell mass and function (1,2). The unique peptide component of islet amyloid is islet amyloid polypeptide (IAPP), or amylin (3,4), which is a normal product of the ␤-cell that is cosecreted with insulin in response to nutrient stimulation (5). The human IAPP (hIAPP) molecule, unlike rodent IAPP, is capable of forming amyloid fibrils because of species-specific differences in the amino acid sequence (6 -8). Using hIAPP transgenic models of islet amyloid formation, we and others (9 -16) have shown that hIAPP expression and/or amyloid deposition is associated with both impaired ␤-cell function (manifest primarily as hyperglycemia) and reduced ␤-cell mass.The regulation of IAPP production and secretion has long been studied and shown to closely mirror that of insulin. Thus, in conditions associated with insulin resistance, and specifically hyperinsulinemia, IAPP levels are elevated (17)(18)(19). In the face of ␤-cell dysfunction, this can potentially lead to aggregation and fibril formation (20). A factor that may further accelerate amyloidogenesis is reduced IAPP and/or amyloid degradation. Studies by Bennett et al. (21,22) have demonstrated that insulindegrading enzyme degrades ...

Neprilysin Impedes Islet Amyloid Formation by Inhibition of Fibril Formation Rather Than Peptide Degradation

“…At this time, the specific localization of neprilysin within the islet has not been clearly defined. Previously, using an immunohistochemical approach, we showed that islet neprilysin localized to both ␤-cells and non-␤-cells and is largely membranous with some cytoplasmic localization (8). Reports describing neprilysin in non-islet tissues also document that it is predominantly plasma membrane-bound; however, there is evidence in neurons that neprilysin is also localized in secretory vesicles (41).…”

“…Neprilysin Enzymatic Activity Assay-Islet neprilysin enzyme activity was determined fluorometrically as previously described (8).…”

“…Real-time Quantitative Reverse Transcription-PCR-Gene expression of neprilysin, hIAPP, mIAPP, and insulin in isolated islets was determined with real-time quantitative reverse transcription-PCR performed using the TaqMan system (ABI Prism 7000, Applied Biosystems, Foster City, CA) as previously described (8). TaqMan Assays on Demand neprilysin (Mm00485028_m1), hIAPP (Hs00169095_m1), mIAPP (Mm00439403_m1), and insulin II (Mm00731595_gH) gene expression mixes were from Applied Biosystems.…”

“…Given that amyloidosis in type 2 diabetes and Alzheimer disease shares some similarities (13), and our previous observation of a relationship between islet amyloid deposition and neprilysin levels in vivo (8), we sought to determine whether neprilysin can indeed inhibit islet amyloid formation and its associated ␤-cell toxicity in vitro and, if so, by what mechanism. We used isolated islets from our human IAPP (hIAPP) transgenic mice, an amyloidogenic model that we developed (14) because mouse IAPP (mIAPP) does not form amyloid (15)(16)(17).…”

“…Recently we demonstrated that another amyloid-degrading enzyme, neprilysin, is present and active in islets and may play a role in islet amyloid accumulation (8). Neprilysin is a 90-to 110-kDa zinc metallopeptidase with a short cytoplasmic NH 2 terminus, a transmembrane hydrophobic region, and a large extracellular domain containing the active site.…”