Gunilla T. Westermark

Islet Amyloid Polypeptide, Islet Amyloid, and Diabetes Mellitus

Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

Co-localization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets.

SummaryIslet amyloid polypeptide is a novel 37 amino-acid-residues polypeptide which has been isolated from amyloid deposits in an insulinoma, and in human and cat islets of Langerhans. The molecule has 46% homology with the calcitonin gene-related peptide. Light microscopy examination of the pancreas shows that islet amyloid polypeptide immunoreactivity is restricted to the islet B cells. The present study utilized a rabbit antiserum against a synthetic peptide corresponding to positions 20–29 of islet amyloid polypeptide, a sequence without any amino-acid identity with calcitonin gene-related peptide. By applying the immunogold technique at the ultrastructural level, it was shown that both insulin and islet amyloid polypeptide immunoreactivity occurs in the central granular core of the human B cell secretory granules, while the A cells remain unlabelled. The demonstration that islet amyloid polypeptide is a granular protein of the B cells may indicate that it is released together with insulin. Further studies are necessary to evaluate the functional role of islet amyloid polypeptide.

Transmissibility of systemic amyloidosis by a prion-like mechanism.

The generation of amyloid fibrils from an amyloidogenic polypeptide occurs by a nucleation-dependent process initiated in vitro by seeding the protein solution with preformed fibrils. This phenomenon is evidenced in vivo by the fact that amyloid protein A (AA) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an i.v. injection of protein extracted from AA amyloid-laden mouse tissue. Heretofore, the chemical nature of this “amyloid enhancing factor” (AEF) has not been definitively identified. Here we report that the active principle of AEF extracted from the spleen of mice with silver nitrate-induced AA amyloidosis was identified unequivocally as the AA fibril itself. Further, we demonstrated that this material was extremely potent, being active in doses <1 ng, and that it retained its biologic activity over a considerable length of time. Notably, the AEF was also effective when administered orally. Our studies have provided evidence that AA and perhaps other forms of amyloidosis are transmissible diseases, akin to the prion-associated disorders.

Staining methods for identification of amyloid in tissue.

Publisher Summary The staining reaction given by amyloid after treatment with iodine was often used in the earlier studies of amyloidosis, and amyloid is still identified by its characteristic histological staining reactions. Despite the enormous amount of knowledge now known regarding the molecular nature of amyloid, histological staining methods are crucial for the diagnosis of amyloidosis and are also used commonly in amyloid research. Also, the introduction of modern immunohistochemical techniques has made it possible to identify normal and abnormal components in tissue. Immunohistochemistry (often used interchangeably with immunocytochemistry) has become an important tool in amyloid research. Amyloid was first recognized by its tinctorial properties, which were elicited when amyloid-laden tissues were treated with iodine at the autopsy table. This reaction is now known to depend on the presence of minor carbohydrate components in the amyloid deposits. Iodine reacts with the amyloid, giving it a mahogany-like color that changes to blue when sulfuric acid is subsequently added. The staining properties of amyloid with rosaniline dyes (e.g., methyl violet and cresyl violet), which were the main staining methods for amyloid before Congo red staining was introduced in the 1920s, are also based on the presence of these same carbohydrate components. Because of their low sensitivity and lack of specificity, these methods are not commonly used any longer. Most, if not all, dyes used for the identification of amyloid are compounds developed for use by the textile industry. This includes the dye Congo red, which was introduced as the first direct cotton dye in 1884. Much of the background knowledge regarding the properties of these amyloid-associated dyes comes from textile staining.

Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism

Secondary, or amyloid protein A (AA), amyloidosis is a complication of chronic inflammatory diseases, both infectious and noninfectious. AA constitutes the insoluble fibrils, which are deposited in different organs, and is a major N-terminal part of the acute phase protein serum AA. It is not known why only some patients with chronic inflammation develop AA amyloidosis. Nucleation is a widely accepted mechanism in amyloidogenesis. Preformed amyloid-like fibrils act as nuclei in amyloid fibril formation in vitro, and AA amyloid fibrils and synthetic amyloid-like fibrils also may serve as seed for fibril formation in vivo. In addition to amyloid fibrils, there is a variety of similar nonmammalian protein fibrils with β-pleated structure in nature. We studied three such naturally occurring protein fibrils: silk from Bombyx mori, Sup35 from Saccharomyces cerevisiae, and curli from Escherichia coli. Our results show that these protein fibrils exert amyloid-accelerating properties in the murine experimental AA amyloidosis, suggesting that such environment factors may be important risk factors in amyloidogenesis.

Islet amyloid polypeptide-like immunoreactivity in the islet B cells of Type 2 (non-insulin-dependent) diabetic and non-diabetic individuals

SummaryA novel peptide, islet amyloid polypeptide (IAPP), with structural resemblance to calcitonin gene-related peptide has recently been purified from amyloid deposits in an insulinoma and from islets of Langerhans. By immunohistochemical methods, using antisera to a synthetic undecapeptide of IAPP and to insulin, we show that freshly fixed islet B cells in man, guinea pig, rat, mouse and hamster exhibit strong IAPP-immunoreactivity while A cells are unreactive. In human autopsy material, all of 11 non-diabetic individuals had IAPP immunoreactivity of the islets. In comparison 8 of the 13 patients with Type 2 (non-insulin-dependent) diabetes had no IAPP immunoreactive cells. The proportion of islet cells having IAPP immunoreactivity exceeded 10% in only 1 of the 5 remaining diabetic patients while in all 13 patients substantially more than 10% of the islet cells contained immunoreactive insulin. IAPP-positive amyloid deposits were found in 20–99% of the islets in 12 of the Type 2 diabetic patients while 6 of 11 non-diabetic subjects had amyloid in 3–11% of their islets. In islets with IAPP-immunoreactive amyloid, very few IAPP-cells were seen despite a strong reaction of the B cells with antiserum to insulin. This study shows that IAPP is a normal islet B cell component and that IAPP immunoreactivity in B cells is diminished in Type 2 diabetes while IAPP is deposited as amyloid fibrils in the islets of Langerhans. Although the function of IAPP is unknown, its occurrence in the islet B cells and its structural relation to calcitonin gene-related peptide makes a hormonal nature probable. The present study indicates an altered expression or metabolic fate of IAPP in Type 2 diabetes.

Islet Amyloid Polypeptide in Patients with Pancreatic Cancer and Diabetes

BACKGROUND The diabetes mellitus that occurs in patients with pancreatic cancer is characterized by marked insulin resistance that declines after tumor resection. Islet amyloid polypeptide (IAPP), a hormonal factor secreted from the pancreatic beta cells, reduces insulin sensitivity in vivo and glycogen synthesis in vitro. In this study, we examined the relation between IAPP and diabetes in patients with pancreatic cancer. METHODS We measured IAPP in plasma from 30 patients with pancreatic cancer, 46 patients with other cancers, 23 patients with diabetes, and 25 normal subjects. IAPP immunoreactivity and IAPP messenger RNA were studied in pancreatic cancers, pancreatic tissue adjacent to cancers, and normal pancreatic tissue. RESULTS Plasma IAPP concentrations were elevated in the patients with pancreatic cancer as compared with the normal subjects (mean [+/- SD], 22.3 +/- 13.6 vs. 8.0 +/- 5.0 pmol per liter; P < 0.001), normal in the patients with other cancers, and normal or low in the patients with diabetes. Among the patients with pancreatic cancer, the concentrations were 25.0 +/- 8.7 pmol per liter in the 7 patients with diabetes who required insulin, 31.4 +/- 12.6 pmol per liter in the 11 patients with diabetes who did not require insulin, and 12.2 +/- 2.4 pmol per liter in the 9 patients with normal glucose tolerance (3 patients had impaired glucose tolerance; their mean plasma IAPP concentration was 11.7 +/- 5.5 pmol per liter). Plasma IAPP concentrations decreased after surgery in the seven patients with pancreatic cancer who were studied before and after subtotal pancreatectomy (28.9 +/- 16.4 vs. 5.6 +/- 3.4 pmol per liter, P = 0.01). Pancreatic cancers contained IAPP, but the concentrations were lower than in normal pancreatic tissue (17 +/- 16 vs. 183 +/- 129 pmol per gram, P < 0.001). In samples from the patients with both pancreatic cancer and diabetes, immunostaining for IAPP was reduced in islets of pancreatic tissue surrounding the tumor; in situ hybridization studies suggested that transcription occurred normally in these islets. CONCLUSIONS Plasma IAPP concentrations are elevated in patients with pancreatic cancer who have diabetes. Since IAPP may cause insulin resistance, its overproduction may contribute to the diabetes that occurs in these patients.

Effects of beta cell granule components on human islet amyloid polypeptide fibril formation

Formation of amyloid‐like fibrils in a solution of human islet amyloid polypeptide (hIAPP) with and without the presence of other β‐cell granule components was studied in vitro. Insulin at less than equimolar concentration strongly inhibited hIAPP fibrillogenesis. Proinsulin had a weaker inhibitory effect while C‐peptide, Ca2+ and Zn2+ each individually enhanced fibril formation. C‐peptide combined with Ca2+ had an inhibitory effect. Since IAPP was found almost exclusively in the halo fractions of isolated islet secretory granules, primarily the concentrations of C‐peptide, Ca2+ and possibly proinsulin may be crucial for the native state of IAPP. It is concluded that an imbalance between fibril formation enhancers and inhibitors may be of importance in the pathogenesis of amyloid in the islets of Langerhans.

β-Cell Loss and β-Cell Apoptosis in Human Type 2 Diabetes Are Related to Islet Amyloid Deposition

Amyloid deposition and reduced β-cell mass are pathological hallmarks of the pancreatic islet in type 2 diabetes; however, whether the extent of amyloid deposition is associated with decreased β-cell mass is debated. We investigated the possible relationship and, for the first time, determined whether increased islet amyloid and/or decreased β-cell area quantified on histological sections is correlated with increased β-cell apoptosis. Formalin-fixed, paraffin-embedded human pancreas sections from subjects with (n = 29) and without (n = 39) diabetes were obtained at autopsy (64 ± 2 and 70 ± 4 islets/subject, respectively). Amyloid and β cells were visualized by thioflavin S and insulin immunolabeling. Apoptotic β cells were detected by colabeling for insulin and by TUNEL. Diabetes was associated with increased amyloid deposition, decreased β-cell area, and increased β-cell apoptosis, as expected. There was a strong inverse correlation between β-cell area and amyloid deposition (r = -0.42, P < 0.001). β-Cell area was selectively reduced in individual amyloid-containing islets from diabetic subjects, compared with control subjects, but amyloid-free islets had β-cell area equivalent to islets from control subjects. Increased amyloid deposition was associated with β-cell apoptosis (r = 0.56, P < 0.01). Thus, islet amyloid is associated with decreased β-cell area and increased β-cell apoptosis, suggesting that islet amyloid deposition contributes to the decreased β-cell mass that characterizes type 2 diabetes.

Conjugated Polyelectrolytes—Conformation-Sensitive Optical Probes for Staining and Characterization of Amyloid Deposits

Specific markers for diseases associated with protein aggregate depositions are of great interest. Here we report the use of conjugated polyelectrolytes as conformation‐sensitive optical probes for histological labeling of amyloid deposits in ex vivo tissue samples—amyloid light chains in primary systemic amyloidosis, islet amyloid polypeptide in human pancreas, and Aβ amyloid in Alzheimers disease. Under suitable conditions, these probes bind specifically to amyloid deposits, and this is seen as an orange‐red emission from the polyelectrolyte. Furthermore, the probes emit light of different colors when bound to different amyloid deposits or other intracellular structures. This phenomenon is most probably due to differences in the protein conformation in these structures. Hence, different protein conformations will generate geometric alterations of the bound polyelectrolyte backbone, affording different emissions from the bound probe. Conformation‐sensitive probes thus provide a direct link between spectral signal and protein conformation. Finally, the probes also proved useful for ex vivo fluorescence imaging by multiphoton excitation.