Timothy D. O'Brien

Islet amyloid, islet-amyloid polypeptide, and diabetes mellitus

Islet-amyloid deposits, which are a common feature of Type II diabetes mellitus, are derived from the polymerization of a putative hormone identified as IAPP. IAPP is synthesized by normal islet beta cells and probably is cosecreted with insulin. Although the physiologic function of IAPP and its role in the pathogenesis of Type II diabetes mellitus are just beginning to be unraveled, IAPP may play an important part in the development of this most common form of diabetes mellitus by opposing the action of insulin in peripheral tissues. The polymerization of IAPP to form extracellular islet-amyloid deposits may further contribute to the development of Type II diabetes mellitus by destroying islet cells and by disrupting the passage of glucose and hormones to and from them. Substantial evidence indicates that the propensity of IAPP to polymerize and form extracellular amyloid deposits in only certain species (e.g., humans, cats, and raccoons) is directly associated with an intrinsically amyloidogenic part of the molecule--i.e., positions 20 through 29 of IAPP. The inherent amyloidogenicity of IAPP in these species may be further facilitated by increased beta-cell production of IAPP, leading to a high local concentration that predisposes to polymerization. The latter possibility is supported by studies demonstrating that IAPP production by islet beta cells is increased in normoglycemic cats with impaired glucose tolerance. Although increased production of IAPP may initially cause insulin resistance, prolonged overproduction of IAPP may ultimately impair insulin secretion by leading to the progressive deposition of insoluble islet amyloid, a finding apparent in most subjects with overt diabetes. If, as these studies suggest, increased IAPP production is linked to the development of Type II diabetes mellitus, further studies must address the genetic and nongenetic factors that influence this important biologic change in humans and some animal species.

Evidence for Proteotoxicity in β Cells in Type 2 Diabetes: Toxic Islet Amyloid Polypeptide Oligomers Form Intracellularly in the Secretory Pathway

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in beta cells and islet amyloid derived from islet amyloid polypeptide (IAPP), a protein co-expressed with insulin by beta cells. It is increasingly appreciated that the toxic form of amyloidogenic proteins is not amyloid but smaller membrane-permeant oligomers. Using an antibody specific for toxic oligomers and cryo-immunogold labeling in human IAPP transgenic mice, human insulinoma and pancreas from humans with and without T2DM, we sought to establish the abundance and sites of formation of IAPP toxic oligomers. We conclude that IAPP toxic oligomers are formed intracellularly within the secretory pathway in T2DM. Most striking, IAPP toxic oligomers appear to disrupt membranes of the secretory pathway, and then when adjacent to mitochondria, disrupt mitochondrial membranes. Toxic oligomer-induced secretory pathway and mitochondrial membrane disruption is a novel mechanism to account for cellular dysfunction and apoptosis in T2DM.

Islet Amyloid Polypeptide and Insulin Secretion From Isolated Perfused Pancreas of Fed, Fasted, Glucose-Treated, and Dexamethasone-Treated Rats

Rats from four experimental treatment groups, including fed controls, 24- to 30-h fasted, dexamethasone-treated, and intraperitoneal glucose-treated, were used to assess the effects of these treatments on the immunohistochemically detectable islet amyloid polypeptide (IAPP) content in the pancreatic islets. Isolated perfused pancreases from additional animals in these groups were used to assess insulin and IAPP secretion and relative amounts of these hormones secreted into the perfusate under low-glucose (2.75 mM) and high-glucose (16.7 mM) conditions. Insulin and IAPP concentrations in the perfusate were measured by radioimmunoassays. Titration of immunohistochemical staining revealed the highest levels of IAPP in the dexamethasone- and glucose-treated groups, followed by the fed controls; the least amount was observed in the fasted group. In the perfusion experiments, the dexamethasone-treated group had significantly higher IAPP secretion than did all of the other groups under stimulation with 16.7 mM glucose. In addition, both dexamethasone treatment and glucose treatment increased the relative amount of IAPP to insulin secretion during 16.7 mM glucose stimulation in comparison with fed controls and fasted groups. Fasting tended to have the opposite effect and significantly decreased the relative amount of IAPP to insulin secreted under stimulation with 16.7 mM glucose. In all groups, IAPP and insulin secretion were generally parallel, which is consistent with their colocalization in the β-cell secretory vesicle and co-release after glucose stimulation. However, significant differences in the insulin-IAPP ratios between experimental groups is consistent with the hypothesis that production of IAPP and insulin are regulated differently in the β-cell. The increased secretion of IAPP in severe hyperglycemia may also facilitate the formation of IAPP-derived islet amyloid deposits, thus contributing to progressive worsening of the diabetic state.

Islet Amyloid Polypeptide: A Review of Its Biology and Potential Roles in the Pathogenesis of Diabetes Mellitus

Islet amyloidosis (IA) is the principal lesion in the endocrine pancreas of human beings with non-insulin-dependent diabetes mellitus (NIDDM) and in the similar forms of diabetes mellitus in domestic cats and macaques. As such, the delineation of the pathogenesis of this form of amyloidosis may be crucial to the understanding of the development and progression of NIDDM. Islet amyloid polypeptide (IAPP) is a recently discovered polypeptide that is the principal constituent of IA in human beings, cats, and macaques. IAPP is produced by the pancreatic β-cells and is co-packaged with insulin in the β-cell secretory vesicles. Immunohistochemical and physiologic evidence supports the notion that the β-cells are heterogenous with respect to their relative contents of insulin and IAPP. Therefore, although IAPP is co-secreted with insulin in response to a variety of well-known insulin secretogogues, the molar ratio of these two proteins that is released from the islets may vary, depending upon the glucose concentration and prevailing metabolic milieu. IAPP is highly conserved among mammalian species and has about 45% homology to another neuropeptide, calcitonin gene-related peptide. IAPP is encoded by a single-copy gene located, in the human being, on chromosome 12. IAPP is expressed as a 93 (murine)–89 (human)-amino acid prepropolypeptide that is processed enzymatically, resulting in the removal of amino- and carboxy-terminal propeptide segments. The 20–29 region of the IAPP molecule is most important in the ability of IAPP to form amyloid fibrils. The role of IAPP and IA in the pathogenesis of human NIDDM and similar forms of diabetes mellitus in cats and macaques may involve several possible mechanisms, including 1) direct physical/chemical damage to β-cells, resulting in necrosis and loss of functional islet tissue, 2) biologic activities of IAPP that oppose those of insulin or abnormally suppress insulin secretion, and 3) interference by IA deposits of passage of insulin out of β-cells and/or entrance of glucose and other secretogogues into the islet. The roles of each of these possible mechanisms have yet to be demonstrated. In addition, the physiological significance of the apparent IAPP deficiency in both insulin-dependent diabetes mellitus and NIDDM is currently unknown.

Multipotent Adult Progenitor Cells from Swine Bone Marrow

We show that multipotent adult progenitor cells (MAPCs) can be derived from both postnatal and fetal swine bone marrow (BM). Although swine MAPC (swMAPC) cultures are initially mixed, cultures are phenotypically homogenous by 50 population doublings (PDs) and can be maintained as such for more than 100 PDs. swMAPCs are negative for CD44, CD45, and major histocompatibility complex (MHC) classes I and II; express octamer binding transcription factor 3a (Oct3a) mRNA and protein at levels close to those seen in human ESCs (hESCs); and have telomerase activity preventing telomere shortening even after 100 PDs. Using quantitative‐reverse transcription‐polymerase chain reaction (Q‐RT‐PCR), immunofluorescence, and functional assays, we demonstrate that swMAPCs differentiate into chondrocytes, adipocytes, osteoblasts, smooth muscle cells, endothelium, hepatocyte‐like cells, and neuron‐like cells. Consistent with what we have shown for human and rodent MAPCs, Q‐RT‐PCR demonstrated a significant upregulation of transcription factors and other lineage‐specific transcripts in a time‐dependent fashion similar to development. When swMAPCs were passaged for 3–6 passages at high density (2,000–8,000 cells per cm2), Oct3a mRNA levels were no longer detectable, cells acquired the phenotype of mesenchymal stem cells (CD44+, MHC class I+), and could differentiate into typical mesenchymal lineages (adipocytes, osteoblasts, and chondroblasts), but not endothelium, hepatocyte‐like cells, or neuron‐like cells. Even if cultures were subsequently replated at low density (approximately 100–500 cells per cm2) for >20 PDs, Oct3a was not re‐expressed, nor were cells capable of differentiating to cells other than mesenchymal‐type cells. This suggests that the phenotype and functional characteristics of swMAPCs may not be an in vitro culture phenomenon.

Islet amyloid polypeptide — a novel controversy in diabetes research

The discovery of a previously unknown polypeptide in the islet Beta cells was unexpecled. This putative hormone, named islet amyloid polypeptided (IAPP) or amylin, has beer implicated in the normal regulation of glucose melabolism and has beer proposed to have a role in the pathogenesis of Type 2 (non-insulin-dependent) diabetes mellitus. IAPP is therefore of great interest in the field of diabetes research at present

Treatment With Growth Hormone and Dexamethasone in Mice Transgenic for Human Islet Amyloid Polypeptide Causes Islet Amyloidosis and β-Cell Dysfunction

Islet amyloid derived from islet amyloid polypeptide (IAPP) is a well-recognized feature of type II diabetes. However, the mechanism of islet amyloidogenesis is unknown. In vitro studies suggest that amino acid residues 20–29 in human, but not mouse, IAPP confer amyloidogenicity consistent with the absence of spontaneous islet amyloidosis in mice. Several clinical and in vitro studies suggest that increased synthetic rates of IAPP predispose to IAPP-amyloidosis. In the present study, we sought to test the hypothesis that pharmacological induction of insulin resistance in a mouse transgenic (TG) for human IAPP would induce islet amyloid and β-cell dysfunction. TG and non-transgenic (N-TG) control mice were treated with both rat growth hormone (12 μg/day) and dexamethasone (0.24 mg/day) (dex/GH) or received no treatment for 4 weeks, after which animals were killed to examine islet morphology. Treatment with dex/GH caused hyperglycemia (7.3 ± 0.4 vs. 5.2 ± 0.1 mmol/l, TG vs. N-TG, P < 0.001) associated with a decreased plasma insulin concentration (595 ± 51 vs. 996 ± 100 pmol/1, TG vs. N-TG, P < 0.05) in TG versus control mice. Islet amyloid was induced in treated TG mice but not in control mice. Islet amyloid was identified in both intra- and extracellular deposits, the former being associated with evidence of β-cell degeneration. We conclude that dex/GH treatment in mice TG for human IAPP induces IAPP-derived islet amyloid, hyperglycemia, and islet dysfunction. The present model recapitulates the islet morphology and phenotype of type II diabetes.

A Feline Model of Experimentally Induced Islet Amyloidosis

The study of the pathogenesis of islet amyloidosis and its relationship to the development and progression of type 2 diabetes mellitus has been hampered by the lack of an experimentally inducible animal model. The domestic cat, by virtue of the fact that it is one of the few species that spontaneously develop a form of diabetes mellitus that closely resembles human type 2 diabetes, including the formation of amyloid deposits derived from islet amyloid polypeptide (IAPP), was considered to be an excellent candidate species in which to attempt to develop a nontransgenic animal model for this disease process. To develop the model, 8 healthy domestic cats were given a 50% pancreatectomy, which was followed by treatment with growth hormone and dexamethasone. Once a stable diabetic state was established, cats were randomly assigned to groups treated with either glipizide or insulin at doses appropriate to control hyperglycemia. Cats were maintained on this treatment regimen for 18 months and then euthanized. Based on light microscopic examination of Congo red-stained sections of pancreas, all cats were negative for the presence of islet amyloid at the time of pancreatectomy. At the end of the study all 4 glipizide-treated cats had islet amyloid deposits, whereas only 1 of 4 insulin-treated cats had detectable amyloid. In addition, the glipizide treated cats had threefold higher basal and fivefold higher glucose-stimulated plasma IAPP concentrations than insulin-treated cats, suggesting an association between elevated IAPP secretion and islet amyloidosis. Blood-glycosylated hemoglobin concentrations were not significantly different between the two treatment groups. This study documents for the first time an inducible model of islet amyloidosis in a nontransgenic animal.

Immunohistochemical morphometry of pancreatic endocrine cells in diabetic, normoglycaemic glucose-intolerant and normal cats

The anatomical distribution and volume fractions of pancreatic A cells (glucagon), B cells (insulin) and D cells (somatostatin) were evaluated by an immunoperoxidase technique in 6 diabetic cats, 6 normoglycaemic glucose-intolerant cats and 6 normal control cats. Islets lacking A cells were observed in some sections from the right lobe of the pancreas which correlated with a significantly lower A cell volume fraction in the right pancreatic lobe. Endocrine cell volume fractions in normoglycaemic glucose-intolerant cats were not significantly different from controls. Thus, a reduction in B cell volume fraction was not necessary for the occurrence of impaired glucose tolerance in these cats. However, the reduction of B cell volume fraction in the 2 normoglycaemic glucose-intolerant cats with insular amyloidosis may in part explain the more severely impaired glucose tolerance previously observed in these cats. Insular amyloidosis in our feline diabetics, as in human type II diabetics, was associated with a significant decrease in A and B cell volume fractions. In both human type II and feline diabetes mellitus, however, the reduction in B cell mass does not appear sufficient alone to lead to diabetes mellitus. Therefore, amyloid replacement of functional endocrine cells does not appear to be the primary diabetogenic event in feline diabetes mellitus, but may contribute to progression of the condition due to loss of functional B cell reserves. We thus postulate that a B cell defect precedes deposition of islet amyloid and that these amyloid deposits may thus provide an important biochemical clue to specific B cell derangements occurring in adult-onset diabetics.

Reversal of New-Onset Diabetes through Modulating Inflammation and Stimulating β-Cell Replication in Nonobese Diabetic Mice by a Dipeptidyl Peptidase IV Inhibitor

Inhibition of dipeptidyl peptidase IV (DPP-IV) activity by NVP-DPP728, a DPP-IV inhibitor, improves the therapeutic efficacy of glucagon-like peptide-1 (GLP-1). CD26 is a membrane-associated glycoprotein with DPP-IV activity and is expressed on lymphocytes. We investigated the effect of NVP-DPP728 on reversing new-onset diabetes in nonobese diabetic (NOD) mice and modulating the inflammatory response and stimulating beta-cell regeneration. New-onset diabetic NOD mice were treated with NVP-DPP728 for 2, 4, and 6 wk. Blood glucose level was monitored. Regulatory T cells in thymus and secondary lymph nodes, TGF-beta1 and GLP-1 in plasma, and the insulin content in the pancreas were measured. Immunostaining for insulin and bromodeoxyuridine (BrdU) were performed. The correlation of beta-cell replication with inflammation was determined. In NVP-DPP728-treated NOD mice, diabetes could be reversed in 57, 74, and 73% of mice after 2, 4, and 6 wk treatment, respectively. Insulitis was reduced and the percentage of CD4(+)CD25(+)FoxP3(+) regulatory T cells was increased in treated NOD mice with remission. Plasma TGF-beta1 and GLP-1, the insulin content, and both insulin(+) and BrdU(+) beta-cells in pancreas were also significantly increased. No significant correlations were found between numbers of both insulin(+) and BrdU(+) beta-cells in islets and beta-cell area or islets with different insulitis score in NOD mice with remission of diabetes. In conclusion, NVP-DPP728 treatment can reverse new-onset diabetes in NOD mice by reducing insulitis, increasing CD4(+)CD25(+)FoxP3(+) regulatory T cells, and stimulating beta-cell replication. beta-Cell replication is not associated with the degree of inflammation in NVP-DPP728-treated NOD mice.