The role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

Pancreatic ß cell-selective zinc transporter 8 insufficiency accelerates diabetes associated with islet amyloidosis.

GWAS have shown that the common R325W variant of SLC30A8 (ZnT8) increases the risk of type 2 diabetes (T2D). However, ZnT8 haploinsufficiency is protective against T2D in humans, counterintuitive to earlier work in humans and mouse models. Therefore, whether decreasing ZnT8 activity is beneficial or detrimental to ß cell function, especially under conditions of metabolic stress, remains unknown. In order to examine whether the existence of human islet amyloid polypeptide (hIAPP), a coresident of the insulin granule, affects the role of ZnT8 in regulating ß cell function, hIAPP-expressing transgenics were generated with reduced ZnT8 (ZnT8B+/- hIAPP) or null ZnT8 (ZnT8B-/- hIAPP) expression specifically in ß cells. We showed that ZnT8B-/- hIAPP mice on a high-fat diet had intensified amyloid deposition and further impaired glucose tolerance and insulin secretion compared with control, ZnT8B-/-, and hIAPP mice. This can in part be attributed to impaired glucose sensing and islet cell synchronicity. Importantly, ZnT8B+/- hIAPP mice were also glucose intolerant and had reduced insulin secretion and increased amyloid aggregation compared with controls. These data suggest that loss of or reduced ZnT8 activity in ß cells heightened the toxicity induced by hIAPP, leading to impaired ß cell function and glucose homeostasis associated with metabolic stress.

Amyloid formation reduces protein kinase B phosphorylation in primary islet ß-cells which is improved by blocking IL-1ß signaling.

Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced ß-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of ß-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two ex vivo models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet ß-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1ß (IL-1ß) signaling in islets can restore the changes in ß-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1ß signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). ß-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1ß levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced ß-cell phospho-PKB levels and increased islet IL-1ß levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved ß-cell survival. Furthermore, inhibition of IL-1ß signaling by treatment with anakinra or exenatide increased ß-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in ß-cells which is associated with elevated islet IL-1ß levels. Inhibitors of amyloid or amyloid-induced IL-1ß production may provide a new approach to restore phospho-PKB levels thereby enhance ß-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation.

Amyloid formation disrupts the balance between interleukin-1ß and interleukin-1 receptor antagonist in human islets.

OBJECTIVES: ß-cell dysfunction and apoptosis associated with islet inflammation play a key role in the pathogenesis of type 2 diabetes (T2D). Growing evidence suggests that islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to islet inflammation and ß-cell death in T2D. We recently showed the role of interleukin-1ß (IL-1ß)/Fas/caspase-8 apoptotic pathway in amyloid-induced ß-cell death. In this study, we used human islets in culture as an ex vivo model of amyloid formation to: (1) investigate the effects of amyloid on islet levels of the natural IL-1 receptor antagonist (IL-1Ra); (2) examine if modulating the IL-1ß/IL-1Ra balance can prevent amyloid-induced ß-cell Fas upregulation and apoptosis. METHODS: Isolated human islets (n = 10 donors) were cultured in elevated glucose (to form amyloid) with or without a neutralizing human IL-1ß antibody for up to 7 days. Parallel studies were performed with human islets in which amyloid formation was prevented by adeno-siRNA-mediated suppression of hIAPP expression (as control). ß-cell levels of IL-1Ra, Fas, apoptosis as well as islet function, insulin- and amyloid-positive areas, and IL-1Ra release were assessed. RESULTS: Progressive amyloid formation in human islets during culture was associated with alterations in IL-1Ra. Islet IL-1Ra levels were higher at early stages but were markedly reduced at later stages of amyloid formation. Furthermore, IL-1Ra release from human islets was reduced during 7-day culture in a time-dependent manner. These changes in IL-1Ra production and release from human islets during amyloid formation adversely correlated with islet IL-1ß levels, ß-cell Fas expression and apoptosis. Treatment with IL-1ß neutralizing antibody markedly reduced amyloid-induced ß-cell Fas expression and apoptosis, thereby improving islet ß-cell survival and function during culture. CONCLUSIONS: These data suggest that amyloid formation impairs the balance between IL-1ß and IL-1Ra in islets by increasing IL-1ß production and reducing IL-1Ra levels thereby promoting ß-cell dysfunction and death. Restoring the IL-1ß/IL-1Ra ratio may provide an effective strategy to protect islet ß-cells from amyloid toxicity in T2D.

Dual role of interleukin-1ß in islet amyloid formation and its ß-cell toxicity: Implications for type 2 diabetes and islet transplantation.

AIMS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell failure in type 2 diabetes, cultured and transplanted islets. We previously showed that biosynthetic hIAPP aggregates induce ß-cell Fas upregulation and activation of the Fas apoptotic pathway. We used cultured human and hIAPP-expressing mouse islets to investigate: (1) the role of interleukin-1ß (IL-1ß) in amyloid-induced Fas upregulation; and (2) the effects of IL-1ß-induced ß-cell dysfunction on pro-islet amyloid polypeptide (proIAPP) processing and amyloid formation. RESEARCH DESIGN AND METHODS: Human and h IAPP -expressing mouse islets were cultured to form amyloid without or with the IL-1 receptor antagonist (IL-1Ra) anakinra, in the presence or absence of recombinant IL-1ß. Human islets in which amyloid formation was prevented (amyloid inhibitor or Ad-prohIAPP-siRNA) were cultured similarly. ß-cell function, apoptosis, Fas expression, caspase-8 activation, islet IL-1ß, ß-cell area, ß-/α-cell ratio, amyloid formation, and (pro)IAPP forms were assessed. RESULTS: hIAPP aggregates were found to increase IL-1ß levels in cultured human islets that correlated with ß-cell Fas upregulation, caspase-8 activation and apoptosis, all of which were reduced by IL-1Ra treatment or prevention of amyloid formation. Moreover, IL-1Ra improved culture-induced ß-cell dysfunction and restored impaired proIAPP processing, leading to lower amyloid formation. IL-1ß treatment potentiated impaired proIAPP processing and increased amyloid formation in cultured human and h IAPP -expressing mouse islets, which were prevented by IL-1Ra. CONCLUSIONS: IL-1ß plays a dual role by: (1) mediating amyloid-induced Fas upregulation and ß-cell apoptosis; (2) inducing impaired proIAPP processing thereby potentiating amyloid formation. Blocking IL-1ß may provide a new strategy to preserve ß cells in conditions associated with islet amyloid formation.

IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc.

The role of caspase-8 in amyloid-induced beta cell death in human and mouse islets.

AIMS/HYPOTHESIS: Reduced beta cell mass due to increased beta cell apoptosis is a key defect in type 2 diabetes. Islet amyloid, formed by the aggregation of human islet amyloid polypeptide (hIAPP), contributes to beta cell death in type 2 diabetes and in islet grafts in patients with type 1 diabetes. In this study, we used human islets and hIAPP-expressing mouse islets with beta cell Casp8 deletion to (1) investigate the role of caspase-8 in amyloid-induced beta cell apoptosis and (2) test whether caspase-8 inhibition protects beta cells from amyloid toxicity. METHODS: Human islet cells were cultured with hIAPP alone, or with caspase-8, Fas or amyloid inhibitors. Human islets and wild-type or hIAPP-expressing mouse islets with or without caspase-8 expression (generated using a Cre/loxP system) were cultured to form amyloid. Caspase-8 and -3 activation, Fas and FLICE inhibitory protein (FLIP) expression, islet beta cell and amyloid area, IL-1ß levels, and the beta:alpha cell ratio were assessed. RESULTS: hIAPP treatment induced activation of caspase-8 and -3 in islet beta cells (via Fas upregulation), resulting in apoptosis, which was markedly reduced by blocking caspase-8, Fas or amyloid. Amyloid formation in cultured human and hIAPP-expressing mouse islets induced caspase-8 activation, which was associated with Fas upregulation and elevated islet IL-1ß levels. hIAPP-expressing mouse islets with Casp8 deletion had comparable amyloid, IL-1ß and Fas levels with those expressing hIAPP and Casp8, but markedly lower beta cell apoptosis, higher beta:alpha cell ratio, greater beta cell area, and enhanced beta cell function. CONCLUSIONS/INTERPRETATION: Beta cell Fas upregulation by endogenously produced and exogenously applied hIAPP aggregates promotes caspase-8 activation, resulting in beta cell apoptosis. The prevention of amyloid-induced caspase-8 activation enhances beta cell survival and function in islets.

Three-dimensional scaffolds reduce islet amyloid formation and enhance survival and function of cultured human islets.

Islet transplantation provides a promising approach for treatment of type 1 diabetes mellitus. Amyloid formation and loss of extracellular matrix are two nonimmune factors contributing to death of isolated human islets. We tested the effects of two types of three-dimensional scaffolds, collagen matrix (CM) and fibroblast-populated collagen matrix (FPCM), on amyloid formation, viability, and function of isolated islets. Islets from cadaveric donors were cultured in FPCM, CM, or two-dimensional plate (2D) for 7 days. After 7 days, compared with the 2D culture condition, CM and FPCM markedly reduced amyloid formation of cultured islets and decreased apoptotic ß-cell rate by ∼75%. IL-1ß and Fas levels were also reduced in scaffold-embedded islets. Furthermore, ß/α cell ratios were increased by ∼18% and ∼36% in CM- and FPCM-embedded islets, respectively. Insulin content and insulin response to elevated glucose were also enhanced by both three-dimensional scaffolds. Moreover, culture in CM and FPCM (but not 2D) preserved insulin, GLUT-2, and PDX-1 mRNA expression. FPCM-embedded islets had significantly higher insulin response and lower amyloid formation than CM-embedded islets. These findings suggest that three-dimensional scaffolds reduce amyloid formation and improve viability and function of human islets in vitro, and that CM and fibroblasts have additive effects in enhancing islet function and reducing amyloid formation. Using this strategy is likely to improve outcome in human islet transplantation.

Cleavage of protein kinase D after acute hypoinsulinemia prevents excessive lipoprotein lipase-mediated cardiac triglyceride accumulation.

OBJECTIVE: During hypoinsulinemia, when cardiac glucose utilization is impaired, the heart rapidly adapts to using more fatty acids. One means by which this is achieved is through lipoprotein lipase (LPL). We determined the mechanisms by which the heart regulates LPL after acute hypoinsulinemia. RESEARCH DESIGN AND METHODS: We used two different doses of streptozocin (55 [D-55] and 100 [D-100] mg/kg) to induce moderate and severe hypoinsulinemia, respectively, in rats. Isolated cardiomyocytes were also used for transfection or silencing of protein kinase D (PKD) and caspase-3. RESULTS: There was substantial increase in LPL in D-55 hearts, an effect that was absent in severely hypoinsulinemic D-100 animals. Measurement of PKD, a key element involved in increasing LPL, revealed that only D-100 hearts showed an increase in proteolysis of PKD, an effect that required activation of caspase-3 together with loss of 14-3-3zeta, a binding protein that protects enzymes against degradation. In vitro, phosphomimetic PKD colocalized with LPL in the trans-golgi. PKD, when mutated to prevent its cleavage by caspase-3 and silencing of caspase-3, was able to increase LPL activity. Using a caspase inhibitor (Z-DEVD) in D-100 animals, we effectively lowered caspase-3 activity, prevented PKD cleavage, and increased LPL vesicle formation and translocation to the vascular lumen. This increase in cardiac luminal LPL was associated with a striking accumulation of cardiac triglyceride in Z-DEVD-treated D-100 rats. CONCLUSIONS After severe hypoinsulinemia, activation of caspase-3 can restrict LPL translocation to the vascular lumen. When caspase-3 is inhibited, this compensatory response is lost, leading to lipid accumulation in the heart.

Small interfering RNA-mediated suppression of proislet amyloid polypeptide expression inhibits islet amyloid formation and enhances survival of human islets in culture.

OBJECTIVE: Islet amyloid, formed by aggregation of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of beta-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance beta-cell survival and function. RESEARCH DESIGN AND METHODS: Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, beta-cell apoptosis, and beta-cell function. RESULTS: Thioflavine S-positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 +/- 4 vs. +Ad-siRNA, 153 +/- 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 +/- 33 vs. +Ad-siRNA, 285 +/- 21 percent basal, P < 0.05). CONCLUSIONS: These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and beta-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.

Impaired NH2-terminal processing of human proislet amyloid polypeptide by the prohormone convertase PC2 leads to amyloid formation and cell death.

Islet amyloid, formed by aggregation of islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of the pancreas in type 2 diabetes and may contribute to the progressive loss of beta-cells in this disease. We tested the hypothesis that impaired processing of the IAPP precursor proIAPP contributes to amyloid formation and cell death. GH3 cells lacking the prohormone convertase 1/3 (PC1/3) and IAPP and with very low levels of prohormone convertase 2 (PC2) were transduced with adenovirus (Ad) expressing human or rat (control) proIAPP linked to green fluorescent protein, with or without Ad-PC2 or Ad-PC1/3. Expression of human proIAPP increased the number of transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells 96 h after transduction (+hIAPP 8.7 +/- 0.4% vs. control 3.0 +/- 0.4%; P < 0.05). COOH-terminal processing of human proIAPP by PC1/3 increased (hIAPP+PC1/3 10.4 +/- 0.7%; P < 0.05), whereas NH(2)-terminal processing of proIAPP by addition of PC2 markedly decreased (hIAPP+PC2 5.5 +/- 0.5%; P < 0.05) the number of apoptotic GH3 cells. Islets from mice lacking PC2 and with beta-cell expression of human proIAPP (hIAPP(+/+)/PC2(-/-)) developed amyloid associated with beta-cell death during 2-week culture. Rescue of PC2 expression by ex vivo transduction with Ad-PC2 restored NH(2)-terminal processing to mature IAPP and decreased both the extent of amyloid formation and the number of TUNEL-positive cells (-PC2 26.5 +/- 4.1% vs. +PC2 16.1 +/- 4.3%; P < 0.05). These findings suggest that impaired NH(2)-terminal processing of proIAPP leads to amyloid formation and cell death and that accumulation of the NH(2)-terminally extended human proIAPP intermediate may be a critical initiating step in amyloid formation.

Processing of pro-islet amyloid polypeptide in the constitutive and regulated secretory pathways of beta cells.

Islet amyloid is a pathologic characteristic of the pancreas in type 2 diabetes comprised mainly of the beta-cell peptide islet amyloid polypeptide (IAPP; amylin). We used a pulse-chase approach to investigate the kinetics of processing and secretion of the IAPP precursor, proIAPP, in beta cells. By only 20 min after synthesis, a COOH-terminally processed proIAPP intermediate (approximately 6 kDa) was already present in beta cells. Formation of this NH2-terminally extended intermediate was not prevented by arresting secretory pathway transport at the trans-Golgi network (TGN) by either brefeldin A or temperature blockade, suggesting that this initial cleavage step occurs in the TGN before entry of (pro)IAPP into granules. Mature IAPP (approximately 4 kDa) was not detected until 60 min of chase, suggesting that NH2-terminal cleavage occurs in granules. Cells chased in low glucose without Ca2+ or with diazoxide, to block regulated release, secreted both proIAPP (approximately 8 kDa) and a partially processed form (approximately 6 kDa) via the constitutive secretory pathway. Stimulation of regulated secretion resulted in secretion primarily of mature IAPP as well as low levels of both unprocessed (approximately 8 kDa) and partially processed (approximately 6 kDa) proIAPP. We conclude that normal processing of proIAPP is a two-step process initiated by cleavage at its COOH terminus (likely by prohormone convertase 1/3 in the TGN) followed by cleavage at its NH2 terminus (by prohormone convertase 2 in granules) to form IAPP. Both proIAPP and its NH2-terminally extended intermediate appear to be normal secretory products of the beta cell that can be released via either the regulated or constitutive secretory pathways.

Role of carboxypeptidase E in processing of pro-islet amyloid polypeptide in {beta}-cells.

Islet amyloid polypeptide (IAPP; amylin) is a peptide hormone that is cosecreted with insulin from beta-cells. Impaired processing of proIAPP, the IAPP precursor, has been implicated in islet amyloid formation in type 2 diabetes. We previously showed that proIAPP is processed to IAPP by the prohormone convertases PC1/3 and PC2 at its carboxyl (COOH) and amino (NH(2)) termini, respectively. In this study, we investigated the role of carboxypeptidase E (CPE) in the processing of proIAPP using mice lacking active CPE (Cpe(fat)/Cpe(fat)) and NIT-2 cells, a beta-cell line derived from their islets. Western blot analysis demonstrated that an approximately 6-kDa NH(2)-terminally unprocessed form of proIAPP was elevated approximately 86% in islets from Cpe(fat)/Cpe(fat) mice, compared with wild type. This increase was independent of the development of hyperglycemia (8 wk male) or obesity (18 wk female). Impaired proIAPP processing was associated with a decrease in PC2 (but not PC1/3) and both the 21- and 27-kDa forms of the PC2 chaperone protein 7B2, suggesting that PC2-mediated processing of proIAPP at its NH(2) terminus was impaired in the absence of CPE. Formation of COOH-terminally amidated (pro)IAPP was reduced approximately 75% in NIT-2, compared with NIT-1 beta-cells, supporting a direct role for CPE in maturation of IAPP by removal of its COOH-terminal dibasic residues, the step essential for IAPP amidation. We conclude that lack of CPE in islet beta-cells results in a marked decrease in processing of proIAPP at its NH(2) (but not COOH) terminus that is associated with attenuated levels of PC2 and (pro)7B2 and a great reduction in formation of mature amidated IAPP.

Extended life span is associated with insulin resistance in a transgenic mouse model of insulinoma secreting human islet amyloid polypeptide.

Pancreatic amyloid is found in patients with insulinomas and type 2 diabetes. To study mechanisms of islet amyloidogenesis, we produced transgenic mice expressing the unique component of human islet amyloid, human islet amyloid polypeptide (hIAPP). These mice develop islet amyloid after 12 mo of high-fat feeding. To determine whether we could accelerate the rate of islet amyloid formation, we crossbred our hIAPP transgenic animals with RIP-Tag mice that develop islet tumors and die at 12 wk of age from hypoglycemia. At 12 wk of age, this new line of hIAPPxRIP-Tag mice was heavier (29.7 +/- 1.0 vs. 25.0 +/- 1.3 g, P < 0.05) and had increased plasma glucose levels (4.6 +/- 0.4 vs. 2.9 +/- 0.6 mmol/l, P < 0.05) compared with littermate RIP-Tag mice. However, the hIAPPxRIP-Tag mice did not display islet amyloid or amyloid fibrils despite high circulating hIAPP levels (24.6 +/- 7.0 pmol/l). Interestingly, hIAPPxRIP-Tag mice had a longer life span than RIP-Tag mice (121 +/- 8 vs. 102 +/- 5 days, P < 0.05). This increase in life span in hIAPPxRIP-Tag was positively correlated with body weight (r = 0.48, P < 0.05) and was associated with decreased insulin sensitivity compared with RIP-Tag mice. hIAPPxRIP-Tag mice did not develop amyloid during their 4-mo life span, suggesting that increased hIAPP secretion is insufficient for islet amyloid formation within such a short time. However, hIAPPxRIP-Tag mice did have an increase in life span that was associated with insulin resistance, suggesting that hIAPP has extrapancreatic effects, possibly on peripheral glucose metabolism.

Role of beta-cell prohormone convertase (PC)1/3 in processing of pro-islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) (amylin), the major component of islet amyloid, is produced by cleavage at the COOH- and NH(2)-termini of its precursor, proIAPP, likely by the beta-cell prohormone convertases (PC) 1/3 and PC2. Mice lacking PC2 can process proIAPP at its COOH- but not its NH(2)-terminal cleavage site, suggesting that PC1/3 is capable of initiating proIAPP cleavage at its COOH-terminus. To determine the precise role of PC1/3 in proIAPP processing, Western blot analysis was performed on islets isolated from mice lacking PC1/3 (PC1/3(-/-)). These islets contained not only fully processed IAPP as in PC1/3(+/+) islets, but also elevated levels of a COOH-terminally unprocessed intermediate form, suggesting impaired processing at the COOH-terminus. Next, GH3 cells that do not normally express proIAPP or detectable levels of PC1/3 or PC2 were cotransduced with adenoviruses expressing rat proIAPP and either PC2 or PC1/3. As expected, in GH3 cells transduced to express only proIAPP, no processing was observed. Coexpression of proIAPP and PC2 resulted in production of mature IAPP, whereas in cells that coexpressed proIAPP and PC1/3 only a 6-kDa intermediate was produced. We conclude that PC1/3 is important for processing of proIAPP at the COOH-terminus, but in its absence, PC2 can initiate complete processing of proIAPP to IAPP by cleaving the precursor at either its NH(2)- or COOH-terminal cleavage sites.

Islet amyloid polypeptide and type 2 diabetes.

Type 2 diabetes is associated with progressive beta-cell failure manifest as a decline in insulin secretion and increasing hyperglycemia. A growing body of evidence suggests that beta-cell failure in type 2 diabetes correlates with the formation of pancreatic islet amyloid deposits, indicating that islet amyloid may have an important role in beta-cell loss in this disease. Islet amyloid polypeptide (IAPP; amylin), the major component of islet amyloid, is co-secreted with insulin from beta-cells. In type 2 diabetes, this peptide aggregates to form amyloid fibrils that are toxic to beta-cells. The mechanism(s) responsible for islet amyloid formation in type 2 diabetes is still unclear but it appears that an increase in the secretion of IAPP, per se, is not sufficient. Other factors, such as impairment in the processing of proIAPP, the IAPP precursor, have been proposed to contribute to the development of islet amyloid deposits. Inhibitors of islet amyloid fibril formation might prevent the progression to beta-cell failure in type 2 diabetes and should therefore be considered as a therapeutic approach to treat this disease.

Mechanisms by which bis(maltolato)oxovanadium(IV) normalizes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in streptozotocin-diabetic rats in vivo.

Vanadium treatment normalizes plasma glucose levels in streptozotocin-diabetic rats in vivo, but the mechanism(s) involved are still unclear. Here, we tested the hypothesis that the in vivo effects of vanadium are mediated by changes in gluconeogenesis. Diabetic rats were treated with bis(maltolato)oxovanadium(IV) (BMOV) in the drinking water (0.75-1 mg/ml, 4 wk) or, for comparison, with insulin implants (4 U/d) for the final week of study. As with insulin, BMOV lowered plasma glucose and normalized phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA in the liver and kidney of diabetic rats. To determine the importance of reducing hyperglycemia per se, diabetic rats were treated either with a single ED(50) dose of BMOV (0.1 mmol/kg, ip) or with phlorizin (900 mg/kg.d, 5 d). BMOV rapidly restored PEPCK and G-6-Pase mRNA and normalized plasma glucose in responsive (50%) diabetic rats but had no effect on the nonresponsive hyperglycemic rats. Phlorizin corrected plasma glucose but had no effect on PEPCK mRNA and only partially normalized G-6-Pase mRNA. In conclusion, 1) BMOV inhibits PEPCK mRNA expression and activity by rapid mechanisms that are not reproduced simply by correction of hyperglycemia; and 2) BMOV inhibits G-6-Pase expression by complex mechanisms that depend, in part, on correction of hyperglycemia.