Loss of coupling between calcium influx, energy consumption and insulin secretion associated with development of hyperglycaemia in the UCD-T2DM rat model of type 2 diabetes.

AIMS/HYPOTHESIS: Previous studies on isolated islets have demonstrated tight coupling between calcium (Ca(2+)) influx and oxygen consumption rate (OCR) that is correlated with insulin secretion rate (ISR). To explain these observations, we have proposed a mechanism whereby the activation of a highly energetic process (Ca(2+)/metabolic coupling process [CMCP]) by Ca(2+) mediates the stimulation of ISR. The aim of the study was to test whether impairment of the CMCP could play a role in the development of type 2 diabetes. METHODS: Glucose- and Ca(2+)-mediated changes in OCR and ISR in isolated islets were compared with the time course of changes of plasma insulin concentrations observed during the progression to hyperglycaemia in a rat model of type-2 diabetes (the University of California at Davis type 2 diabetes mellitus [UCD-T2DM] rat). Islets were isolated from UCD-T2DM rats before, 1 week, and 3 weeks after the onset of hyperglycaemia. RESULTS: Glucose stimulation of cytosolic Ca(2+) and OCR was similar for islets harvested before and 1 week after the onset of hyperglycaemia. In contrast, a loss of decrement in islet OCR and ISR in response to Ca(2+) channel blockade coincided with decreased fasting plasma insulin concentrations observed in rats 3 weeks after the onset of hyperglycaemia. CONCLUSIONS/INTERPRETATION: These results suggest that phenotypic impairment of diabetic islets in the UCD-T2DM rat is downstream of Ca(2+) influx and involves unregulated stimulation of the CMCP. The continuously elevated levels of CMCP induced by chronic hyperglycaemia in these islets may mediate the loss of islet function.

Rosiglitazone treatment does not decrease amyloid deposition in transplanted islets from transgenic mice expressing human islet amyloid polypeptide.

In human islet transplantation, insulin independence decreases over time. We previously showed that amyloid deposition following transplantation of islets from human islet amyloid polypeptide (hIAPP) transgenic mice resulted in ß-cell loss and that rosiglitazone treatment decreased islet amyloid deposition and preserved ß-cell area in the endogenous pancreas of hIAPP transgenic mice. Thus, we sought to determine if rosiglitazone treatment decreases islet amyloid deposition and the associated ß-cell loss after islet transplantation. Streptozocin-diabetic mice were transplanted with 100 islets from hIAPP transgenic (T) mice or nontransgenic (NT) littermates under the kidney capsule and received either rosiglitazone (R) in drinking water or plain drinking water (C). The resultant groups (NTC [n = 11], NTR [n = 9], TC [n = 14], and TR [n = 10]) were followed for 12 weeks after which the graft was removed and processed for histology. Amyloid was detected in nearly all T islet grafts (TC = 13/14, TR = 10/10) but not in NT grafts. Rosiglitazone did not alter amyloid deposition (% graft area occupied by amyloid; TC: 2.15 ± 0.7, TR: 1.72 ± 0.66; P = .86). % ß-cell/graft area was decreased in the TC grafts compared to NTC (56.2 ± 3.1 vs 73.8 ± 1.4; P < .0001) but was not different between TC and TR groups (56.2 ± 3.1 vs 61.0 ± 2.9; P = .34). Plasma glucose levels before and after transplantation did not differ between NTC and TC groups and rosiglitazone did not affect plasma glucose levels post-islet transplantation. Rosiglitazone did not decrease amyloid deposition in hIAPP transgenic islet grafts. Therefore, rosiglitazone treatment of recipients of amyloid forming islets may not improve transplantation outcomes.

cJUN N-terminal kinase (JNK) activation mediates islet amyloid-induced beta cell apoptosis in cultured human islet amyloid polypeptide transgenic mouse islets.

AIMS/HYPOTHESIS: Aggregation of human islet amyloid polypeptide (hIAPP) as islet amyloid is associated with increased beta cell apoptosis and reduced beta cell mass in type 2 diabetes. Islet amyloid formation induces oxidative stress, which contributes to beta cell apoptosis. The cJUN N-terminal kinase (JNK) pathway is a critical mediator of beta cell apoptosis in response to stress stimuli including oxidative stress and exogenous application of hIAPP. We determined whether amyloid formation by endogenous hIAPP mediates beta cell apoptosis through JNK activation and downstream signalling pathways. METHODS: hIAPP transgenic and non-transgenic mouse islets were cultured for up to 144 h in 16.7 mmol/l glucose to induce islet amyloid in the presence or absence of the amyloid inhibitor Congo Red or a cell-permeable JNK inhibitor. Amyloid, beta cell apoptosis, JNK signalling and activation of downstream targets in the intrinsic and extrinsic apoptotic pathways were measured. RESULTS: JNK activation occurred with islet amyloid formation in hIAPP transgenic islets after 48 and 144 h in culture. Neither high glucose nor the hIAPP transgene alone was sufficient to activate JNK independent of islet amyloid. Inhibition of islet amyloid formation with Congo Red reduced beta cell apoptosis and partially decreased JNK activation. JNK inhibitor treatment reduced beta cell apoptosis without affecting islet amyloid. Islet amyloid increased mRNA levels of markers of the extrinsic (Fas, Fadd) and intrinsic (Bim [also known as Bcl2l11]) apoptotic pathways, caspase 3 and the anti-apoptotic molecule Bclxl (also known as Bcl2l1) in a JNK-dependent manner. CONCLUSIONS/INTERPRETATION: Islet amyloid formation induces JNK activation, which upregulates predominantly pro-apoptotic signals in both extrinsic and intrinsic pathways, resulting in beta cell apoptosis.

Exendin-4 increases islet amyloid deposition but offsets the resultant beta cell toxicity in human islet amyloid polypeptide transgenic mouse islets.

AIMS/HYPOTHESIS: In type 2 diabetes, aggregation of islet amyloid polypeptide (IAPP) into amyloid is associated with beta cell loss. As IAPP is co-secreted with insulin, we hypothesised that IAPP secretion is necessary for amyloid formation and that treatments that increase insulin (and IAPP) secretion would thereby increase amyloid formation and toxicity. We also hypothesised that the unique properties of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 to maintain or increase beta cell mass would offset the amyloid-induced toxicity. METHODS: Islets from amyloid-forming human IAPP transgenic and control non-transgenic mice were cultured for 48 h in 16.7 mmol/l glucose alone (control) or with exendin-4, potassium chloride (KCl), diazoxide or somatostatin. Human IAPP and insulin release, amyloid deposition, beta cell area/islet area, apoptosis and AKT phosphorylation levels were determined. RESULTS: In control human IAPP transgenic islets, amyloid formation was associated with increased beta cell apoptosis and beta cell loss. Increasing human IAPP release with exendin-4 or KCl increased amyloid deposition. However, while KCl further increased beta cell apoptosis and beta cell loss, exendin-4 did not. Conversely, decreasing human IAPP release with diazoxide or somatostatin limited amyloid formation and its toxic effects. Treatment with exendin-4 was associated with an increase in AKT phosphorylation compared with control and KCl-treated islets. CONCLUSIONS/INTERPRETATION: IAPP release is necessary for islet amyloid formation and its toxic effects. Thus, use of insulin secretagogues to treat type 2 diabetes may result in increased islet amyloidogenesis and beta cell death. However, the AKT-associated anti-apoptotic effects of GLP-1 receptor agonists such as exendin-4 may limit the toxic effects of increased islet amyloid.

Toxic oligomers and islet beta cell death: guilty by association or convicted by circumstantial evidence?

Type 2 diabetes is a progressive disease characterised by islet amyloid deposits in the majority of patients. Amyloid formation is considered a significant factor in deterioration of islet function and reduction in beta cell mass, and involves aggregation of monomers of the normally soluble beta cell peptide, human islet amyloid polypeptide (hIAPP) into oligomers, fibrils and, ultimately, mature amyloid deposits. Despite extensive in vitro studies, the process of hIAPP aggregation in vivo is poorly understood, though it is widely reported to promote cytotoxicity. Recently, studies have suggested that only the early stages of fibril assembly, and in particular small hIAPP oligomers, are responsible for beta cell cytotoxicity. This challenges the prior concept that newly formed fibrils and/or mature fibrillar amyloid are cytotoxic. Herein, evidence both for and against the toxic hIAPP oligomer hypothesis is presented; from this, it is apparent that what exactly causes beta cell death when hIAPP aggregates remains debatable. Moreover, substantially more work with more specific reagents and techniques than are currently available will be required to identify conclusively the toxic species resulting from hIAPP aggregation. Keeping an open mind on the nature of the cytotoxic insult has implications for therapeutic developments and clinical care in type 2 diabetes.

Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress.

AIMS/HYPOTHESIS: Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of human IAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of human IAPP. METHODS: Islets from human IAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to 7 days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreases from human IAPP transgenic and non-transgenic mice and humans with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of Bip, Atf4 and Chop (also known as Ddit3) and alternate splicing of Xbp1 mRNA, or in pancreases by immunostaining for immunoglobulin heavy chain-binding protein (BIP), C/EBP homologous protein (CHOP) and X-box binding protein 1 (XBP1). RESULTS: Amyloid formation in human IAPP transgenic islets was associated with reduced beta cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from human IAPP transgenic mice or humans was not associated with upregulation of ER stress markers. CONCLUSIONS/INTERPRETATION: Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of human IAPP.

The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.

The critical role of the beta cell in the pathogenesis of type 2 diabetes is now well established. When examined in patients with type 2 diabetes and individuals at increased risk, reductions in beta cell mass and abnormalities of beta cell function can both be demonstrated. The question of whether one alone is sufficient or both are necessary for the development of hyperglycaemia has been debated. Based on human and animal studies, it appears that neither alone is sufficient. Rather, for glucose to rise to the level at which diabetes would be diagnosed, defects in beta cell mass and in beta cell function are required.

Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis.

AIMS/HYPOTHESIS: Islet amyloid in type 2 diabetes contributes to loss of beta cell mass and function. Since islets are susceptible to oxidative stress-induced toxicity, we sought to determine whether islet amyloid formation is associated with induction of oxidative stress. METHODS: Human islet amyloid polypeptide transgenic and non-transgenic mouse islets were cultured for 48 or 144 h with or without the antioxidant N-acetyl-L: -cysteine (NAC) or the amyloid inhibitor Congo Red. Amyloid deposition, reactive oxygen species (ROS) production, beta cell apoptosis, and insulin secretion, content and mRNA were measured. RESULTS: After 48 h, amyloid deposition was associated with increased ROS levels and increased beta cell apoptosis, but no change in insulin secretion, content or mRNA levels. Antioxidant treatment prevented the rise in ROS, but did not prevent amyloid formation or beta cell apoptosis. In contrast, inhibition of amyloid formation prevented the induction of oxidative stress and beta cell apoptosis. After 144 h, amyloid deposition was further increased and was associated with increased ROS levels, increased beta cell apoptosis and decreased insulin content. At this time-point, antioxidant treatment and inhibition of amyloid formation were effective in reducing ROS levels, amyloid formation and beta cell apoptosis. Inhibition of amyloid formation also increased insulin content. CONCLUSIONS/INTERPRETATION: Islet amyloid formation induces oxidative stress, which in the short term does not mediate beta cell apoptosis, but in the longer term may feed back to further exacerbate amyloid formation and contribute to beta cell apoptosis.

Amyloid formation results in recurrence of hyperglycaemia following transplantation of human IAPP transgenic mouse islets.

AIMS/HYPOTHESIS: Islet transplantation is a potential cure for diabetes; however, rates of graft failure remain high. The aim of the present study was to determine whether amyloid deposition is associated with reduced beta cell volume in islet grafts and the recurrence of hyperglycaemia following islet transplantation. METHODS: We transplanted a streptozotocin-induced mouse model of diabetes with 100 islets from human IAPP (which encodes islet amyloid polypeptide) transgenic mice that have the propensity to form islet amyloid (n = 8-12) or from non-transgenic mice that do not develop amyloid (n = 6-10) in sets of studies that lasted 1 or 6 weeks. RESULTS: Plasma glucose levels before and for 1 week after transplantation were similar in mice that received transgenic or non-transgenic islets, and at that time amyloid was detected in all transgenic grafts and, as expected, in none of the non-transgenic grafts. However, over the 6 weeks following transplantation, plasma glucose levels increased in transgenic but remained stable in non-transgenic islet graft recipients (p < 0.05). At 6 weeks, amyloid was present in 92% of the transgenic grafts and in none of the non-transgenic grafts. Beta cell volume was reduced by 30% (p < 0.05), beta cell apoptosis was twofold higher (p < 0.05), and beta cell replication was reduced by 50% (p < 0.001) in transgenic vs non-transgenic grafts. In summary, amyloid deposition in islet grafts occurs prior to the recurrence of hyperglycaemia and its accumulation over time is associated with beta cell loss. CONCLUSIONS/INTERPRETATION: Islet amyloid formation may explain, in part, the non-immune loss of beta cells and recurrence of hyperglycaemia following clinical islet transplantation.

Resistin is not associated with insulin sensitivity or the metabolic syndrome in humans.

AIMS/HYPOTHESIS: The aim of this study was to further elucidate the relationship between resistin and insulin sensitivity, body fat distribution and the metabolic syndrome in humans. METHODS: We measured plasma resistin levels in 177 non-diabetic subjects (75 male, 102 female; age 32-75 years). BMI, waist circumference, blood pressure, lipids, glucose, plasminogen-activator inhibitor 1 (PAI-1), adiponectin and leptin levels were also measured. The insulin sensitivity index (S(I)) was quantified using Bergman's minimal model. Intra-abdominal fat (IAF) and subcutaneous fat (SQF) areas were quantified by CT scan. Presence of metabolic syndrome criteria was determined using the National Cholesterol Education Program Adult Treatment Panel III guidelines. RESULTS: When subjects were divided into categories based on BMI (< or > or =27.5 kg/m(2)) and S(I) (< or > or = 7 x 10(-5) min(-1) [pmol/l](-1)), resistin levels did not differ between the lean, insulin-sensitive (n=53, 5.36+/-0.3 ng/ml), lean, insulin-resistant (n=67, 5.70+/-0.4 ng/ml) and obese, insulin-resistant groups (n=48, 5.94+/-0.4 ng/ml; ANOVA p=0.65). Resistin correlated with age (r=-0.22, p<0.01), BMI (r=0.16, p=0.03) and SQF (r=0.19, p=0.01) but not with S(I) (p=0.31) or IAF (p=0.52). Resistin did not correlate with the number of metabolic syndrome criteria or any of the individual metabolic syndrome criteria. In contrast, adiponectin, PAI-1 and leptin each correlated with IAF, SQF and S(I). Additionally, the number of metabolic syndrome criteria correlated with adiponectin (r=-0.32, p<0.001), leptin (r=0.31, p<0.001) and PAI-1 (r=0.26, p=0.001). CONCLUSIONS/INTERPRETATION: In contrast to other adipokines, resistin is only weakly associated with body fat and is unlikely to be a major mediator of insulin resistance or the metabolic syndrome in humans.

Dietary-fat-induced obesity in mice results in beta cell hyperplasia but not increased insulin release: evidence for specificity of impaired beta cell adaptation.

AIMS/HYPOTHESIS: Increased dietary fat intake is associated with obesity and insulin resistance, but studies have shown that the subsequent increase in insulin release is not appropriate for this obesity-induced insulin resistance. We therefore sought to determine whether the impaired beta cell adaptation is due to inadequate expansion of the beta cell population or to a lack of an adaptive increase in insulin release. METHODS: Male mice were fed diets containing increasing amounts of fat (15, 30 or 45% of energy intake) for 1 year, after which islet morphology and secretory function were assessed. RESULTS: Increased dietary fat intake was associated with a progressive increase in body weight (p<0.001). Fractional beta cell area (total beta cell area/section area) was increased with increasing dietary fat (1.36+/-0.39, 2.46+/-0.40 and 4.93+/-1.05%, p<0.001), due to beta cell hyperplasia, and was positively and highly correlated with body weight (r2=0.68, p<0.005). In contrast, insulin release following i.p. glucose did not increase with increasing dietary fat (118+/-32, 108+/-47 and 488+/-200 pmol/l per mmol/l, p=0.07) and did not correlate with body weight (r2=0.11). When this response was examined relative to fractional beta cell area (insulin release/fractional beta cell area), it did not increase but rather tended to decrease with increasing dietary fat (157+/-55, 43+/-13 and 97+/-53 [pmol/l per mmol/l]/%, p=0.06) and did not correlate with body weight (r2=0.02). CONCLUSIONS/INTERPRETATION: Long-term fat feeding is associated with an increase in the beta cell population but an inadequate functional adaptation. Thus, a functional rather than a morphological abnormality appears to underlie dietary-fat-induced beta cell dysfunction.

The effect of apolipoprotein E deficiency on islet amyloid deposition in human islet amyloid polypeptide transgenic mice.

AIMS/HYPOTHESIS: Islet amyloid deposits are present in over 85% of Type 2 diabetic patients and have been suggested to be pathogenic. The mechanism that converts islet amyloid polypeptide (IAPP), the unique component of these deposits, into amyloid fibrils in vivo is not known. The amino acid sequence of IAPP is critical but insufficient for beta-pleated sheet formation. As apolipoprotein E (apoE), another component of islet amyloid deposits, plays a critical role in amyloid formation in Alzheimer's disease, we hypothesised that apoE could play an important role in islet amyloid formation. METHODS: Transgenic mice expressing the human form of IAPP ( hIAPP (+/0)) were crossbred with apoE deficient ( apoE (-/-)) mice and followed for 12 months, at which time the prevalence and severity of islet amyloid, as well as plasma glucose, hIAPP, immunoreactive insulin (IRI) and lipid concentrations were measured. RESULTS: The prevalence and severity of islet amyloid after one year of follow up were comparable among hIAPP (+/0) mice that were apoE (+/+), apoE (+/-) or apoE (-/-). Differences in glucose tolerance, lipid abnormalities or changes in pancreatic content or plasma concentrations of hIAPP and/or IRI did not account for these findings. CONCLUSION/INTERPRETATION: Our data shows that, unlike in the localized amyloidosis in the brain characteristic of Alzheimer's disease, apoE is not critical for islet amyloid formation in a transgenic mouse model of Type 2 diabetes mellitus. These results indicate that the mechanisms of localised amyloid formation probably vary among different amyloid-associated disorders. Therefore, therapeutic strategies targeting apoE might not apply equally to patients with different amyloid associated diseases.

Islet amyloid develops diffusely throughout the pancreas before becoming severe and replacing endocrine cells.

Islet amyloid occurs in >90% of type 2 diabetic patients and may play a role in the pathogenesis of this disease. To determine whether islet amyloid occurs diffusely throughout the pancreas, whether it affects islets equally, and whether it decreases islet endocrine cells, we characterized islet amyloidosis by computerized fluorescence microscopy in transgenic mice that develop typical islet amyloid. These mice produce the unique amyloidogenic component of human islet amyloid, human islet amyloid polypeptide (hIAPP). The prevalence of amyloid (number of islets containing amyloid/total number of islets x 100) and the severity of amyloid (Sigmaamyloid area/Sigmaislet area x 100) were found to be uniform throughout the pancreas. Furthermore, a high prevalence of amyloid was observed in islets when the severity of amyloid was only 1.5% of the islet area, suggesting a diffuse distribution of amyloid from the very early stages of islet amyloidosis. In 12 hIAPP transgenic mice with an amyloid severity of 9.6 +/- 3.4%, the proportion of islets composed of beta- and delta-cells was reduced in the transgenic mice compared with 6 nontransgenic mice that do not develop amyloid (beta-cells: 62.9 +/- 3.1% vs. 75.5 +/- 0.9%, P = 0.02; delta-cells: 2.8 +/- 0.5% vs. 4.4 +/- 0.4%, P = 0.05), whereas the proportion of islets composed of alpha-cells did not significantly differ between the two groups of mice. In the individual islets in these transgenic mice, amyloid severity was inversely correlated with beta-cell, (r = -0.59, P < 0.0001), alpha-cell (r = -0.32, P < 0.0001), and delta-cell (r = -0.25, P < 0.0001) areas. In conclusion, islet amyloidosis occurs uniformly throughout the pancreas, affecting all islets before becoming severe. A reduction in islet endocrine mass starts at this early stage of islet amyloid development and progresses as amyloid mass increases.

Oophorectomy promotes islet amyloid formation in a transgenic mouse model of Type II diabetes.

AIMS/HYPOTHESIS: In Type II (non-insulin-dependent) diabetes mellitus, amyloid depletes islet mass. We previously found that 81% of male human islet amyloid polypeptide (IAPP) transgenic mice but only 11% of female mice developed islet amyloid, suggesting that either testosterone promotes or ovarian products protect against amyloid deposition. METHODS: We did a bilateral oophorectomy or sham procedure in female human IAPP transgenic mice (n = 11 and n = 8, respectively) and in female non-transgenic mice (n = 7 and n = 9, respectively) at 6-8 weeks of age. Animals were followed for 1 year on a 9% fat (w/w) diet. Before we killed them we measured, fasting plasma human IAPP and did an intraperitoneal glucose tolerance test. Pancreatic content of IAPP and immunoreactive insulin (IRI) were estimated and pancreata were analysed for islet amyloid. RESULTS: No amyloid was detected in either the sham-operated transgenic mice or, as expected, in both groups of non-transgenic mice. In strong contrast, 7 of 11 (64%) oophorectomized mice developed islet amyloid (p < 0.05). Amyloid deposition in the oophorectomized transgenic mice was not associated with any differences in incremental body weight, fasting human IAPP concentrations or glucose tolerance between the groups. Furthermore, pancreatic content of mouse IAPP, human IAPP and immunoreactive insulin did not differ between groups. CONCLUSION/INTERPRETATION: Oophorectomy is associated with an enhancement of islet amyloid formation in the absence of changes in glucose tolerance, circulating IAPP or pancreatic content of IRI, mouse or human IAPP. Thus, the early stages of islet amyloidogenesis seem to be independent of glucose tolerance, with ovarian products having a protective role.

The constitutive secretory pathway is a major route for islet amyloid polypeptide secretion in neonatal but not adult rat islet cells.

Islet amyloid polypeptide (IAPP or amylin) is a normal secretory product of the pancreatic beta-cell that is cosecreted with insulin and is the major constituent of islet amyloid deposits in individuals with type 2 diabetes or insulinomas. We have previously reported that glucose stimulates IAPP, but not insulin secretion, from neonatal rat beta-cells when regulated secretion is prevented by use of calcium-free media, suggesting that IAPP secretion occurs via a constitutive secretory pathway. To directly test this hypothesis, we examined the effects of 2 substances-brefeldin A (BFA) and cycloheximide (CHX)-that are predicted to selectively block constitutive secretion on the release of IAPP-like immunoreactivity (IAPP-LI) and immunoreactive insulin (IRI) from neonatal rat islet cell monolayer cultures. When regulated release was prevented by use of calcium-free media, glucose-stimulated IAPP-LI release was nearly abolished by blocking constitutive release with 10 microg/ml BFA (mean +/- SD: 8.7 +/- 7.7 vs. 29.3 +/- 14.3 pmol/l; n = 5; P < 0.05), an inhibitor of constitutive vesicle formation. Similarly, calcium-independent, glucose-stimulated IAPP-LI secretion was markedly suppressed when new protein synthesis was blocked by administration of 20 microg/ml CHX (4.6 +/- 2.1 vs. 29.5 +/- 14.0 pmol/l; n = 5; P < 0.005). Secretion of IRI was low in the absence of calcium, and neither BFA nor CHX had any further effect. When calcium was added to the incubation media to allow regulated secretion of both IRI and IAPP-LI, both BFA (47.7 micro 8.7 vs. 80.7 micro 10.3 pmol/l; P < 0.001) and CHX (37.3 +/- 5.8 vs. 73.3 +/- 6.2 pmol/l; n = 5; P < 0.0001) inhibited glucose-stimulated IAPP-LI secretion by approximately 40%, but again had no inhibitory effect on IRI secretion. These data indicate that approximately 40% of glucose-stimulated IAPP-LI release occurs via a constitutive secretory pathway in neonatal rat islet cells. By contrast, in adult rat islets, glucose-stimulated IAPP-LI release was almost abolished in the absence of calcium (86 +/- 3% inhibition; P < 0.05) and unaffected by addition of BFA (275 +/- 28 vs. 205 +/- 89 pmol/l; NS) or CHX (160 +/- 20 vs. 205 +/- 89 pmol/l; NS), suggesting that constitutive secretion of IAPP does not occur in mature beta-cells. Collectively, these data suggest that a significant proportion of glucose-stimulated IAPP secretion from neonatal, but not adult, rat islet cells occurs via a constitutive secretory pathway.

Processing of synthetic pro-islet amyloid polypeptide (proIAPP) 'amylin' by recombinant prohormone convertase enzymes, PC2 and PC3, in vitro.

Islet amyloid polypeptide (IAPP), amylin, is the constituent peptide of pancreatic islet amyloid deposits which form in islets of Type 2 diabetic subjects. Human IAPP is synthesized as a 67-residue propeptide in islet beta-cells and colocalized with insulin in beta-cell granules. The mature 37-amino acid peptide is produced by proteolysis at pairs of basic residues at the C- and N-termini of the mature peptide. To determine the enzymes responsible for proteolysis and their activity at the potential cleavage sites, synthetic human proIAPP was incubated (0.5-16 h) with recombinant prohormone convertases, PC2 or PC3 at appropriate conditions of calcium and pH. The products were analysed by MS and HPLC. Proinsulin was used as a control and was cleaved by both recombinant enzymes resulting in intermediates. PC3 was active initially at the N-terminal-IAPP junction and later at the C-terminus, whereas initial PC2 activity was at the IAPP-C-terminal junction. Processing at the basic residues within the C-terminal flanking peptide rarely occurred. There was no evidence for substantial competition for the processing enzymes when the combined substrates proinsulin and proIAPP were incubated with both PC2 and PC3. As proinsulin cleavage is sequential in vivo (PC3 active at the B-chain-C-peptide junction, followed by PC2 at A chain-C-peptide junction), these data suggest that proteolysis of proIAPP and proinsulin is coincident in secretory granules and increased proinsulin secretion in diabetes could be accompanied by increased production of proIAPP.

Thrombocytopenia possibly caused by structurally related third-generation cephalosporins.

Thrombocytopenia is defined as a decrease in the platelet count to less than 100 x 10(9)/L and it is the most commonly reported drug-induced blood dyscrasia. Heparin is the most commonly reported cause of drug-induced thrombocytopenia with a reported incidence between one and ten percent. Thrombocytopenia induced by cephalosporins has been reported but is relatively rare. This report does not completely document that two third-generation cephalosporins caused platelet counts to fall less than 100 x 10(9)/L in the patient described but there was no other explanation available. Platelet counts began to fall with the institution of third-generation cephalosporins and began to rise when these agents were stopped. In order to document that thrombocytopenia was induced by the third-generation cephalosporins a rechallenge would have been necessary; this was not considered to be safe in this patient. A review of the literature is presented describing similar cases of cephalosporin-induced thrombocytopenia.