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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.

Deposition of islet amyloid polypeptide (IAPP) as amyloid in the pancreatic islet occurs in approximately 90% of individuals with Type 2 diabetes and is associated with decreased islet beta-cell mass and function. Human IAPP (hIAPP), but not rodent IAPP, is amyloidogenic and toxic to islet beta-cells. In addition to IAPP, islet amyloid deposits contain other components, including heparan sulfate proteoglycans (HSPGs). The small molecule 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-alpha-D-xylo-hexopyranose (WAS-406) inhibits HSPG synthesis in hepatocytes and blocks systemic amyloid A deposition in vivo. To determine whether WAS-406 inhibits localized amyloid formation in the islet, we incubated hIAPP transgenic mouse islets for up to 7 days in 16.7 mM glucose (conditions that result in amyloid deposition) plus increasing concentrations of the inhibitor. WAS-406 at doses of 0, 10, 100, and 1,000 microM resulted in a dose-dependent decrease in amyloid deposition (% islet area occupied by amyloid: 0.66 +/- 0.14%, 0.10 +/- 0.06%, 0.09 +/- 0.07%, and 0.004 +/- 0.003%, P < 0.001) and an increase in beta-cell area in hIAPP transgenic islets (55.0 +/- 2.6 vs. 60.6 +/- 2.2% islet area for 0 vs. 100 microM inhibitor, P = 0.05). Glycosaminoglycan, including heparan sulfate, synthesis was inhibited in both hIAPP transgenic and nontransgenic islets (the latter is a control that does not develop amyloid), while O-linked protein glycosylation was also decreased, and WAS-406 treatment tended to decrease islet viability in nontransgenic islets. Azaserine, an inhibitor of the rate-limiting step of the hexosamine biosynthesis pathway, replicated the effects of WAS-406, resulting in reduction of O-linked protein glycosylation and glycosaminoglycan synthesis and inhibition of islet amyloid formation. In summary, interventions that decrease both glycosaminoglycan synthesis and O-linked protein glycosylation are effective in reducing islet amyloid formation, but their utility as pharmacological agents may be limited due to adverse effects on the islet.

Islet amyloid contributes to the loss of beta-cell mass in type 2 diabetes. To examine the roles of glucose and time on amyloid formation, we developed a rapid in vitro model using isolated islets from human islet amyloid polypeptide (hIAPP) transgenic mice. Islets from hIAPP transgenic and non-transgenic mice were cultured for up to 7 days with either 5.5, 11.1, 16.7 or 33.3mmol/l glucose. At various time-points throughout the culture period, islets were harvested for determination of amyloid and beta-cell areas, and for measures of cell viability, insulin content, and secretion. Following culture of hIAPP transgenic islets in 16.7 or 33.3mmol/l glucose, amyloid formation was significantly increased compared to 5.5 or 11.1mmol/l glucose culture. Amyloid was detected as early as day 2 and increased in a time-dependent manner so that by day 7, a decrease in the proportion of beta-cell area in hIAPP transgenic islets was evident. When compared to non-transgenic islets after 7-day culture in 16.7mmol/l glucose, hIAPP transgenic islets were 24% less viable, had decreased beta-cell area and insulin content, but displayed no change in insulin secretion. Thus, we have developed a rapid in vitro model of light microscopy-visible islet amyloid formation that is both glucose- and time-dependent. Formation of amyloid in this model is associated with reduced cell viability and beta-cell loss but adequate functional adaptation. It thus enables studies investigating the mechanism(s) underlying the amyloid-associated loss of beta-cell mass in type 2 diabetes.

Islet amyloid contributes to loss of beta-cell mass and function in type 2 diabetes. It is poorly understood how the building block of amyloid, islet amyloid polypeptide (IAPP), misfolds and accumulates within the islet to contribute to cellular dysfunction. We sought to determine whether neprilysin, an amyloid-degrading enzyme, is present in islets and plays a role in the accumulation of amyloid fibrils. Human IAPP (hIAPP) transgenic mice, a model of islet amyloid in which primarily male mice develop amyloid by 12 months of age, were studied at 10 weeks and 6 months of age, enabling investigation of islet changes before and during early amyloidogenesis. Neprilysin was present in islets, including beta-cells, and islet neprilysin mRNA and activity were found to decline with age in nontransgenic mice as well as in hIAPP transgenic female mice. In contrast, neprilysin mRNA and activity did not decrease in amyloid-prone hIAPP transgenic male mice at 6 months compared with nontransgenic mice and female hIAPP transgenic mice. Islet amyloid was detected in 43% of the 6-month-old hIAPP transgenic male mice only, suggesting the sustained elevation of islet neprilysin in these mice was a compensatory mechanism aimed at preventing amyloid accumulation. In keeping with amyloid formation, the proportion of insulin-positive area to islet area was significantly reduced in 6-month-old hIAPP transgenic male mice, which also displayed mild fasting hyperglycemia compared with age-matched transgenic female and nontransgenic mice. Together, these findings demonstrate that neprilysin is a factor associated with islet amyloid accumulation and subsequent deterioration of beta-cell function in hIAPP transgenic male mice.

Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes. In obese individuals, adipose tissue releases increased amounts of non-esterified fatty acids, glycerol, hormones, pro-inflammatory cytokines and other factors that are involved in the development of insulin resistance. When insulin resistance is accompanied by dysfunction of pancreatic islet beta-cells - the cells that release insulin - failure to control blood glucose levels results. Abnormalities in beta-cell function are therefore critical in defining the risk and development of type 2 diabetes. This knowledge is fostering exploration of the molecular and genetic basis of the disease and new approaches to its treatment and prevention.

Objective: We sought to determine whether a history of gestational diabetes mellitus (GDM) further increases the risk of cardiovascular disease (CVD) in parous women with first-degree relatives with type 2 diabetes., Research Design and Methods: Women with (n = 332) and without (n = 663) a history of GDM were compared regarding 1) the revised National Cholesterol Education Program Adult Treatment Panel III metabolic syndrome criteria, 2) the prevalence of type 2 diabetes, and 3) self-reported CVD., Results: Women with prior GDM were younger (48.6 +/- 0.7 vs. 52.4 +/- 0.6 years [means +/- SE];P < 0.001) and less likely to be postmenopausal (48.3 vs. 57.9%; P < 0.005). Although both groups were obese (BMI 34.4 +/- 1.2 vs. 33.7 +/- 0.6 kg/m(2)), women with prior GDM were more likely to have metabolic syndrome (86.6 vs. 73.5%; P < 0.001) and type 2 diabetes (93.4 vs. 63.3%; P < 0.001). Moreover, they had a higher prevalence of CVD (15.5 vs. 12.4%; adjusted odds ratio 1.85 [95% CI 1.21-2.82];P = 0.005) that occurred at a younger age (45.5 +/- 2.2 vs. 52.5 +/- 1.9 years;P = 0.02) and was independent of metabolic syndrome (1.74 [1.10-2.76]; P = 0.02) and type 2 diabetes (1.56 [1.002-2.43];P < 0.05)., Conclusions: Among women with a family history of type 2 diabetes, those with prior GDM were even more likely to not only have CVD risk factors, including metabolic syndrome and type 2 diabetes, but also to have experienced CVD events, which occurred at a younger age. Thus, women with both a family history of type 2 diabetes and personal history of GDM may be especially suitable for early interventions aimed at preventing or reducing their risk of CVD and diabetes.

Objective: To determine whether the hyperbolic relationship between insulin sensitivity and the acute insulin response to glucose (AIRg) exists in subjects with impaired fasting glucose (IFG) or decreased glucose tolerance., Research Design and Methods: We studied 219 healthy subjects (88 male and 131 female subjects, aged 26-75 years) with fasting plasma glucose (FPG) <6.11 mmol/l. Subjects underwent an intravenous glucose tolerance test to determine the insulin sensitivity index (Si), AIRg, and the glucose disappearance constant (Kg), the latter a measure of intravenous glucose tolerance., Results: Si and AIRg were inversely related for the entire cohort, and this relationship was not significantly different from hyperbolic. The inverse relationship between Si and AIRg was not significantly different when compared between groups based on fasting glucose (normal fasting glucose [NFG], FPG <5.56 mmol/l vs. IFG, FPG 5.56-6.11 mmol/l) or by the Kg quartile. However, the curve relating Si and AIRg was left shifted in the IFG compared with NFG group (P < 0.001) and was progressively more left shifted with decreasing Kg (P < 0.001), consistent with decreasing beta-cell function. These changes were not observed for the curves relating Si and fasting insulin, suggesting that in the fasting state beta-cell function is maintained even in patients with mild IFG. Finally, the disposition index (DI) (Si x AIRg) was calculated as a measure of beta-cell function. The DI progressively decreased with increasing FPG, even in the group of subjects classified as NFG., Conclusions: The inverse relationship between insulin sensitivity and AIRg is consistent with a hyperbola not only in subjects with normal glucose tolerance but also with mild IFG or decreased Kg. Based on a hyperbolic relationship, a decrease in beta-cell function can be detected as FPG increases, even in patients who are normal glucose tolerant.

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.

Genetic background is important in determining susceptibility to metabolic abnormalities such as insulin resistance and beta-cell dysfunction. Islet amyloid is associated with reduced beta-cell mass and function and develops in the majority of our C57BL/6J x DBA/2J (F(1)) male human islet amyloid polypeptide (hIAPP) transgenic mice after 1 yr of increased fat feeding. To determine the relative contribution of each parental strain, C57BL/6J (BL6) and DBA/2J (DBA2), to islet amyloid formation, we studied male hIAPP mice on each background strain (BL6, n = 13; and DBA2 n = 11) and C57BL/6J x DBA/2J F(1) mice (n = 17) on a 9% (wt/wt) fat diet for 1 yr. At the end of 12 mo, islet amyloid deposition was quantified from thioflavin S-stained pancreas sections. The majority of mice in all groups developed islet amyloid (BL6: 91%, F(1): 76%, DBA2: 100%). However, the prevalence (%amyloid-positive islets; BL6: 14 +/- 3%, F(1): 44 +/- 8%, DBA2: 49 +/- 9%, P < 0.05) and severity (%islet area occupied by amyloid; BL6: 0.03 +/- 0.01%, F(1): 9.2 +/- 2.9%, DBA2: 5.7 +/- 2.3%, p < or = 0.01) were significantly lower in BL6 than F(1) and DBA2 mice. Increased islet amyloid severity was negatively correlated with insulin-positive area per islet, in F(1) (r(2) = 0.75, P < 0.001) and DBA2 (r(2) = 0.87, P < 0.001) mice but not BL6 mice (r(2) = 0.07). In summary, the extent of islet amyloid formation in hIAPP transgenic mice is determined by background strain, with mice expressing DBA/2J genes (F(1) and DBA2 mice) being more susceptible to amyloid deposition that replaces beta-cell mass. These findings underscore the importance of genetic and environmental factors in studying metabolic disease.

The increasing production of genetically-modified mouse models has necessitated studies to determine the inherent physiological characteristics of commonly used mouse strains. In this study we examined insulin secretory function in response to an intravenous bolus of glucose or glucose plus arginine in anesthetized C57BL/6, DBA/2 and 129T2 mice fed either a control or high fat diet for 6 weeks. The results show that 129T2 mice had higher fasting plasma glucose levels and lower fasting plasma insulin levels compared with C57BL/6 and DBA/2 mice regardless of diet. Furthermore, 129T2 mice were glucose intolerant and secreted significantly less insulin in response to glucose and glucose plus arginine irrespective of diet compared with the other two strains of mice. DBA/2 mice hypersecreted insulin in response to glucose and glucose plus arginine compared with C57BL/6 and 129T2 mice. Moreover while first phase insulin secretion was appropriately increased in response to the high fat diet in C57BL/6 and 129T2 mice, this was not the case for DBA/2 mice. Mean islet area was decreased in response to a high fat diet in DBA/2 mice, while there was no dietary effect on the other two strains. This study highlights the inherent genetic differences that exist among seemingly normal strains of mice that are commonly used to make transgenic and knockout mice. Understanding these differences will provide researchers with the information to choose the appropriate genetic background on which to express their particular genetic alteration.

Islet amyloid deposition in type 2 diabetes is associated with reduced beta-cell mass. Therefore, interventions aimed at reducing islet amyloid formation may help preserve beta-cell mass in type 2 diabetes. Rosiglitazone and metformin act by different mechanisms to improve insulin sensitivity and thereby reduce beta-cell secretory demand, resulting in decreased release of insulin and islet amyloid polypeptide (IAPP), the unique constituent of islet amyloid deposits. We hypothesized that this reduced beta-cell secretory demand would lead to reduced islet amyloid formation. Human IAPP (hIAPP) transgenic mice, a model of islet amyloid, were treated for 12 months with rosiglitazone (1.5 mg.kg(-1).day(-1), n = 19), metformin (1 g.kg(-1).day(-1), n = 18), or control (n = 17). At the end of the study, islet amyloid prevalence (percent islets containing amyloid) and severity (percent islet area occupied by amyloid), islet mass, beta-cell mass, and insulin release were determined. Islet amyloid prevalence (44 +/- 8, 13 +/- 4, and 11 +/- 3% for control, metformin-, and rosiglitazone-treated mice, respectively) and severity (9.2 +/- 3.0, 0.22 +/- 0.11, and 0.10 +/- 0.05% for control, metformin-, and rosiglitazone-treated mice, respectively) were markedly reduced with both rosiglitazone (P < 0.001 for both measures) and metformin treatment (P < 0.001 for both measures). Both treatments were associated with reduced insulin release assessed as the acute insulin response to intravenous glucose (2,189 +/- 857, 621 +/- 256, and 14 +/- 158 pmol/l for control, metformin-, and rosiglitazone-treated mice, respectively; P < 0.05 for metformin vs. control and P < 0.005 for rosiglitazone vs. control), consistent with reduced secretory demand. Similarly, islet mass (33.4 +/- 7.0, 16.6 +/- 3.6, and 12.2 +/- 2.1 mg for control, metformin-, and rosiglitazone-treated mice, respectively) was not different with metformin treatment (P = 0.06 vs. control) but was significantly lower with rosiglitazone treatment (P < 0.05 vs. control). When the decreased islet mass was accounted for, the islet amyloid-related decrease in beta-cell mass (percent beta-cell mass/islet mass) was ameliorated in both rosiglitazone- and metformin-treated animals (57.9 +/- 3.1, 64.7 +/- 1.4, and 66.1 +/- 1.6% for control, metformin-, and rosiglitazone-treated mice, respectively; P < 0.05 for metformin or rosiglitazone vs. control). In summary, rosiglitazone and metformin protect beta-cells from the deleterious effects of islet amyloid, and this effect may contribute to the ability of these treatments to alleviate the progressive loss of beta-cell mass and function in type 2 diabetes.

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 prevalence of glucose intolerance and type 2 diabetes increases with age. To determine whether the hyperbolic relationship between insulin sensitivity and the insulin response is affected by age and whether the decline in beta-cell function with age is related to increases in intra-abdominal fat or age per se, we studied 220 healthy subjects with fasting glucose <6.1 mmol/l (89 men and 131 women, aged 26-75 years, BMI 18.7-40.4 kg/m(2)). The insulin sensitivity index (S(i)) and the acute insulin response to glucose (AIRg) were determined, and from these beta-cell function was estimated as the disposition index (S(i) x AIRg). Intra-abdominal fat and subcutaneous fat areas were quantified by computed tomography. S(i) (5.40 +/- 0.5 vs. 7.86 +/- 0.7 x10(-5) min(-1)/[pmol/l]), P < 0.01) was decreased and intra-abdominal fat (117 +/- 10 vs. 81 +/- 9 cm(2), P < 0.05) was increased in the oldest (age 60-75 years) versus the youngest (age 26-44 years) quartile. The hyperbolic relationship between S(i) and AIRg was present independent of age; thus, beta-cell function measured as the disposition index (1,412 +/- 120 vs. 2,125 +/- 150 x10(-5) min(-1), P < 0.01) was lower in the oldest versus the youngest quartile. In multiple regression, intra-abdominal fat (r = -0.470, P < 0.001) but not age was associated with S(i), but both intra-abdominal fat (r = -0.198, P = 0.003) and age (r = -0.131, P = 0.05) were correlated with the disposition index. These data suggest that although intra-abdominal fat is a strong determinant of insulin sensitivity and beta-cell function, age has an independent effect on beta-cell function that may contribute to the increased prevalence of type 2 diabetes in older populations.

Islet amyloid deposition is a pathogenic feature of type 2 diabetes, and these deposits contain the unique amyloidogenic peptide islet amyloid polypeptide. Autopsy studies in humans have demonstrated that islet amyloid is associated with loss of beta-cell mass, but a direct role for amyloid in the pathogenesis of type 2 diabetes cannot be inferred from such studies. Animal studies in both spontaneous and transgenic models of islet amyloid formation have shown that amyloid forms in islets before fasting hyperglycemia and therefore does not arise merely as a result of the diabetic state. Furthermore, the extent of amyloid deposition is associated with both loss of beta-cell mass and impairment in insulin secretion and glucose metabolism, suggesting a causative role for islet amyloid in the islet lesion of type 2 diabetes. These animal studies have also shown that beta-cell dysfunction seems to be an important prerequisite for islet amyloid formation, with increased secretory demand from obesity and/or insulin resistance acting to further increase islet amyloid deposition. Recent in vitro studies suggest that the cytotoxic species responsible for islet amyloid-induced beta-cell death are formed during the very early stages of islet amyloid formation, when islet amyloid polypeptide aggregation commences. Interventions to prevent islet amyloid formation are emerging, with peptide and small molecule inhibitors being developed. These agents could thus lead to a preservation of beta-cell mass and amelioration of the islet lesion in type 2 diabetes.

The underlying pathophysiology of the metabolic syndrome is the subject of debate, with both insulin resistance and obesity considered as important factors. We evaluated the differential effects of insulin resistance and central body fat distribution in determining the metabolic syndrome as defined by the National Cholesterol Education Program (NCEP) Adult Treatment Panel III. In addition, we determined which NCEP criteria were associated with insulin resistance and central adiposity. The subjects, 218 healthy men (n = 89) and women (n = 129) with a broad range of age (26-75 years) and BMI (18.4-46.8 kg/m2), underwent quantification of the insulin sensitivity index (Si) and intra-abdominal fat (IAF) and subcutaneous fat (SCF) areas. The metabolic syndrome was present in 34 (15.6%) of subjects who had a lower Si [median: 3.13 vs. 6.09 x 10(-5) min(-1)/(pmol/l)] and higher IAF (166.3 vs. 79.1 cm2) and SCF (285.1 vs. 179.8 cm2) areas compared with subjects without the syndrome (P < 0.001). Multivariate models including Si, IAF, and SCF demonstrated that each parameter was associated with the syndrome. However, IAF was independently associated with all five of the metabolic syndrome criteria. In multivariable models containing the criteria as covariates, waist circumference and triglyceride levels were independently associated with Si and IAF and SCF areas (P < 0.001). Although insulin resistance and central body fat are both associated with the metabolic syndrome, IAF is independently associated with all of the criteria, suggesting that it may have a pathophysiological role. Of the NCEP criteria, waist circumference and triglycerides may best identify insulin resistance and visceral adiposity in individuals with a fasting plasma glucose <6.4 mmol/l.

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.

Pancreatic islet amyloid deposits in type 2 diabetes are associated with decreased islet beta-cell function. They contain both amylin (islet amyloid polypeptide), the beta-cell-derived unique fibrillogenic component, and heparan sulfate proteoglycans (HSPGs). We hypothesized that beta-cell HSPGs contribute to islet amyloidogenesis. [35S]Sulfate-labeled proteoglycans from islet-derived beta-TC3 cell cultures eluted from diethylaminoethyl Sephacel at 0.35M NaCl. Chromatography on Sepharose CL-4B and SDS-PAGE analysis revealed distinct populations of proteoglycans. Medium HSPGs eluted at K(av) approximately 0.18 and 0.50 with glycosaminoglycan chains of approximately 28 and 19 kDa, respectively. A third population containing chondroitin/dermatan sulfate eluted at K(av) approximately 0.70 with glycosaminoglycan chains of approximately 10 kDa. A single size class of heparan and chondroitin/dermatan sulfate proteoglycans in the cell layer eluted at K(av) approximately 0.40 with glycosaminoglycan chains of approximately 19 kDa. Medium and cell layer proteoglycans bound exclusively to fibrillogenic amylin, as determined by gel mobility shift assays, indicating a possible role for beta-cell-derived proteoglycans in islet amyloid formation.

Transgenic mice expressing the amyloidogenic human islet amyloid polypeptide (hIAPP) in their islet beta-cells are a model of islet amyloid formation as it occurs in type 2 diabetes. Our hIAPP transgenic mice developed islet amyloid when fed a breeder chow but not regular chow. Because the breeder chow contained increased amounts of fat, we hypothesized that increased dietary fat enhances islet amyloid formation. To test this hypothesis, we fed male hIAPP transgenic and nontransgenic control mice diets containing 15% (low fat), 30% (medium fat), or 45% (high fat) of calories derived from fat for 12 months, and we measured islet amyloid, islet endocrine cell composition, and beta-cell function. Increased dietary fat in hIAPP transgenic mice was associated with a dose-dependent increase in both the prevalence (percentage of islets containing amyloid deposits; 34 +/- 8, 45 +/- 8, and 58 +/- 10%, P < 0.05) and severity (percentage of islet area occupied by amyloid; 0.8 +/- 0.5, 1.0 +/- 0.5, and 4.6 +/- 2.5%, P = 0.05) of islet amyloid. In addition, in these hIAPP transgenic mice, there was a dose-dependent decrease in the proportion of islet area comprising beta-cells, with no significant change in islet size. In contrast, nontransgenic mice adapted to diet-induced obesity by increasing their islet size more than twofold. Increased dietary fat was associated with impaired insulin secretion in hIAPP transgenic (P = 0.05) but not nontransgenic mice. In summary, dietary fat enhances both the prevalence and severity of islet amyloid and leads to beta-cell loss and impaired insulin secretion. Because both morphologic and functional defects are present in hIAPP transgenic mice, this would suggest that the effect of dietary fat to enhance islet amyloid formation might play a role in the pathogenesis of the islet lesion of type 2 diabetes in humans.

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.

First-degree relatives of individuals with type 2 diabetes are at increased risk of developing hyperglycemia. To examine the prevalence and pathogenesis of abnormal glucose homeostasis in these subjects, 531 first-degree relatives with no known history of diabetes (aged 44.1 +/- 0.7 years; BMI 29.0 +/- 0.3 kg/m(2)) underwent an oral glucose tolerance test (OGTT). Newly identified diabetes was found in 19% (n = 100), and impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT) was found in 36% (n = 191). Thus, only 45% (n = 240) had normal glucose tolerance (NGT). The homeostasis model assessment of insulin resistance (HOMA-IR) was used to estimate insulin sensitivity; beta-cell function was quantified as the ratio of the incremental insulin to glucose responses over the first 30 min during the OGTT (DeltaI(30)/DeltaG(30)). This latter measure was also adjusted for insulin sensitivity as it modulates beta-cell function ([DeltaI(30)/DeltaG(30)]/HOMA-IR). Decreasing glucose tolerance was associated with increasing insulin resistance (HOMA: NGT 12.01 +/- 0.54 pmol/mmol; IFG/IGT 16.14 +/- 0.84; diabetes 26.99 +/- 2.62; P < 0.001) and decreasing beta-cell function (DeltaI(30)/DeltaG(30): NGT 157.7 +/- 9.7 pmol/mmol; IFG/IGT 100.4 +/- 5.4; diabetes 57.5 +/- 7.3; P < 0.001). Decreasing beta-cell function was also identified when adjusting this measure for insulin sensitivity ([DeltaI(30)/DeltaG(30)]/HOMA-IR). In all four ethnic groups (African-American, n = 55; Asian-American, n = 66; Caucasian, n = 217; Hispanic-American, n = 193), IFG/IGT and diabetic subjects exhibited progressively increasing insulin resistance and decreasing beta-cell function. The relationships of insulin sensitivity and beta-cell function to glucose disposal, as measured by the incremental glucose area under the curve (AUCg), were examined in the whole cohort. Insulin sensitivity and AUCg were linearly related so that insulin resistance was associated with poorer glucose disposal (r(2) = 0.084, P < 0.001). In contrast, there was a strong inverse curvilinear relationship between beta-cell function and AUCg such that poorer insulin release was associated with poorer glucose disposal (log[DeltaI(30)/DeltaG(30)]: r(2) = 0.29, P < 0.001; log[(DeltaI(30)/DeltaG(30))/HOMA-IR]: r(2) = 0.45, P < 0.001). Thus, abnormal glucose metabolism is common in first-degree relatives of subjects with type 2 diabetes. Both insulin resistance and impaired beta-cell function are associated with impaired glucose metabolism in all ethnic groups, with beta-cell function seeming to be more important in determining glucose disposal.

Obesity is associated with insulin resistance, particularly when body fat has a central distribution. However, insulin resistance also frequently occurs in apparently lean individuals. It has been proposed that these lean insulin-resistant individuals have greater amounts of body fat than lean insulin-sensitive subjects. Alternatively, their body fat distribution may be different. Obesity is associated with elevated plasma leptin levels, but some studies have suggested that insulin sensitivity is an additional determinant of circulating leptin concentrations. To examine how body fat distribution contributes to insulin sensitivity and how these variables are related to leptin levels, we studied 174 individuals (73 men, 101 women), a priori classified as lean insulin-sensitive (LIS, n = 56), lean insulin-resistant (LIR, n = 61), and obese insulin-resistant (OIR, n = 57) based on their BMI and insulin sensitivity index (S(I)). Whereas the BMI of the two lean groups did not differ, the S(I) of the LIR subjects was less than half that of the LIS group. The subcutaneous and intra-abdominal fat areas, determined by computed tomography, were 45 and 70% greater in the LIR subjects (P < 0.001) and 2.5- and 3-fold greater in the OIR group, as compared with the LIS group. Fasting plasma leptin levels were moderately increased in LIR subjects (10.8 +/- 7.1 vs. 8.1 +/- 6.4 ng/ml in LIS subjects; P < 0.001) and doubled in OIR subjects (21.9 +/- 15.5 ng/ml; P < 0.001). Because of the confounding effect of body fat, we examined the relationships between adiposity, insulin sensitivity, and leptin concentrations by multiple regression analysis. Intra-abdominal fat was the best variable predicting insulin sensitivity in both genders and explained 54% of the variance in S(I). This inverse relationship was nonlinear (r = -0.688). On the other hand, in both genders, fasting leptin levels were strongly associated with subcutaneous fat area (r = 0.760) but not with intra-abdominal fat. In line with these analyses, when LIS and LIR subjects were matched for subcutaneous fat area, age, and gender, they had similar leptin levels, whereas their intra-abdominal fat and insulin sensitivity remained different. Thus, accumulation of intra-abdominal fat correlates with insulin resistance, whereas subcutaneous fat deposition correlates with circulating leptin levels. We conclude that the concurrent increase in these two metabolically distinct fat compartments is a major explanation for the association between insulin resistance and elevated circulating leptin concentrations in lean and obese subjects.

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.

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.

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.

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 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.