Islet amyloid contributes to loss of β-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 β-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 β-cell function in hIAPP transgenic male mice.
Islet amyloid is a pathological characteristic of the endocrine pancreas in type 2 diabetes thought to contribute to reduced β-cell mass and function (1,2). The unique peptide component of islet amyloid is islet amyloid polypeptide (IAPP), or amylin (3,4), which is a normal product of the β-cell that is cosecreted with insulin in response to nutrient stimulation (5). The human IAPP (hIAPP) molecule, unlike rodent IAPP, is capable of forming amyloid fibrils because of species-specific differences in the amino acid sequence (6–8). Using hIAPP transgenic models of islet amyloid formation, we and others (9–16) have shown that hIAPP expression and/or amyloid deposition is associated with both impaired β-cell function (manifest primarily as hyperglycemia) and reduced β-cell mass.
The regulation of IAPP production and secretion has long been studied and shown to closely mirror that of insulin. Thus, in conditions associated with insulin resistance, and specifically hyperinsulinemia, IAPP levels are elevated (17–19). In the face of β-cell dysfunction, this can potentially lead to aggregation and fibril formation (20). A factor that may further accelerate amyloidogenesis is reduced IAPP and/or amyloid degradation. Studies by Bennett et al. (21,22) have demonstrated that insulin-degrading enzyme degrades exogenous IAPP, so that inhibition of insulin-degrading enzyme activity increases amyloid formation and IAPP-induced cytotoxicity. Although the data are intriguing, these studies are somewhat limited in that they used a model in which IAPP was applied extracellularly to cultured cells.
Recent evidence has implicated another closely related enzyme, neprilysin (also known as neutral endopeptidase, CD10 [a neutrophil cluster-differentiation antigen], or CALLA [common acute lymphoblastic leukemia antigen]), in amyloid degradation. Neprilysin is a 90- to 110-kDa zinc metallopeptidase with a short cytoplasmic NH2 terminus, a transmembrane hydrophobic region and a large extracellular domain containing the active site. Neprilysin is widely expressed, being most abundant in the kidney but also previously identified in the exocrine pancreas of guinea pigs (23). It has broad specificity, and so its physiological role depends on its cellular localization, although it functions primarily as an ectoenzyme catalyzing peptide hydrolysis at the extracellular face of the plasma membrane. Of note, neprilysin has been implicated in degradation of amyloid-β (Aβ), the unique peptide component of Alzheimer’s amyloid (24–26), and the amyloid observed in Alzheimer’s disease and type 2 diabetes have many similarities (20).
To date, there are no studies investigating the role of neprilysin in pancreatic islets. There is, however, one study where neprilysin activity was detected in RINm5F plasma cell membranes (27). RINm5F cells are derived from a rat insulinoma and are commonly used as a pancreatic β-cell model. The study showed that when incubated with RINm5F cell plasma membranes, IAPP was hydrolyzed, although the methodology used did not conclusively identify neprilysin as the enzyme responsible for this peptidase activity. Thus, we hypothesized that neprilysin was present in pancreatic islets and that it plays a role in preventing islet amyloid formation via its degradative activity. To test this hypothesis, we used a hIAPP transgenic mouse model that expresses hIAPP in pancreatic β-cells. In this mouse model, 81% of male mice and only 11% of female mice develop light microscopy–visible islet amyloid after 12–16 months on a 9% fat diet (12). Although we have shown that ovarian products protect against amyloid deposition (28), the specific mechanism has yet to be established.
RESEARCH DESIGN AND METHODS
Transgenic mice.
Hemizygous transgenic mice expressing hIAPP in their pancreatic β-cells (29) were generated by breeding hIAPP transgenic C57BL/6 female mice with DBA/2J wild-type male mice. Transgenic status was determined by PCR of genomic DNA using primers directed against the hIAPP transgene, as previously described (30). Mice were fed a diet containing 18% kcal from fat (9% fat by weight, Purina autoclavable mouse diet no. 5021), which is a moderate-fat diet that has been previously shown to induce islet amyloid deposition primarily in male mice (12). hIAPP transgenic mice and nontransgenic littermate controls were studied at 10 weeks and 6 months of age. The study was approved by the institutional animal care and use committee at the Seattle Veterans Affairs Puget Sound Health Care System.
Isolation of pancreatic islets.
Islets were isolated from the pancreata of female and male mice by collagenase digestion using methods previously described (31). The glucose concentration in the culture media was 11.1 mmol/l. Islets were cultured overnight at 37°C in a humidified atmosphere of 95% air/5% CO2 to allow them to recover from the isolation procedure before being lysed for various measures. For determination of neprilysin levels, islets were pooled from two mice so that both mRNA and activity could be measured from the same sample, allowing for the level of mRNA expression to be directly correlated with enzymatic activity from the same population of islets (n = 4 per group, with each n comprising islets from two mice).
Real-time quantitative RT-PCR.
Gene expression of mouse neprilysin in isolated pancreatic islets was determined with real-time quantitative RT-PCR using the TaqMan system (ABI Prism 7000; Applied Biosystems, Foster City, CA). Total RNA was extracted from isolated islets using a High Pure RNA isolation kit (Roche Applied Sciences, Indianapolis, IN) according to the manufacturer’s instructions. cDNA was then synthesized with RT using standard techniques (high-capacity cDNA archive kit no. 4322171; Applied Biosystems) with random hexamers, dNTPs (deoxyribonucleotide triphosphates), and total RNA. For quantitative RT-PCR, the 25-μl PCR mixture contained: 2 μl template, 9.25 μl H2O, 12.5 μl 2× TaqMan Universal PCR Master Mix, and 1.25 μl TaqMan MGB probe Assays on Demand neprilysin gene expression mix (no. Mm00485028 m1; Applied Biosystems). TaqMan eukaryotic 18S rRNA (no. Hs99999901 s1; Applied Biosystems) was used as endogenous control. The PCR was performed at 50°C for 2 min and then at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Each sample was run in triplicate.
Western blotting analysis.
Isolated islets were treated with lysis buffer (20 mmol/l Tris-HCl, pH 7.5, 150 mmol/l NaCl, 1% Nonidet P-40), sonicated on ice, and centrifuged at 10,000g for 15 min. Supernatants were collected, and 40 μg of protein (as determined by the bicinchoninic acid assay) was fractionated by 10% SDS-PAGE under reducing conditions. Equal protein loading was confirmed by Coomassie Blue staining. Protein was transferred to polyvinylidene difluoride membranes, which were probed with antibody as previously described (31). Both the primary antibody (polyclonal rabbit anti-neprilysin; Chemicon, Temecula, CA) and the secondary antibody polyclonal (goat anti-rabbit IgG coupled to horseradish peroxidase; DAKO, Glostrup, Denmark) were diluted 1:1,000. For specificity control, primary antibody was replaced with normal rabbit serum (1:1,000). Immunoreactive bands were detected by enhanced chemiluminescence as described by the manufacturer (Perkin-Elmer Life Sciences, Boston, MA).
Neprilysin enzymatic activity assay.
Islets were lysed as described for Western blotting, and neprilysin enzyme activity was determined fluorometrically as previously described (23). Briefly, 50 μg of protein was incubated at 37°C with 80 μmol/l glutaryl-ala-ala-phe-4-methoxy-2-naphthylamine (Glu-MNA) and 0.1 μg/μl aminopeptidase M (microsomal from porcine kidney). Glu-MNA was broken down by neprilysin in the islet lysates to Phe-4-methoxy-2-naphthylamine, which was further cleaved by aminopeptidase M to generate the fluorescent product methoxy-2-naphthylamine (MNA). The specific neprilysin inhibitor DL-[N-(3-mercapto-2-benzylpropanoyl)]glycine (DL-thiorphan) (32) was used to differentiate neprilysin enzyme activity from nonspecific endopeptidase activity. Fluorescence was measured at 360-nm excitation and 460-nm emission and compared with an MNA standard curve. Mouse kidney lysates were used as a positive control because neprilysin is most abundant in the kidney.
Plasma and pancreatic peptide measurements.
Plasma for glucose, insulin, and hIAPP-like immunoreactivity was obtained from overnight-fasted animals under pentobarbital anesthesia (100 mg/kg i.p.) before death. At time of death, a small portion of the pancreas was snap frozen for homogenization in 50% (vol/vol) isopropanol/1% (vol/vol) trifluoroacetic acid and subsequent measurement of pancreatic hIAPP-like immunoreactivity, mouse IAPP-like immunoreactivity, and protein content.
Assays.
Plasma glucose was determined using a glucose oxidase method. Plasma insulin levels were measured by radioimmunoassay (30). Plasma and pancreatic hIAPP-like immunoreactivity were quantified by enzyme immunoabsorbance assay, with F024 and F002 as the capture and detection antibodies (kind gift of Amylin Pharmaceuticals, San Diego, CA). Pancreatic mouse IAPP-like immunoreactivity was determined using a previously described radioimmunoassay for rodent IAPP (30). Total protein for determinations of pancreatic hIAPP-like immunoreactivity and mouse IAPP-like immunoreactivity content were assessed using a bicinchoninic acid kit (Pierce Biotechnology, Rockford, IL).
Histological assessment of islet amyloid and insulin.
At the time of death, pancreata were excised and fixed in 4% (wt/vol) phosphate-buffered paraformaldehyde (PFA) and embedded in paraffin. Then, 5-μm sections were cut and costained with thioflavin S to visualize amyloid deposits and with anti-insulin antibody (1:2,000; Sigma, St. Louis, MO) followed by Cy3-conjugated anti-mouse immunoglobulins to visualize islet β-cells. To visualize cell nuclei, sections were counterstained with the nuclear dye Hoechst 33258 (2 μg/ml; Sigma). Histological assessments were made on an average of 18 islets per mouse on three sections representing the head, body, and tail of the pancreas. We have previously shown this sampling technique to be sufficiently representative of a whole mouse pancreas (33). The proportion of insulin area to islet area was determined using a computer-based quantitative method as reported previously (33), with islet area being determined morphometrically by manually outlining each islet when viewed under fluorescence at excitation 480 nm and emission 505 nm (channel used for thioflavin S staining).
Histological assessment of neprilysin localization.
Isolated islets were fixed in 4% (wt/vol) PFA, embedded in agar, and refixed in PFA. Then, 5-μm sections were cut and treated with 0.05% (vol/vol) trypsin for antigen retrieval. After blocking endogenous peroxidase activity with 0.3% (vol/vol) hydrogen peroxide and nonspecific immunoreactivity with 10% (vol/vol) normal goat serum, islet sections were reacted overnight with mouse monoclonal anti-neprilysin antibody (1:80 dilution; Novocastra Labs, Newcastle, U.K.). Polyclonal goat anti-mouse IgG coupled to horseradish peroxidase (1:1,000) was then applied, followed by tyramide signal amplification using a kit according to manufacturer’s instructions (NEN Life Sciences, Boston, MA). Sections were subsequently incubated overnight with mouse anti-insulin antibody (1:1,000) followed by Alexa 488–conjugated anti-mouse immunoglobulins. To visualize cell nuclei, sections were counterstained with Hoechst 33258 (2 μg/ml). For negative controls, primary antibodies were omitted. In addition, the neprilysin staining pattern was confirmed using another anti-neprilysin antibody (polyclonal rabbit anti-mouse neprilysin, 1:100; Alpha Diagnostic International, San Antonio, TX), which yielded results identical to those of the monoclonal mouse anti-neprilysin antibody. Antibody specificity was confirmed by preabsorbing the antibody with excess peptide.
Statistical analyses.
Data are the means ± SE for the number of experiments indicated. Statistical significance was determined using the nonparametric Mann-Whitney U test. P < 0.05 was considered statistically significant.
RESULTS
Identification of neprilysin in mouse islets.
Islets from 10-week-old female and male nontransgenic and hIAPP transgenic mice were isolated and lysed for detection of neprilysin protein (n = 6 per group). Figure 1A shows a representative Western blot with a band migrating at ∼90 kDa corresponding to neprilysin. Mouse kidney was used as a positive control because neprilysin is most abundant in the kidney, and so, as expected, islets contained less neprilysin protein than kidney.
Double-immunofluorescence staining of islets from 10-week-old female and male nontransgenic and hIAPP transgenic mice was used to determine whether neprilysin is localized to the β-cells. Figure 1B shows a representative hIAPP transgenic islet with neprilysin staining in both β-cells and non-β-cells. The staining pattern is largely membranous with some cytoplasmic staining also. In a subpopulation of islet cells, neprilysin staining is more intense. An identical pattern was seen in female and male islets and in nontransgenic islets.
Body weight, plasma measurements, and pancreatic peptide content.
Body weight was significantly greater in hIAPP transgenic and nontransgenic male mice compared with age-matched females at 10 weeks and 6 months of age (Table 1). Consistent with increased body weight, fasting plasma insulin levels were increased in male versus female mice regardless of genotype at both ages. Fasting plasma hIAPP levels and pancreatic content were similar between 10-week-old hIAPP transgenic male and female mice; plasma hIAPP levels tended to be higher, and hIAPP pancreatic content was significantly higher in male versus female hIAPP transgenic mice at 6 months of age. Pancreatic mouse IAPP content mirrored hIAPP content such that the hIAPP-to–mouse IAPP ratio was not different between any of the groups. Fasting plasma glucose levels were similar between hIAPP transgenic male and female mice at 10 weeks of age. In contrast, 6-month-old amyloid-prone hIAPP transgenic mice exhibited fasting hyperglycemia compared with hIAPP transgenic female and nontransgenic male and female mice at 6 months of age.
Detection of amyloid in hIAPP transgenic islets at 10 weeks and 6 months of age and impact on β-cell area.
Thioflavin S staining was used to detect islet amyloid in pancreatic sections from 10- and 6-month-old hIAPP transgenic mice. No amyloid was detected in any of the 10-week-old mice. Similarly, no amyloid was detected in any of the 6-month-old female hIAPP transgenic mice (n = 8). In contrast, in 6-month-old male hIAPP transgenic mice, 43% of the mice studied exhibited islet amyloid (n = 7). The amount of islet amyloid seen at 6 months of age was small, occupying on average <1% of total islet area.
To determine whether the amyloid seen at 6 months of age in hIAPP transgenic male mice impacts β-cell area, pancreas sections from 6-month-old transgenic and nontransgenic mice were immunolabeled for insulin, and then the insulin-positive area as a proportion of total islet area was quantified. As shown in Fig. 2, islets from amyloid-prone 6-month-old male hIAPP transgenic mice displayed reduced β-cell area compared with those from female hIAPP transgenic mice and from both male and female nontransgenic mice (P < 0.01).
Effect of age and sex on neprilysin levels in mouse islets.
Because the 6-month-old hIAPP transgenic male mice exhibited light microscopy–visible amyloid, we sought to determine how this influenced islet neprilysin levels. Ten-week-old mice were used for comparison, as well as nontransgenic littermates at both 10 weeks and 6 months of age. Neprilysin mRNA and activity levels are shown in Fig. 3A and B, respectively. In islets isolated from 10-week-old nontransgenic male and female mice, neprilysin mRNA and activity levels did not differ. Similarly, islet neprilysin mRNA and activity did not differ among 10-week-old hIAPP transgenic male and female mice, nor when compared with age-matched nontransgenic mice. At 6 months of age, neprilysin mRNA levels were significantly decreased in islets isolated from nontransgenic male and female mice compared with neprilysin levels at 10 weeks of age (P < 0.05 for both sexes). Similarly, neprilysin activity was significantly reduced in 6-month-old versus 10-week-old male and female nontransgenic mice (P < 0.05 for both sexes). Six-month-old hIAPP transgenic female mice also exhibited a decrease in both neprilysin mRNA and activity compared with 10-week-old transgenic female mice (P < 0.05). In contrast, in islets from amyloid-prone hIAPP transgenic male mice, neprilysin mRNA and activity levels did not decrease by 6 months of age, exhibiting neprilysin levels comparable to 10-week-old mice. Thus, neprilysin mRNA and activity levels were elevated in 6-month-old hIAPP transgenic male mice compared with age-matched hIAPP transgenic female mice and age-matched nontransgenic mice.
DISCUSSION
Islet amyloid is present in ∼80% of people with type 2 diabetes (17,34), replacing β-cell mass and being associated with impaired β-cell function (1,2). The process of amyloid formation is progressive, and therefore amyloid is typically observed in parallel with worsening β-cell function in type 2 diabetes and also with aging, a condition known to be associated with reduced β-cell function (2,35,36). The mechanism(s) for islet amyloidogenesis is currently unknown; however, data suggest that one possible mechanism may involve changes in enzymes like neprilysin that may degrade either the primary constituent (IAPP) or other components of islet amyloid (e.g., heparan sulfate proteoglycans, apolipoprotein E, serum amyloid P component). This possibility is suggested by findings of reduced neprilysin mRNA and protein levels in high–amyloid plaque–bearing regions of brain from patients with Alzheimer’s disease (37,38), another amyloid-associated disease with many similarities to amyloid in type 2 diabetes (20). We therefore sought to determine whether neprilysin is present and active in islets and whether changes in neprilysin activity are associated with amyloid deposition.
To our knowledge, this is the first report describing production as well as activity of the amyloid-degrading enzyme neprilysin in pancreatic islets, although previously neprilysin activity was detected in a pancreatic β-cell line (27). Our work confirms neprilysin expression in β-cells and shows that neprilysin also exists in non–β-cells of the islet, consistent with the fact that it has previously been detected in a wide range of cells/tissues (39). Although the present data are limited to mouse islets, we have preliminary data (not shown) demonstrating expression of neprilysin in human islets. That neprilysin in islets may be of functional importance is illustrated by the fact that it is able to hydrolyze various proteins produced in pancreatic islet cells like glucagon (40) and angiotensin II (41). Therefore, neprilysin may regulate a number of processes in the islet. Furthermore, it is particularly suited to function in the islet as an amyloid-degrading enzyme because the active site is extracellular and we have shown amyloid fibrils accumulate in the extracellular space between β-cells and capillaries (12).
In the current study, we used a transgenic mouse model that is capable of producing the human form of IAPP in pancreatic β-cells. Earlier studies of these mice revealed that as with human islet amyloid formation (35), the deposition of amyloid increased with age and was associated with the development of hyperglycemia (12). In addition, amyloid was observed predominantly in male mice. Here, we have shown that in vivo at 10 weeks of age, hIAPP transgenic mice do not exhibit light microscopy–visible islet amyloid; however, by 6 months of age, 43% of the transgenic males studied had some amyloid, albeit at much lower levels than typically seen in these mice at 12 months of age. In line with these age- and sex-associated changes in amyloid, we observed changes in neprilysin.
Islet neprilysin levels in 10-week-old mice did not differ between males and females or between genotypes, consistent with the absence of islet amyloid in all mice. At 6 months of age, islet neprilysin levels in the transgenic female mice and nontransgenic mice of both sexes were decreased compared with 10-week-old mice. These findings are in keeping with the well-documented observation that neprilysin levels in the brain decline with age (42–46). In 6-month-old hIAPP transgenic male mice, neprilysin remained at the same level as seen in 10-week-old transgenic male mice (i.e., not decreasing with age as would be expected) and as seen in the nontransgenic mice and transgenic female mice. We believe the sustained elevation of neprilysin in our 6-month-old amyloid-prone hIAPP transgenic male mice represents an injury response that is mediated by the elevated hIAPP levels and is aimed at degrading islet amyloid to reduce its accumulation. This response is similar to that described in a study of neprilysin expression in a transgenic mouse model of Alzheimer’s-related amyloid (47). At an age before the onset of severe amyloid plaque deposition, injection of synthetic fibrillar Aβ into the mouse brains caused an upregulation of neprilysin and clearance of Aβ (47). At an older age, when brains already contained abundant plaques, injection of synthetic fibrillar Aβ also increased neprilysin expression, but this was insufficient to clear the Aβ or existing amyloid (48). Thus, we speculate that it is likely that in islets of 12-month-old hIAPP transgenic male mice, neprilysin levels may still be elevated, but they would be inadequate for clearance of the already severe islet amyloid deposits.
That islets from hIAPP transgenic female mice had lower neprilysin levels compared with transgenic males at 6 months of age is in keeping with them not having visible islet amyloid and with our previous observation of amyloid being a rare finding in hIAPP transgenic female mice (12,28). It is possible that this is related to estrogen’s effect to regulate neprilysin activity, although this seems unlikely. Studies in Sprague-Dawley rats have shown that oophorectomy results in decreased neprilysin activity in brain, kidney, and lung and that estrogen replacement restores neprilysin levels back to those seen in sham surgery control rats (49). In the current study, the finding that the nontransgenic males and females had similar levels of neprilysin at either 10 weeks or 6 months of age excludes an effect of estrogen on regulation of islet neprilysin activity and, in fact, suggests that amyloidogenesis in hIAPP transgenic mice drives the difference in islet neprilysin levels.
Despite elevated neprilysin in hIAPP transgenic male compared with female mice at 6 months of age, amyloid deposits were still detected in the male islets. This was associated with significantly greater fasting plasma glucose levels in the 6-month-old hIAPP transgenic male mice and therefore suggests that amyloid may be an early event contributing to impaired β-cell function and subsequent hyperglycemia. Chronic hyperglycemia is associated with oxidative stress and promotes abnormal posttranslational protein modification. Neprilysin has been shown to be oxidatively modified by 4-hydroxy-2-nonenal (50) and other reactive oxygen species, leading to enhanced susceptibility to proteolytic degradation and inactivation (51). Therefore, though not decreased in the hIAPP transgenic male mice at 6 months of age, neprilysin activity may still be inappropriately low because of oxidative stress–mediated inactivation, which could render the enzyme less effective in degrading islet amyloid.
Because we and others have shown that islet amyloid deposition is associated with reduced β-cell mass (10,11,13–16), insulin-positive islet area was determined as a surrogate for β-cell area. Despite the relatively small amount of amyloid (<1% of islet area) detected in islets from 6-month-old hIAPP transgenic male mice (compared with 9% of islet area at 12 months of age) (10), β-cell area was significantly reduced compared with transgenic female (by ∼11%) and nontransgenic control mice. This reduction in β-cell area seems paradoxical given the amount of amyloid detected using light microscopy; however, it has been suggested that the small IAPP oligomeric form of amyloid, detectable by electron microscopy, is more toxic than mature amyloid fibrils (16,52). hIAPP transgenic males at 6 months of age exhibited higher levels of plasma and pancreatic hIAPP than female transgenic mice, equating to a potentially greater capacity to form toxic oligomers that are not visible by the light microscopy techniques we used.
In summary, we have shown that the amyloid-degrading enzyme neprilysin is present in pancreatic islets and that aging is associated with reductions in neprilysin mRNA and activity. However, amyloid-prone hIAPP transgenic male islets do not exhibit this age-related decline, possibly indicating a compensatory mechanism aimed at reducing the magnitude of islet amyloid deposition. These studies, for the first time, provide insight into the possible role of neprilysin in the degradation of islet amyloid and suggest that development of approaches aimed at reducing and/or preventing the progressive β-cell failure associated with type 2 diabetes could include approaches that involve modulating neprilysin activity.
. | 10 weeks old . | . | 6 months old . | . | . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|
. | Transgenic male . | Transgenic female . | Transgenic male . | Transgenic female . | Nontransgenic male . | Nontransgenic female . | ||||
n | 7 | 9 | 5 | 9 | 4 | 7 | ||||
Body weight (g) | 26.3 ± 1.6 | 19.5 ± 0.5* | 45.6 ± 2.5* | 29.6 ± 1.7† | 43.9 ± 4.0* | 32.4 ± 1.1 | ||||
Fasting glucose (mmol/l) | 9.59 ± 0.51 | 9.10 ± 0.32 | 14.37 ± 1.79* | 9.40 ± 0.37† | 9.00 ± 0.44† | 10.90 ± 0.62† | ||||
Fasting insulin (pmol/l) | 196.6 ± 25.6 | 91.7 ± 14.3* | 305.5 ± 80.5 | 65.0 ± 8.1† | 383.9 ± 97.1 | 68.1 ± 7.9 | ||||
Fasting hIAPP (pmol/l) | 11.8 ± 1.8 | 11.8 ± 1.3 | 60.8 ± 10.6 | 43.1 ± 10.0 | ND | ND | ||||
Pancreatic hIAPP (pmol/mg protein) | 5.5 ± 1.2 | 7.2 ± 1.0 | 12.0 ± 1.8* | 5.3 ± 2.0† | ND | ND | ||||
Pancreatic mIAPP (pmol/mg protein) | 4.1 ± 0.7 | 5.3 ± 1.1 | 8.1 ± 1.2* | 4.6 ± 1.5 | 5.7 ± 2.1 | 2.5 ± 0.4 | ||||
Pancreatic hIAPP-to-mIAPP ratio | 1.3 ± 0.3 | 1.6 ± 0.1 | 1.5 ± 0.2 | 1.2 ± 0.2 | ND | ND |
. | 10 weeks old . | . | 6 months old . | . | . | . | ||||
---|---|---|---|---|---|---|---|---|---|---|
. | Transgenic male . | Transgenic female . | Transgenic male . | Transgenic female . | Nontransgenic male . | Nontransgenic female . | ||||
n | 7 | 9 | 5 | 9 | 4 | 7 | ||||
Body weight (g) | 26.3 ± 1.6 | 19.5 ± 0.5* | 45.6 ± 2.5* | 29.6 ± 1.7† | 43.9 ± 4.0* | 32.4 ± 1.1 | ||||
Fasting glucose (mmol/l) | 9.59 ± 0.51 | 9.10 ± 0.32 | 14.37 ± 1.79* | 9.40 ± 0.37† | 9.00 ± 0.44† | 10.90 ± 0.62† | ||||
Fasting insulin (pmol/l) | 196.6 ± 25.6 | 91.7 ± 14.3* | 305.5 ± 80.5 | 65.0 ± 8.1† | 383.9 ± 97.1 | 68.1 ± 7.9 | ||||
Fasting hIAPP (pmol/l) | 11.8 ± 1.8 | 11.8 ± 1.3 | 60.8 ± 10.6 | 43.1 ± 10.0 | ND | ND | ||||
Pancreatic hIAPP (pmol/mg protein) | 5.5 ± 1.2 | 7.2 ± 1.0 | 12.0 ± 1.8* | 5.3 ± 2.0† | ND | ND | ||||
Pancreatic mIAPP (pmol/mg protein) | 4.1 ± 0.7 | 5.3 ± 1.1 | 8.1 ± 1.2* | 4.6 ± 1.5 | 5.7 ± 2.1 | 2.5 ± 0.4 | ||||
Pancreatic hIAPP-to-mIAPP ratio | 1.3 ± 0.3 | 1.6 ± 0.1 | 1.5 ± 0.2 | 1.2 ± 0.2 | ND | ND |
Data are means ± SE.
P < 0.05 vs. 10-week-old transgenic male;
P < 0.05 vs. 6-month-old transgenic males. mIAPP, mouse IAPP; ND, not determined.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Article Information
This work was supported by research funding from the Department of Veterans Affairs. S.Z. was supported by an American Diabetes Association Mentor-Based Fellowship.
We thank Breanne Barrow, Rahat Bhatti, Mike Peters, Robin Vogel, Jira Wade, Melissah Watts, Shani Wilbur, and Joshua Willard for excellent technical support.