Transgene expression in hProCpepGFP and hProC(A7)Y-CpepGFP mice.
Two transgenic lines were used. The hProCpepGFP mouse carries a GFP-labeled proinsulin (17
), whereas the hProC(A7)Y-CpepGFP mouse carries a similar GFP-labeled proinsulin bearing the misfolding-inducing Akita
mutation. In islets isolated from the two mouse lines, we examined an abundance of mRNAs encoding these GFP-labeled preproinsulins and the endogenous Ins2
mRNA levels (Ins2
represents the majority of total insulin transcripts in mouse islets [20
]). Using qPCR (normalized to β-actin mRNA) with concentration standards of hProCpepGFP plasmid DNA, we confirmed that the levels of hProCpepGFP and hProC(A7)Y-CpepGFP mRNA represented only 5.5 and 4.2%, respectively, of that measured for endogenous Ins2
We examined pancreatic cryosections from neonatal male hProCpepGFP and hProC(A7)Y-CpepGFP mice (sex determined by Y-chromosome PCR) using intrinsic GFP fluorescence and insulin immunofluorescence. At postnatal day 1, hProCpepGFP and hProC(A7)Y-CpepGFP male mice demonstrated GFP fluorescence (either properly folded or misfolded mutant proinsulin) in essentially all of the cells that were immunolabeled for endogenous insulin + proinsulin, i.e., in the vast majority of β-cells (; quantified in ). Thus, the data in indicate that the two animal models demonstrate comparable levels and patterns of transgene mRNA and protein expression across the β-cell population in vivo.
FIG. 7. Quantification of the distribution of endogenous and GFP-labeled proinsulin + insulin among the cells within islets of neonatal and adult transgenic male mice. Islet β-cells were scored for cells in which both endogenous and GFP-labeled proinsulin (more ...)
Most male hProC(A7)Y-CpepGFP transgenic mice avoid frank diabetes.
We compared body weights and random glucose levels of hProC(A7)Y-CpepGFP transgenic mice with those of hProCpepGFP mice that are phenotypically identical to C57BL/6 littermates (17
). Mean body weight of hProC(A7)Y-CpepGFP males was similar to that of normal control subjects, and mean random glucose of hProC(A7)Y-CpepGFP males (between 8:00 a.m.
and 12:00 p
.) was only modestly elevated and significantly lower than hProCpepGFP Akita
males (). A histogram of individual males of each genotype showed that random blood glucose levels in the vast majority (>85%) of hProC(A7)Y-CpepGFP males had a distribution largely overlapping with that of normal controls (). Although a small subset of males (~10% of total) exhibited random hyperglycemia, the results indicate that the majority of animals with transgenic expression of hProC(A7)Y-CpepGFP (driven by the Ins1
promoter) do not have frank diabetes (), i.e., a considerably milder phenotype than Akita
mice who bear the same mutation in one of the endogenous Ins2
FIG. 2. Blood glucose levels in male mice (median age 3.5 months). A: Random blood glucose was obtained by tail snip from males of the different genotypes indicated. Mean random glucose of hProC(A7)Y-CpepGFP transgenic males was significantly higher than that (more ...)
To identify subtler defects in islet performance in hProC(A7)Y-CpepGFP males, we subjected the animals to intraperitoneal glucose tolerance testing after an overnight fast. Although fasting glucose values were similar to those of hProCpepGFP and C57BL/6 control males, the hProC(A7)Y-CpepGFP males had a significantly higher mean blood glucose at 30 min poststimulation than control subjects (309 ± 32.6 vs. 253.1 ± 16.1 mg/dL; P = 0.03). Although a significant difference was not sustained at 120 min postchallenge, the results indicate that hProC(A7)Y-CpepGFP mice have impaired glucose tolerance ().
Homozygous knockout of the endogenous Ins2
gene (with homozygous expression of wild-type Ins1
still remaining) itself does not cause diabetes (20
). Therefore, we were interested to use homozygous Ins2
knockout to examine the effect of increasing the relative expression of mutant hProC(A7)Y-CpepGFP (transgene) to wild-type insulin. In this background, mice with transgenic expression of wild-type hProCpepGFP were normoglycemic, whereas mice with transgenic expression of hProC(A7)Y-CpepGFP developed hyperglycemia that was particularly severe when both Ins2
alleles were missing, including the cohort of female animals (, bottom
). Thus, although the majority of mice exhibit sufficient compensation to accommodate low-level expression of misfolded proinsulin, these animals are nevertheless predisposed to diabetes, and the relative expression levels of misfolded and wild-type proinsulin dictate the phenotype ().
FIG. 3. Mean random glucose of transgenic male and female mice partially or completely devoid of endogenous Ins2. Transgenic mice were first mated with complete Ins2 knockout animals to generate hProCpepGFP or hProC(A7)Y-CpepGFP, Ins2+/− animals (first (more ...)
In nondiabetic hProC(A7)Y-CpepGFP mice, the production and maturation of endogenous proinsulin is perturbed by transgenic expression of misfolded mutant proinsulin.
To determine whether pancreatic insulin content was comparable in nondiabetic hProC(A7)Y-CpepGFP transgenic mice and nonmutant control subjects, we measured total insulin + proinsulin (endogenous and transgenic) in isolated pancreatic islets. By RIA, islet insulin + proinsulin content per cell was significantly less than that measured for hProCpepGFP transgenic controls (), despite that all islets were derived from nondiabetic animals. In the small subset of diabetic hProC(A7)Y-CpepGFP males, the insulin + proinsulin content was lower still (<5% of control).
We also used an independent assay to follow proinsulin (and insulin) content in isolated islets incubated for 48 h in either 4 or 16.7 mmol/L glucose. Islet lysates were analyzed by reducing SDS-PAGE, electrotransferred to nitrocellulose, and immunoblotted with either anti-GFP (to follow the transgene product) or anti-insulin (to follow the endogenous product). In islets from both hProCpepGFP and hProC(A7)Y-CpepGFP mice, protein encoded by the transgene was up-regulated in response to incubation at high glucose. As expected, hProC(A7)Y-CpepGFP did not exhibit maturation, as measured by its inability to be endoproteolytically converted to CpepGFP, whereas hProCpepGFP was processed in secretory granules to CpepGFP (). Endogenous proinsulin protein expression also was increased by high glucose; however, the amount of proinsulin was markedly less in islets of hProC(A7)Y-CpepGFP transgenic mice (). The immunoblotted insulin band in islets of hProC(A7)Y-CpepGFP transgenic mice did not appear to be greatly affected by the high-glucose incubation; however, we found that when loading amounts that linearly report changes in proinsulin content (, inset), insulin band-intensity changes were rather insensitive and may underestimate the insulin recovery defect in hProC(A7)Y-CpepGFP islets. Indeed by rodent proinsulin–specific RIA, we found that proinsulin content of the islets was 1.26% of total hormone content for both hProCpepGFP and C57BL/6 mice and 1.4% for nondiabetic hProC(A7)Y-CpepGFP mice. Altogether, the data of suggest that the presence of misfolded mutant proinsulin decreases the levels of endogenous bystander proinsulin (), concomitant with a decrease in islet insulin that occurs even in nondiabetic animals ().
Islets of nondiabetic hProC(A7)Y-CpepGFP transgenic mice activate ER stress response.
To determine whether expression of misfolded mutant hProC(A7)Y-CpepGFP below the diabetogenic threshold triggers ER stress response, mRNA levels for BiP and spliced XBP-1 were measured in freshly isolated islets from nondiabetic hProCpepGFP and hProC(A7)Y-CpepGFP transgenic mice. Indeed, islets of hProC(A7)Y-CpepGFP mice had significantly increased BiP mRNA, as well as a tendency to increased splicing of XBP-1 (). Furthermore, hProC(A7)Y-CpepGFP islets also exhibited a trend suggesting increased mRNA for CHOP (), a downstream component of the unfolded protein response pathway that predisposes to cell death (21
). The results suggest that proinsulin misfolding by hProC(A7)Y-CpepGFP does induce ER stress, even in the absence of or before frank diabetes.
FIG. 5. ER stress response activation in islets of hProC(A7)Y-CpepGFP transgenic mice. Freshly isolated islets were extracted for RNA, followed by synthesis of reverse-transcribed cDNAs. Specific primers for BiP, spliced XBP1, total XBP1, and CHOP were used to (more ...)
In hProC(A7)Y-CpepGFP transgenic mice, a subpopulation of β-cells in each islet exhibits misfolded proinsulin accumulation and poor endogenous insulin production.
A recent report of PERK knockout mice has shown that a subpopulation of islet β-cells exhibits an “impacted-ER” phenotype characterized morphologically by an expanded ER with accumulation of proinsulin (4
), consistent with ER crowding (1
). Because of the evidence of ER stress in islets of nondiabetic hProC(A7)Y-CpepGFP mice (), we looked for morphological correlates in the β-cells of these animals. In cryosections of adult male transgenic mice, hProCpepGFP or hProC(A7)Y-CpepGFP protein expression was detected by intrinsic GFP fluorescence, whereas anti-insulin immunostaining was used to reflect endogenous insulin protein.
As in neonatal males (), islets of hProCpepGFP adult males (17
) expressed the GFP-positive transgene product in virtually all β-cells, which were simultaneously positive for endogenous insulin (; quantified in ). Remarkably, in contrast to neonatal animals, islets of nondiabetic hProC(A7)Y-CpepGFP males reproducibly exhibited heterogeneous β-cell subpopulations (). Approximately 40% of the β-cells accumulated the fluorescent, misfolded mutant proinsulin, which, at higher magnification, exhibited an ER distribution pattern (). Within each islet, β-cells rich in hProC(A7)Y-CpepGFP fluorescence tended to be largely devoid of insulin immunostaining; another major subpopulation of β-cells exhibited little GFP fluorescence and immunostained clearly for endogenous insulin; and only a small, third subpopulation accumulated both types of molecules ().
FIG. 6. Distribution of endogenous and GFP-labeled proinsulin + insulin among the cells within islets of adult males. A: Immunostaining with anti-insulin and GFP epifluorescence (performed as described in research design and methods) in an adult hProCpepGFP male. (more ...)
To begin to understand the origin of the heterogeneity of hProC(A7)Y-CpepGFP accumulation within the β-cell population, we also examined hProC(A7)Y-CpepGFP mRNA distribution within the islets by in situ hybridization with a specific complementary oligonucleotide RNA probe. As exemplified in , as much as 60% of transgene mRNA–positive β-cells within the population exhibited little or no hProC(A7)Y-CpepGFP fluorescence. Evidently, the other ~40% subpopulation of β-cells with misfolded proinsulin accumulation and little or no insulin immunostaining ( and ) develops during postnatal life. Even if the larger subpopulation of β-cells that failed to accumulate or had not yet accumulated significant quantities of misfolded proinsulin within the ER () contained all of the islet insulin that is recovered (), the insulin content, even in this subpopulation of β-cells, must still be decreased compared with that of hProCpepGFP control islet β-cells.
As the islets of hProC(A7)Y-CpepGFP mice contained significantly increased BiP mRNA (), we also looked at the distribution of BiP protein compared with islets of hProCpepGFP mice. Although BiP was increased in many β-cells of hProC(A7)Y-CpepGFP mice, a heterogeneous pattern within the islets was observed (, left). BiP tended to be increased in cells that had accumulated hProC(A7)Y-CpepGFP, but BiP was also increased in a few other cells that had not accumulated green fluorescence (, right), suggesting that these cells also were synthesizing increased amounts of misfolded secretory protein.
To examine β-cell heterogeneity at the ultrastructural level, we performed transmission electron microscopy of the islets of nondiabetic hProC(A7)Y-CpepGFP islets. Populations of highly granulated β-cells were readily identified (, β-cell #1). However, side by side with such cells were other β-cells with a highly expanded ER and many fewer (but definite) insulin secretory granules (, β-cell #2). Upon close inspection, dilation of the ER was also seen in the β-cells that retained abundant insulin secretory granules (, β-cell #1). β-Cells with a dramatically expanded ER compartment tended to have unusually small insulin secretory granule profiles (, microgranules). Finally, we were also surprised to discover the unique morphological appearance of a third type of β-cell that also had insulin microgranules but lacked an expanded ER compartment; rather, such cells exhibited a highly shrunken cytoplasm (, β-cells #2 and #3). It is not clear whether this third kind of β-cell has either sufficient hProC(A7)Y-CpepGFP protein or endogenous insulin protein to be detected either by GFP fluorescence or insulin immunofluorescence as was measured in and .
Islets of nondiabetic hProC(A7)Y-CpepGFP mice are hyperplastic.
To further clarify whether the decreased islet insulin is a consequence of a decrease of insulin within β-cells or a decrease in islet size and a resultant decrease of overall β-cell numbers, we examined multiple random pancreatic tail cryosections of 3–6-month-old nondiabetic hProCpepGFP and hProC(A7)Y-CpepGFP male mice. Islets contained within 0.2-cm2 pancreatic cross-sections from three different mice of each genotype were identified, and, by use of GFP fluorescence plus immunostaining with anti-insulin, the islet boundaries were determined and the islet cell nuclei counted by DAPI staining. (Antiglucagon immunostaining was also performed in both sets of mice [not shown], confirming that α-cells did not exceed 10% of islet cells in either mouse line.)
On average, the random cross-sectional islet area of nonmutant hProCpepGFP mice (n = 21 islets) was 2,545 µm2 (±276 SEM), similar to that obtained from C57BL/6 control mice (not shown), whereas random cross-sectional islet area of hProC(A7)Y-CpepGFP mice (n = 25 islets) averaged 4,221 µm2 (±793 SEM), a considerable (65%) increase. On average, each hProCpepGFP islet cross-section contained 40.0 (±6.2 SEM) β-cell profiles, whereas each hProC(A7)Y-CpepGFP islet cross-section contained 62.8 (±23.7 SEM) β-cell profiles. Dividing average islet cross-sectional area by average number of β-cell profiles per cross-section, a rough estimate of average β-cell cross-sectional area was essentially unchanged between the two mouse lines. Thus, rather than β-cell hypertrophy, the data indicate an expansion of β-cell number per islet, suggesting β-cell hyperplasia in compensation for expression of misfolded proinsulin to help these animals avoid diabetes. Even when considering that many islet β-cells accumulating fluorescent misfolded mutant proinsulin have little or no endogenous insulin (), there was not an actual loss of insulin-positive β-cells in nondiabetic hProC(A7)Y-CpepGFP islets, indicating that decreased islet insulin content in these animals () must be caused by a decrease of insulin content per β-cell.