Conditional disruption of Wfs1 in pancreatic islet beta cells and determination of tissue specificity
To generate a conditional Wfs1 mouse model of Wolfram syndrome, we designed a targeting vector that contained loxP sites flanking exon 8 of the gene (Fig. 1a). Exon 8 encodes the transmembrane domain and two-thirds of the protein, and the majority of the mutations in affected individuals are in this exon. Mice containing loxP sequences flanking exon 8 of the gene were crossed with mice expressing Cre recombinase under the control of the rat insulin promoter (RIP2-Cre) .
To confirm the deletion of Wfs1 in pancreatic islets, lysates from animals lacking Wfs1 in insulin-producing cells (βWfs
−/−) and littermate loxP/loxP (control) animals were subjected to immunoblotting using an antibody that recognises an epitope encoded by exon 8 (Fig. 1b). Expression of Wfs1 in non-islet tissues appeared to be preserved (Fig. 1c). These data suggest that the Cre-loxP system resulted in mice with marked deficiency of the Wfs1 protein specifically in pancreatic islets.
General phenotypic characteristics
−/− mice were born at expected Mendelian ratios with no differences in reproductive success. βWfs
−/− animals and control animals had similar weights during the first 16 weeks of life, but at 24 weeks the βWfs
−/− animals had lower weights when compared with control mice (Table 1, p<0.05). The reduction in weight at 24 weeks occurred at a time when the βWfs
−/− mice had developed hyperglycaemia and insulinopenia relative to controls (Table 1).
Measurement of blood glucose, insulin and glucose tolerance
At 8, 12, 16 and 24 weeks of age, mean fasted glucose concentrations did not differ between βWfs
−/− and control animals (Table 1). Fed glucose concentrations were significantly higher in βWfs
−/− animals only at 24 weeks (p<0.05). Fasting insulin levels were significantly reduced in the βWfs
−/− mice at 12 weeks (p<0.05). Fed insulin concentrations were significantly reduced in 12- and 24-week-old βWfs
−/− mice (p<0.05). Both fasting and fed glucose : insulin ratios were elevated in βWfs
−/− mice at 12 and 24 weeks of age (p<0.05), further illustrating the insulinopenia for the degree of hyperglycaemia in these mice.
Glucose tolerance did not differ between the two groups at 8 weeks (Fig. 2a). By 12 weeks, mild glucose intolerance 60 min after glucose injection was observed in βWfs
−/− animals (Fig. 2b). Glucose tolerance in 16-week-old βWfs
−/− animals was significantly impaired 30, 60 and 120 min after glucose injection (Fig. 2c, p<0.05). By 24 weeks, nearly all βWfs
−/− animals exhibited significant glucose intolerance (p<0.01) (Fig. 2d). Moreover, two of 13 animals had developed severe fasting hyperglycaemia by this age, one of which died at 20 weeks of age. The RIP2-Cre mice used in these studies had glucose tolerance that was not different from the loxP/loxP littermate control mice or wild-type mice (data not shown). The results of these experiments indicated that conditional inactivation of Wfs1 in pancreatic beta cells results in progressive glucose intolerance and diabetes.
Impaired glucose-stimulated insulin secretion in βWfs
To further assess beta cell function in βWfs
−/− mice, insulin secretion was evaluated. As seen in Fig. 3a, the glucose-stimulated insulin responses were significantly impaired in βWfs
−/− animals (p<0.05). Comparison of the glucose : insulin ratio at 30 min further illustrated the impairment in insulin secretion in the βWfs
−/− animals (Fig. 3b).
Alterations in islet morphology and beta cell mass
Immunostaining for insulin and non-beta cells (somatostatin, glucagon and pancreatic polypeptide) showed that control animals at 12 and 24 weeks had the characteristic appearance of islets, with abundant beta cells in the centre and a rim of non-beta cells at the periphery (Fig. 4a). In contrast, although the beta cell mass did not differ between βWfs
−/− and control (Fig. 4b), asymmetry and disruption of islet architecture was already apparent and was correlated with the relative hypoinsulinaemia at 12 weeks of age. At 24 weeks, there was further disruption of islet architecture, with marked alteration of the ratio of beta to non-beta cells within the islet. Pancreas weight in 12- and 24-week-old mice was not different between the groups (Table 1). Neither the beta cell : pancreas ratio nor beta cell mass was different at 12 weeks (Table 1 and Fig. 4b). Reduction of the beta cell : pancreas ratio and beta cell mass in βWfs
−/− mice was observed at 24 weeks of age, suggesting that the decrease in beta cell mass was one of the components responsible for diabetes in βWfs
−/− mice (Table 1 and Fig. 4b).
Assessment of apoptosis and proliferation
In the βWfs
−/− mice at 24 weeks of age the apparent beta cell dysfunction could result from impaired proliferation or enhanced apoptosis of beta cells. Pancreatic islets were examined by immunohistochemistry with an antibody to the active subunit of cleaved caspase 3 [23, 24]. A significant increase in caspase 3-positive nuclei in βWfs
−/− animals compared with control animals at 12 weeks of age was noted (p<0.05) (Fig. 5a). This 2.5-fold increase in caspase 3-positive cells in βWfs
−/− animals could be an explanation for the reduced beta cell mass and diabetes noted in this model. The frequency of beta cells that were BrdU- and insulin-positive revealed no differences between control and βWf
−/− animals at 12 weeks or 24 weeks (Fig. 5b).
Increased ER stress in beta cells from βWfs
ER stress was assessed by measuring the relative levels of expression of mRNA encoding the ER stress-associated proteins BiP (Hspa5) and CHOP (Ddit3). Given that markedly fewer beta cells in knockout animals, ER stress mRNA expression was normalised to Slc2a2 mRNA instead of 18S rRNA to reflect more accurately the expression of ER stress proteins, specifically in beta cells. Slc2a2 expression was decreased in proportion to the decrease in beta cell mass when normalised to 18S RNA, suggesting that Slc2a2 expression was not significantly altered (Fig. 5c). When corrected for Slc2a2 expression as a reflection of the percentage of beta cells per islet, Hspa5 expression was significantly elevated in islets from βWfs
−/− animals compared with controls (p<0.05), whereas Ddit3 expression appeared elevated but did not reach statistical significance (Fig. 5c).
To further substantiate the increased levels of ER stress in beta cells from βWfs
−/−, pancreas was examined by electron microscopy (Fig. 6). The beta cells were distinguished from alpha and delta cells by the appearance of the secretory granules. The beta cell granules have a white halo, which is not apparent in alpha or delta cells. Homogeneous distribution of secretory granules was observed in beta cells from control animals (Fig. 6a). Ultrastructural analysis of βWfs
−/− pancreas revealed striking abnormalities in beta cell morphology, with some cells more affected than others (Fig. 6b). Higher magnification more clearly shows that beta cells from control mice exhibit abundant secretory granules, with the normal morphological appearance of the ER (Fig. 6b and c). In contrast, beta cell secretory granules from βWfs
−/− islets appeared to be reduced and the majority of the beta cells exhibited abundant dilated ER (Fig. 6b and c). These abnormalities were more significant in some beta cells from βWfs
−/− mice and were never observed in multiple sections from age-matched control animals.
Studies using MIN6 cells containing knockdown of Wfs1
To further elucidate the molecular mechanisms of deficiency of Wfs1 expression on beta cell survival and insulin secretion, stable cell lines with reduced Wfs1 expression were created using RNA interference in MIN6 insulinoma cells. The reduction in Wfs1 expression was confirmed in two cell lines relative to that in two control cell lines. The controls for these experiments included MIN6 cells stably transfected with pSUPER plasmid containing no additional sequences (MIN6-Con(E)) and a second control containing pSUPER with a sequence identical to Wfs1-specific sequence, except for two mismatched base pairs (MIN6-Con(S)), which served as a scrambled control. Both control lines had no alterations in expression of Wfs1 (Fig. 7a). Cell lines with 50% (WfsKD50) and 70% (WfsKD70) reduction in Wfs1 expression and Wfs1 production (Fig. 7a and b, respectively) were used for these experiments.
Evaluation of apoptosis in cell lines with reduced expression of Wfs1
To confirm the role of apoptosis in the alterations of beta cell mass found in vivo, we next evaluated the in vitro model of Wfs1 deficiency. Apoptosis was measured by the number of annexin V-positive cells counted by flow cytometry. As seen in Fig. 7c, the two knockdown cell lines, WfsKD50 and WfsKD70, exhibited increased apoptosis when cultured under standard conditions (19.8±3.6% and 17.3±1.3% respectively compared with MIN6-Con(E) control, 8.3±0.7%; p<0.05). Thus, the in vitro observations were consistent with the in vivo results and supported the conclusion that Wfs1 is required for pancreatic islet beta cell survival.
Evaluation of ER stress in WfsKD cells
The expression of the gene encoding the molecular chaperone BiP (Hspa5), a protein associated with ER stress, was elevated in both knockdown cell lines (p<0.05) (Fig. 7d). The expression of the gene for CHOP (Ddit3), a protein integral in the apoptosis pathway that accompanies ER stress, was also significantly elevated in the WfsKD70 cells (p<0.05) (Fig. 7e). These data are again consistent with those seen in the in vivo model, further suggesting that ER stress is associated with apoptosis in these models.
Determination of insulin secretion of in WfsKD cells
The in vivo model suggested a defect in insulin secretion (Fig. 3a). The in vitro system allowed the assessment of insulin secretion in the absence of metabolic abnormalities induced by chronic hyperglycaemia in vivo. Following correction for insulin content, there was no difference in glucose-stimulated insulin secretion between knockdown cell lines and controls (data not shown).