Islet β-cell dysfunction has long been thought to precede the development of type 1 diabetes. In both NOD mice and humans, loss of first-phase insulin secretion in response to a glucose load has been observed in the prediabetic period (7
). Importantly, findings from the Diabetes Control and Complications Trial showed that individuals entering the study with residual β-cell function had lower rates of hypoglycemia and diabetic microvascular complications (33
). Elucidating the underlying molecular pathways causing β-cell dysfunction in type 1 diabetes might suggest a therapeutic approach to preserving insulin secretion and thereby reducing diabetes complications. No clear mechanism linking immune cell infiltration and islet dysfunction has been definitively identified to date. In this study, we used a well-established mouse model of type 1 diabetes to demonstrate the activation of the NF-κB and ER stress pathways in β-cells of prediabetic NOD mice. The activation of these pathways may accelerate β-cell death in the prediabetic phase of the disease and thus promote further antigen exposure and T cell activation.
In autoimmune type 1 diabetes, the occurrence of β-cell ER stress has remained largely speculative. Given the striking insulitis seen in NOD mice, it is tempting to speculate a role for proinflammatory cytokines—released locally by invading macrophages and helper T cells—in initiating inflammation in the β-cell (via NF-κB) that eventually leads to the early defects in insulin secretion. The cross-talk between inflammation and ER stress has been the focus of several studies involving β-cell lines and islets, but has never been demonstrated in islets of NOD mice. We show here that NOD islets exhibit striking activation of both NF-κB signaling and ER stress pathways. However, it is difficult to know precisely whether NF-κB signaling leads to ER stress or vice-versa (34
). Nevertheless, a hallmark of ER stress is the phosphorylation of the translational initiation factor eIF2-α (35
), an event that is also seen upon the incubation of β-cell lines with IL-1β or a mixture of proinflammatory cytokines (36
). Phosphorylation of eIF2-α blocks global translational initiation in an effort to mitigate ER protein load (31
). In this study, we show for the first time by polyribosomal profiling analysis and 35
S-Met/Cys uptake that MIN6 β-cells exhibit a decline in global translational initiation in response to chronic (days) exposure to proinflammatory cytokines, an effect that is similar to that observed following thapsigargin exposure.
Precisely how cytokines might cause ER stress is not clear, but some studies suggest that the nitric oxide generated via iNOS downregulates SERCA2B (11
). SERCA2B is an ATP-dependent Ca2+
pump that is partially responsible for transport of Ca2+
into the ER lumen, thereby maintaining a steep ER:cytosolic Ca2+
). Although our studies using iNOS inhibitors did not reverse the translational initiation blockade of prolonged cytokine exposure in vitro, they do not entirely rule out a contributory role for nitric oxide, as defects in the expression of other crucial ER proteins (e.g., Wfs1 and ATF4) and the confluence of other stress-induced proteins (e.g., NAPDH oxidase [40
]) may contribute in the longer-term to translational blockade. ER stress has also been shown to activate NF-κB (34
); thus, a self-perpetuating cascade may be occurring in prediabetic NOD β-cells that serves to promote defects in insulin secretion.
Prior studies have shown that β-cell mass is reduced (~30%) in prediabetic NOD mice compared with NOD-SCID mice, suggesting that reductions in β-cell mass (independently of the defects in glucose-stimulated insulin release we have shown here) may also contribute to the relative glucose intolerance of prediabetic NOD mice (9
). Our study does not address whether this reduction in β-cell mass in prediabetic NOD mice is a direct consequence of ER stress. Interestingly, a recent study by Satoh et al. (14
) showed that global Chop
deletion on the NOD background did not protect against β-cell loss or diabetes development. Moreover, studies using isolated rodent and human islets suggest that ER stress may not directly contribute to β-cell death, but rather to insulin secretory defects (11
). In this respect, our results correlate the extent of ER stress with severity of β-cell secretory deficiency in prediabetic NOD mice.
Finally, a notable finding in our study is the reduction in Pdx1 mRNA and protein levels in 10-week-old NOD mice. Stoffers and colleagues (20
) recently showed that Atf4
are directly activated by Pdx1 in β-cells; both of these genes were reduced in our 8- and 10-week-old NOD animals. Hence, as in type 2 diabetes, Pdx1 levels may serve as a barometer of β-cell stress susceptibility in type 1 diabetes. Interestingly, Pdx1
mRNA levels were also reduced in NOD-SCID mice, which correlated with mild deficiencies in islet glucose responsiveness in vitro and elevated levels of Bip
mRNA, and serum proinsulin compared with CD1 mice. These findings may be referable to an inherent genetic feature of the NOD β-cell that enhances susceptibility to proinflammatory cytokines; indeed, although NOD-SCID mice have dysfunctional T and B cells, their intact macrophage activity and persistent TNF-α secretion (41
) may contribute to mild β-cell dysfunction in these animals.
Our data support the conclusion that, independent of large reductions in β-cell mass, dysfunction of the islet β-cell precedes the onset of frank hyperglycemia in NOD mice. Although further studies are clearly warranted to clarify the precise contribution of the ER stress cascade to β-cell loss in type 1 diabetes, our studies suggest that approaches that reduce sources of ER stress or enhance the ability of the β-cell to intrinsically cope with ER stress may preserve β-cell function in the setting of autoimmunity.