292-OR: Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets

It has become increasingly appreciated that inflammatory cytokines promote islet dysfunction in both type 1 and type 2 diabetes. Aberrant secretion of the insulin precursor proinsulin is observed in patients with both types of diabetes and may contribute to proinsulin autoimmunity in T1D, yet the mechanisms responsible for proinsulin release remain obscure. To investigate the effects of inflammation on proinsulin trafficking, human islets were treated ex vivo with the diabetes associated cytokines, Interleukin-1-beta and Interferon-gamma, provoking a four-fold release of proinsulin. To identify proinsulin interacting proteins responsible for aberrant trafficking, intracellular proinsulin was affinity purified and its interactome defined by mass spectrometry. Cytokines altered the identify or magnitude of proinsulin’s engagement with binding partners at every stage of the canonical proinsulin biosynthetic pathway that we recently defined in normal human islets. Of particular relevance to potential avenues of proinsulin escape, cytokines promoted proinsulin interactions with: 1) antigen presentation loading complex components TAP2 and calnexin 2) stress granule assembly factors G3BP1, Ataxin-2 and IGF2BP2, that are also found in secreted exosomes, and 3) the immature insulin granule marker and T1D autoantigen ICA1/ICA169. Pathway analysis further revealed that cytokine treatment increased proinsulin binding, even if indirectly, to multiple KIF family microtubule motor proteins. Given that stress granules and immature granules are sensitive to microtubule dynamics we tested the role of microtubules in cytokine induced proinsulin release. Microtubule destabilization with Nocodazole significantly increased proinsulin release above that provoked by cytokines alone. Together, the data quantitatively map the cytokine reshaped proinsulin interactome and provide new insight into how an inflammatory environment can promote proinsulin escape from human islets.