Avoiding the loss of functional beta cell mass is critical for preventing or treating diabetes. Currently, the molecular mechanisms underlying beta cell death are partially understood, and there is a need to identify new targets for developing novel therapeutics to treat diabetes. Previously, our group established that Mig6, an inhibitor of EGF signaling, mediates beta cell death under diabetogenic conditions. The objective here was to clarify the mechanisms linking diabetogenic stimuli to beta cell death by investigating Mig6-interacting proteins. Using co-immunoprecipitation and mass spectrometry, we evaluated the binding partners of Mig6 under both normal glucose (NG) and glucolipotoxic (GLT) conditions in beta cells. We identified that Mig6 interacted dynamically with NumbL, whereas Mig6 associated with NumbL under NG, and this interaction was disrupted under GLT conditions. Further, we demonstrated that the siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking the activation of NF-κB signaling. Using co-immunoprecipitation experiments, we observed that NumbL’s interactions with TRAF6, a key component of NFκB signaling, were increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 were dynamic and context-dependent. We proposed a model wherein these interactions activated pro-apoptotic NF-κB signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicated that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

Type 1 Diabetes (T1D) is caused by autoimmune-mediated beta cell destruction. Following beta cell injury, the pancreas attempts to launch a cellular repair and regenerative program, yet it fails to completely restore functional beta cell mass. One component of this regenerative program is epidermal growth factor receptor (EGFR) signaling. However, upon irreparable beta cell damage, EGFR signaling is dampened, disrupting attempts to restore functional beta cell mass and maintain normoglycemia. We previously demonstrated that the negative feedback inhibitor of EGFR, Mitogen-inducible gene 6 (Mig6), is induced by the pro-inflammatory cytokines central to the autoimmune-mediated beta cell destruction. We also established that pro-inflammatory cytokines suppress EGFR activation, and siRNA-mediated suppression of Mig6 restores EGFR signaling. Thus, we hypothesized that pro-inflammatory cytokines induce nitric oxide production and that in turn induced Mig6, disrupting EGFR repair mechanisms. We determined that NO induces Mig6, attenuating EGFR signaling, and NO synthase inhibition blocks the cytokine-mediated induction of Mig6, thereby restoring cytokine-impaired EGFR signaling. To that end, we treated mice lacking pancreatic Mig6 and control mice with a streptozotocin (STZ) to induce beta cell death and diabetes in a way that mimics the onset and progression of T1D. Whereas STZ-treated control mice became hyperglycemic and had reduced beta cell mass, STZ-treated Mig6 pancreas-specific knock out (PKO) mice remained euglycemic and glucose tolerant due to preserved beta cell mass. The restoration of beta cell mass in PKO mice was accompanied by enhanced beta cell proliferation. Thus, our work suggests that Mig6 is a promising target to preserve beta cell mass before overt T1D.

This study investigated the effects of different multiple low doses of streptozotocin (STZ), namely 35 and 55 mg/kg, on the onset and progression of diabetes in mice. Both doses are commonly used in research, and while both induced a loss of beta cell mass, they had distinct effects on whole glucose tolerance, beta cell function and gene transcription. Mice treated with 55 mg/kg became rapidly glucose intolerant, whereas those treated with 35 mg/kg had a slower onset and remained glucose tolerant for up to a week before becoming equally glucose intolerant as the 55 mg/kg group. Beta cell mass loss was similar between the two groups, but the 35 mg/kg-treated mice had improved glucose-stimulated insulin secretion in gold-standard hyperglycemic clamp studies. Transcriptomic analysis revealed that the 55 mg/kg dose caused disruptions in nearly five times as many genes as the 35 mg/kg dose in isolated pancreatic islets. Pathways that were downregulated in both doses were more downregulated in the 55 mg/kg-treated mice, while pathways that were upregulated in both doses were more upregulated in the 35 mg/kg treated mice. Moreover, we observed a differential downregulation in the 55 mg/kg-treated islets of beta cell characteristic pathways, such as exocytosis or hormone secretion. On the other hand, apoptosis was differentially upregulated in 35 mg/kg-treated islets, suggesting different transcriptional mechanisms in the onset of STZ-induced damage in the islets. This study demonstrates that the two STZ doses induce distinctly mechanistic progressions for the loss of functional beta cell mass.

Hyperglycemia in both type 1 and 2 diabetes is toxic to pancreatic beta cells and exacerbates the decline in functional beta cell mass in both diseases. Thus, identifying the molecular mechanism for how this glucotoxicity negatively impacts beta cells is essential for identifying new therapeutic targets to treat both forms of diabetes. We identified that EGF receptor feedback inhibitor Mig6 is induced in islets cultured in glucotoxic conditions and that Mig6 promotes apoptosis in beta cells. To define how the adaptor protein Mig6 promotes apoptosis, we sought to identify its interacting partners. Following immunoprecipitation of flag-tagged Mig6 in Ins1-derived 832/13 cells cultured in normal and toxic conditions, mass spectrometric analysis revealed protein phosphatase 2A (PP2A). PP2A controls many cellular functions ranging from metabolism, cell cycle, DNA replication, growth and apoptosis, and PP2A can be exploited by tumor cells to promote cell survival. Thus, we hypothesized Mig6 interacts with PP2A to regulate beta cell apoptosis. We cultured 832/13 cells and mouse islets in low (5 mM) or high glucose (HG, 20 mM) for up to 48h and validated that glucotoxicity increased methylation and decreased phosphorylation of PP2A (indicating activation). HG also increased expression of Mig6, CHOP (ER stress), γH2AX (DNA damage response), and cleaved caspase 3 (CC3; apoptosis). Compared to controls, siRNA-mediated suppression of Mig6 in 832/13 cells increased methylated PP2A and decreased γH2AX and CC3, suggesting Mig6 antagonizes beta cell survival. We generated mice lacking Mig6 in beta cells (Mig6βKO) by crossing Mig6-floxed mice (Mig6fl/fl) with RIP-Cre mice. Beta cell mass and glucose homeostasis were similar between Mig6βKO and control Mig6fl/fl littermates. Islets from Mig6βKO mice had increased methylated PP2A during HG compared to controls. We speculate the Mig6-PP2A interaction limits PP2A activation; hence, disrupting Mig6-PP2A or inhibiting Mig6 could promote compensatory beta cell survival by activating PP2A. Disclosure A. Rezaeizadeh: None. B.M. Bauer: None. P.T. Fueger: None.

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