Deoxyhypusine synthase (DHPS) uses the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired β cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc–induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.

The pancreatic islet is a complex micro-organ containing numerous cell types, including endocrine, immune, and endothelial cells. The communication of these systems is lost upon isolation of the islets, and therefore the pathogenesis of diabetes can only be fully understood by studying this organized, multicellular environment in vivo . We have developed several adaptable tools to create a versatile platform to interrogate β-cell function in vivo . Specifically, we developed β-cell-selective virally-encoded fluorescent protein biosensors that can be rapidly and easily introduced into any mouse. We then coupled the use of these biosensors with intravital microscopy, a powerful tool that can be used to collect cellular and subcellular data from living tissues. Together, these approaches allowed the observation of in vivo β-cell-specific ROS dynamics using the Grx1-roGFP2 biosensor and calcium signaling using the GcAMP6s biosensor. Next, we utilized abdominal imaging windows (AIW) to extend our in vivo observations beyond single-point terminal measurements to collect longitudinal physiological and biosensor data through repeated imaging of the same mice over time. This platform represents a significant advancement in our ability to study β-cell structure and signaling in vivo , and its portability for use in virtually any mouse model will enable meaningful studies of β-cell physiology in the endogenous islet niche.

Widespread use of pancreatic islet transplantation for treatment of type 1 diabetes (T1D) is currently limited by requirements for long-term immunosuppression, limited donor supply, and poor long-term engraftment and function. Upon isolation from their native microenvironment, islets undergo rapid apoptosis, which is further exacerbated by poor oxygen and nutrient supply following infusion into the portal vein. Identifying alternative strategies to restore critical microenvironmental cues, while maximizing islet health and function, is needed to advance this cellular therapy. We hypothesized that biophysical properties provided through type I oligomeric collagen macroencapsulation are important considerations when designing strategies to improve islet survival, phenotype, and function. Mouse islets were encapsulated at various Oligomer concentrations (0.5 -3.0 mg/ml) or suspended in media and cultured for 14 days, after which viability, protein expression, and function were assessed. Oligomer-encapsulated islets showed a density-dependent improvement in in vitro viability, cytoarchitecture, and insulin secretion, with 3 mg/ml yielding values comparable to freshly isolated islets. For transplantation into streptozotocin-induced diabetic mice, 500 islets were mixed in Oligomer and injected subcutaneously, where rapid in situ macroencapsulation occurred, or injected with saline. Mice treated with Oligomer-encapsulated islets exhibited rapid (within 24 h) diabetes reversal and maintenance of normoglycemia for 14 (immunocompromised), 90 (syngeneic), and 40 days (allogeneic). Histological analysis showed Oligomer-islet engraftment with maintenance of islet cytoarchitecture, revascularization, and no foreign body response. Oligomer-islet macroencapsulation may provide a useful strategy for prolonging the health and function of cultured islets and has potential as a subcutaneous injectable islet transplantation strategy for treatment of T1D.

Loss of functional islet β-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II ( Ins2) DNA as a biomarker of β-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of β-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and β-cell mass, death, and dedifferentiation. Compared with LFD controls, β-cell mass increased during the feeding period in HFD animals, and statistically greater β-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of β-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic β-cell death may be a feature of cellular turnover correlated with changes in glycemia during β-cell mass accrual in obesity, whereas β-cell dedifferentiation may be a feature seen later in established disease.-Tersey, S. A., Levasseur, E. M., Syed, F., Farb, T. B., Orr, K. S., Nelson, J. B., Shaw, J. L., Bokvist, K., Mather, K. J., Mirmira, R. G. Episodic β-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Macrophages and related myeloid cells are innate immune cells that participate in the early islet inflammation of type 1 diabetes (T1D). The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of proinflammatory eicosanoids, but its role and mechanisms in myeloid cells in the pathogenesis of islet inflammation have not been elucidated. Leveraging a model of islet inflammation in zebrafish, we show here that macrophages contribute significantly to the loss of β cells and the subsequent development of hyperglycemia. The depletion or inhibition of 12-LOX in this model resulted in reduced macrophage infiltration into islets and the preservation of β cell mass. In NOD mice, the deletion of the gene encoding 12-LOX in the myeloid lineage resulted in reduced insulitis with reductions in proinflammatory macrophages, a suppressed T cell response, preserved β cell mass, and almost complete protection from the development of T1D. 12-LOX depletion caused a defect in myeloid cell migration, a function required for immune surveillance and tissue injury responses. This effect on migration resulted from the loss of the chemokine receptor CXCR3. Transgenic expression of the gene encoding CXCR3 rescued the migratory defect in zebrafish 12-LOX morphants. Taken together, our results reveal a formative role for innate immune cells in the early pathogenesis of T1D and identify 12-LOX as an enzyme required to promote their prodiabetogenic phenotype in the context of autoimmunity.