Loss of glucose-stimulated ß-cell Nr4a1 expression impairs insulin secretion and glucose homeostasis.

A central aspect of type 2 diabetes is decreased functional ß-cell mass. The orphan nuclear receptor Nr4a1 is critical for fuel utilization, but little is known regarding its regulation and function in the ß-cell. Nr4a1 expression is decreased in type 2 diabetes rodent ß-cells and type 2 diabetes patient islets. We have shown that Nr4a1-deficient mice have reduced ß-cell mass and that Nr4a1 knockdown impairs glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 ß-cells. Here, we demonstrate that glucose concentration directly regulates ß-cell Nr4a1 expression. We show that 11 mM glucose increases Nr4a1 expression in INS-1 832/13 ß-cells and primary mouse islets. We show that glucose functions through the cAMP/PKA/CREB pathway to regulate Nr4a1 mRNA and protein expression. Using Nr4a1-/- animals, we show that Nr4a1 is necessary for GSIS and systemic glucose handling. Using RNA-seq, we define Nr4a1-regulated pathways in response to glucose in the mouse islet, including Glut2 expression. Our data suggest that Nr4a1 plays a critical role in the ß-cells response to the fed state.NEW & NOTEWORTHY Nr4a1 has a key role in fuel metabolism and ß-cell function, but its exact role is unclear. Nr4a1 expression is regulated by glucose concentration using cAMP/PKA/CREB pathway. Nr4a1 regulates Glut2, Ndufa4, Ins1, In2, Sdhb, and Idh3g expression in response to glucose treatment. These results suggest that Nr4a1 is necessary for proper insulin secretion both through glucose uptake and metabolism machinery.

Decreased proliferation of aged rat beta cells corresponds with enhanced expression of the cell cycle inhibitor p27KIP1.

BACKGROUND: Over 400 million people are diabetic. Type 1 and type 2 diabetes are characterized by decreased functional ß-cell mass and, consequently, decreased glucose-stimulated insulin secretion. A potential intervention is transplantation of ß-cell containing islets from cadaveric donors. A major impediment to greater application of this treatment is the scarcity of transplant-ready ß-cells. Therefore, inducing ß-cell proliferation ex vivo could be used to expand functional ß-cell mass prior to transplantation. Various molecular pathways are sufficient to induce proliferation of young ß-cells; however, aged ß-cells are refractory to these proliferative signals. Given that the majority of cadaveric donors fit an aged demographic, defining the mechanisms that impede aged ß-cell proliferation is imperative. RESULTS: We demonstrate that aged rat (5-month-old) ß-cells are refractory to mitogenic stimuli that otherwise induce young rat (5-week-old) ß-cell proliferation. We hypothesized that this change in proliferative capacity could be due to differences in cyclin-dependent kinase inhibitor expression. We measured levels of p16INK4a , p15INK4b , p18INK4c , p19INK4d , p21CIP1 , p27KIP1 and p57KIP2 by immunofluorescence analysis. Our data demonstrates an age-dependent increase of p27KIP1 in rat ß-cells by immunofluorescence and was validated by increased p27KIP1 protein levels by western blot analysis. Interestingly, HDAC1, which modulates the p27KIP1 promoter acetylation state, is downregulated in aged rat islets. These data demonstrate increased p27KIP1 protein levels at 5 months of age, which may be due to decreased HDAC1 mediated repression of p27KIP1 expression. SIGNIFICANCE: As the majority of transplant-ready ß-cells come from aged donors, it is imperative that we understand why aged ß-cells are refractory to mitogenic stimuli. Our findings demonstrate that increased p27KIP1 expression occurs early in ß-cell aging, which corresponds with impaired ß-cell proliferation. Furthermore, the correlation between HDAC1 and p27 levels suggests that pathways that activate HDAC1 in aged ß-cells could be leveraged to decrease p27KIP1 levels and enhance ß-cell proliferation.

CEBPA Overexpression Enhances ß-Cell Proliferation and Survival.

A commonality between type 1 and type 2 diabetes is the decline in functional ß-cell mass. The transcription factor Nkx6.1 regulates ß-cell development and is integral for proper ß-cell function. We have previously demonstrated that Nkx6.1 depends on c-Fos mediated upregulation and the nuclear hormone receptors Nr4a1 and Nr4a3 to increase ß-cell insulin secretion, survival, and replication. Here, we demonstrate that Nkx6.1 overexpression results in upregulation of the bZip transcription factor CEBPA and that CEBPA expression is independent of c-Fos regulation. In turn, CEBPA overexpression is sufficient to enhance INS-1 832/13 ß-cell and primary rat islet proliferation. CEBPA overexpression also increases the survival of ß-cells treated with thapsigargin. We demonstrate that increased survival in response to ER stress corresponds with changes in expression of various genes involved in the unfolded protein response, including decreased Ire1a expression. These data show that CEBPA is sufficient to enhance functional ß-cell mass by increasing ß-cell proliferation and modulating the unfolded protein response.