The effects of β-cell specific knockdown of FoxO1 during diet-induced obesity Oluwabukola A. Ajasa, Jennifer Fiori O’Connell, Michael Rouse, Adria Summers, Isabel Gonzalez-Mariscal, and Josephine M. Egan Laboratory of Clinical Investigation, National Institute on Aging (NIA), Baltimore, MD, USA Abstract Results There is a global epidemic of diabetes with at least 347 million people affected and the rates are still on the rise due to increases in body weight and lack of physical activity. Type 2 diabetes mellitus results from the body’s ineffective use of insulin and comprises approximately 90% of diabetes cases worldwide. FoxO1 is transcription factor that plays an important role in regulation of gluconeogenesis and insulin signaling and is the most abundant FoxO isoform in β-cells. It has been reported to inhibit β-cell replication and neogenesis, protect β-cells against oxidative stress, prevent dedifferentiation of β-cells, and maintain β-cell identity. To elucidate the role of FoxO1 in β-cells during insulin resistance using diet-induced (chronic) obesity, we developed a conditional knockout of the FoxO1 gene in β-cells using a tamoxifen inducible Cre-LoxP system. After 16 weeks on a high fat high sugar diet, we found no significant differences in fasting blood glucose or insulin levels, or response to glucose or insulin tolerance tests in wild type (β-FoxO1+/+) versus knockout (β-FoxO1-/-) mice. These results suggest that deletion of FoxO1 is highly protective to β-cells under stress and FoxO1 may serve as a novel target for preventing insulin resistance and β-cell dysregulation. Figure 1: Effect of β-FoxO1 deficiency in mice given a HFS diet. A) Body weight, B) food intake, and C) water intake were measured every week in β-FoxO1+/+ and β-FoxO1-/- mice administered diets for 16 weeks. ****p<0.0001, ++++p<0.0001 A B C Figure 4: In the treated mice, the knockouts were smaller than the wild type. In regards to the alpha-beta cell ratio, under metabolic stress, the FoxO1 knockout is protective of normal islet composition. Under control conditions, FoxO1 is protective of beta cells in the islets, causing dedifferentiation of beta cells to alpha cells. *p<0.05, **p<0.01 Figure 2: Metabolic parameters were similar between β-FoxO1+/+ and β-FoxO1-/- mice. After 16 weeks of diets, A) fasting blood glucose and B) fasting insulin were measured, and C) HOMA-IR was calculated in β-FoxO1+/+ and β-FoxO1-/- mice. *p<0.05, ***p<0.001, ****p<0.0001 A B C Background Activation of the PI3K/AKT/ FOXO axis in different tissues mediates many of insulin’s effects, such as regulation of growth, glucose/ fat metabolism, stress response, and lifespan FoxO1 is abundant in β-cells and has been reported to: Inhibit β-cell replication and neogenesis Induce protective or pro- apoptotic functions in β- cells in response to oxidative stress depending on the conditions Prevent dedifferentiation of β-cells and maintain β-cell identity In this study, we examined the role of FoxO1 in β-cells during chronic (diet induced obesity) insulin resistance. Figure 5:. FoxO3 is significantly upregulated in FoxO1 knockout mice. FoxO1 plays a role in maintaining insulin secretion during metabolic stress. Insulin secretion was increased in the treated knockout but was not significant. Under normal conditions, FoxO1 was also protective of insulin secretion. *p<0.05, ****p<0.0001 Figure 7: β-FoxO1-/- islets were protected from gluco- and lipotoxicity. Islets were extracted from β-FoxO1+/+ and β-FoxO1-/- mice and treated with low glucose (5mM; LG), high glucose (20mM; HG), or high glucose + palmitate (500µM; HG+Palm) for 24 hours and a cell toxicity assay was performed. *p<0.05, +p<0.05 Conclusions In this study, we examined the role of FoxO1 in β-cells during chronic insulin resistance using a novel conditional FoxO1 knockout mouse. We found: No significant differences in fasting blood glucose or insulin levels, or response to glucose or insulin tolerance tests in β-FoxO1+/+ versus β- FoxO1-/- mice. FoxO1 is important to maintaining insulin secretion in metabolic stress, but may also play a role in maintaining secretion under normal conditions. FoxO3 is significantly upregulated in FoxO1 knockout mice, suggestive of a compensatory role in absence of the more abundance FoxO. FoxO1 may serve as a novel target for preventing insulin resistance and β-cell dysregulation. Figure 4: FoxO1 is present in insulin+ cells β-cells. Post tamoxifen injection, FoxO1 is absent in islets of β-FoxO1-/- mice Methods β-FoxO1+/+ and β-FoxO1-/- mice were given tamoxifen (5 days), wait 4 weeks for knockdown Fed Control (CON) of high fat/ high sugar (HFS) diets for 16 weeks, monitored Sacrificed and pancreas harvested, fixed and cast in paraffin Sectioned and stained for insulin, glucagon, FoxO1/FoxO3, and DAPI Confocal imaging and analysis with ImageJ and Pancreas++ FoxO3, another less abundant form of FoxO in pancreatic beta cells, was also stained for and quantified. Pancreas ++ software was used to quantitate total islet size, α-cell to β–cell ratio, insulin content, in at least 100 images per group Future directions FoxO3, as well as other FoxO4, may play a compensatory role in the absence of FoxO1 Stain for Pdx1 and other proteins involved in FoxO1 regulation, such as NeuroD and MafA. Acknowledgements We gratefully acknowledge Dr. Domenico Accili of Columbia University Medical Center for the floxed FoxO1 mouse and Dr. Louis Philipson of the University of Chicago Medicine for the MIP-Cre mouse. This work was supported by the Intramural Research Program of the NIA.