Rotarod test was performed to assess locomotive coordination of C57/Bl6 WT and βeIF4G1KO mice, over the course of 4 days (n=7, 5) (A). Body weight of 2-months-old male (B) and female (C) Rip-Cre WT, eIF4G1f/+, βeIF4G1HET, eIF4G1f/f and βeIF4G1KO (n=7, 8, 11, 7, 11 males; n=5, 5, 6, 5, 7 females). In vivo glucose tolerance (2 g/kg glucose, i.p.) was performed on 5months-old Male C57/Bl6 WT, eIF4G1f/f and βeIF4G1KO (n=7, 5, 5). Area under curve (AUC) of

The nutrient sensor O-GlcNAc transferase (OGT) catalyzes posttranslational addition of O-GlcNAc onto target proteins, influencing signaling pathways in response to cellular nutrient levels. OGT is highly expressed in pancreatic glucagon-secreting cells (α-cells), which secrete glucagon in response to hypoglycemia. The objective of this study was to determine whether OGT is necessary for the regulation of α-cell mass and function in vivo . We utilized genetic manipulation to produce two α-cell specific OGT-knockout models: a constitutive glucagon-Cre (αOGT KO ) and an inducible glucagon-Cre (i-αOGT KO ), which effectively delete OGT in α-cells. Using approaches including immunoblotting, immunofluorescent imaging, and metabolic phenotyping in vivo , we provide the first insight on the role of O-GlcNAcylation in α-cell mass and function. αOGT KO mice demonstrated normal glucose tolerance and insulin sensitivity but displayed significantly lower glucagon levels during both fed and fasted states. αOGT KO mice exhibited significantly lower α-cell glucagon content and α-cell mass at 6 months of age. In fasting, αOGT KO mice showed impaired pyruvate stimulated gluconeogenesis in vivo and reduced glucagon secretion in vitro . i-αOGT KO mice showed similarly reduced blood glucagon levels, defective in vitro glucagon secretion, and normal α-cell mass. Interestingly, both αOGT KO and i-αOGT KO mice had no deficiency in maintaining blood glucose homeostasis under fed or fasting conditions, despite impairment in α-cell mass and function, and glucagon content. In conclusion, these studies provide a first look at the role of OGT signaling in the α-cell, its effect on α-cell mass, and its importance in regulating glucagon secretion in hypoglycemic conditions.

Protein translation is essential for cell physiology, and dysregulation of this process has been linked to aging-related diseases such as type 2 diabetes. Reduced protein level of a requisite scaffolding protein of the initiation complex, eIF4G1, downstream of nutrients and insulin signaling is associated with diabetes in humans and mice. In the current study, we tested the hypothesis that eIF4G1 is critical for β-cell function and glucose homeostasis by genetically ablating eIF4G1 specifically in β-cells in vivo (βeIF4G1 knockout [KO]). Adult male and female βeIF4G1KO mice displayed glucose intolerance but normal insulin sensitivity. β-Cell mass was normal under steady state and under metabolic stress by diet-induced obesity, but we observed increases in proliferation and apoptosis in β-cells of βeIF4G1KO. We uncovered deficits in insulin secretion, partly due to reduced mitochondrial oxygen consumption rate, glucose-stimulated Ca2+ flux, and reduced insulin content associated with loss of eIF4E, the mRNA 5′ cap-binding protein of the initiation complex and binding partner of eIF4G1. Genetic reconstitution of eIF4E in single β-cells or intact islets of βeIF4G1KO mice recovers insulin content, implicating an unexplored role for eIF4G1/eIF4E in insulin biosynthesis. Altogether these data demonstrate an essential role for the translational factor eIF4G1 on glucose homeostasis and β-cell function.

In this demonstration, spheroids formed from the β-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Protein O-GlcNAcylation is a nutrient and stress-sensitive protein post-translational modification (PTM). The addition of an O-GlcNAc molecule to proteins is catalyzed by O-GlcNAc transferase (OGT), whereas O-GlcNAcase (OGA) enzyme is responsible for removal of this PTM. Previous work showed that OGT is highly expressed in the pancreas, and we demonstrated that hypo-O-GlcNAcylation in β-cells cause severe diabetes in mice. These studies show a direct link between nutrient-sensitive OGT and β-cell health and function. In the current study, we hypothesized that hyper-O-GlcNAcylation may confer protection from β-cell failure in high-fat diet (HFD)-induced obesity. To test this hypothesis, we generated a mouse model with constitutive β-cell OGA ablation (βOGAKO) to specifically increase O-GlcNAcylation in β-cells. Under normal chow diet, young male and female βOGAKO mice exhibited normal glucose tolerance but developed glucose intolerance with aging, relative to littermate controls. No alteration in β-cell mass was observed between βOGAKO and littermate controls. Total insulin content was reduced despite an increase in pro-insulin to insulin ratio in βOGAKO islets. βOGAKO mice showed deficit in insulin secretion in vivo and in vitro. When young animals were subjected to HFD, both male and female βOGAKO mice displayed normal body weight gain and insulin tolerance but developed glucose intolerance that worsened with longer exposure to HFD. Comparable β-cell mass was found between βOGAKO and littermate controls. Taken together, these data demonstrate that the loss of OGA in β-cells reduces β-cell function, thereby perturbing glucose homeostasis. The findings reinforce the rheostat model of intracellular O-GlcNAcylation where too much (OGA loss) or too little (OGT loss) O-GlcNAcylation are both detrimental to the β-cell.