Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven posttranslational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKO Panc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.
During early obesity, pancreatic b cells compensate for increased metabolic demand through a transient phase of insulin hypersecretion that stabilizes blood glucose and forestalls diabetic progression. We find evidence that b cell O-GlcNAcylation, a nutrient-responsive post-translational protein modification regulated by O-GlcNAc transferase (OGT), is critical for coupling hyperlipidemia to b cell functional adaptation during this compensatory prediabetic phase. In mice, islet O-GlcNAcylation rises and falls in tandem with the timeline of secretory potentiation during high-fat feeding while genetic models of b-cell-specific OGT loss abolish hyperinsulinemic responses to lipids, in vivo and in vitro. We identify the endoplasmic reticulum (ER) Ca 2+ ATPase SERCA2 as a b cell O-GlcNAcylated protein in mice and humans that is able to rescue palmitate-stimulated insulin secretion through pharmacological activation. This study reveals an important physiological role for b cell O-GlcNAcylation in sensing and responding to obesity, with therapeutic implications for managing the relationship between type 2 diabetes and its most common risk factor.
Fetal growth restriction, or low birthweight is a strong determinant for eventual obesity and Type 2 diabetes. Clinical studies suggest placental mechanistic target of rapamycin (mTOR) signaling regulate fetal birthweight and the metabolic health trajectory of the offspring. In the current study, we used genetic model with loss of placental mTOR function (mTORKO Placenta ) to test the direct role of mTOR signaling on birthweight and the metabolic health in the adult offspring. mTORKO Placenta animals displayed reduced placental area and total weight, as well as fetal bodyweight at embryonic day (e) 17.5. Birthweight and serum insulin levels were reduced; however, β-cell mass was normal in mTORKO Placenta newborns. Adult mTORKO Placenta offspring, under a metabolic high-fat challenge, displayed exacerbated obesity and metabolic dysfunction compared to littermate controls. Subsequently, we tested whether enhancing placental mTOR complex 1 (mTORC1) signaling, via genetic ablation of TSC2, in utero would improve glucose homeostasis in the offspring. Indeed, increased placental mTORC1 conferred protection from a diet-induced obesity in the offspring. In conclusion, placental mTORC1 serves as a mechanistic link between placental function and programming of obesity and insulin resistance in the adult offspring.
An early hallmark of type 2 diabetes is a failure of proinsulin-to-insulin processing in pancreatic -cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and -cell-specific deletion of the nutrientsensing protein modifier OGlcNAc transferase (OGTKO) causes -cell failure and diabetes, including early development of hyperproinsulinemia. The mechanisms underlying this latter defect are unknown. Here, using several approaches, including site-directed mutagenesis, Click O-GlcNAc labeling, immunoblotting, and immunofluorescence and EM imaging, we provide the first evidence for a relationship between the O-GlcNAcylation of eukaryotic translation initiation factor 4␥1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in OGTKO mice. We first established that OGTKO hyperproinsulinemia is independent of age, sex, glucose levels, and endoplasmic reticulum-CCAAT enhancerbinding protein homologous protein (CHOP)-mediated stress status. Of note, OGT loss was associated with a reduction in -cell-resident CPE, and genetic reconstitution of CPE in OGTKO islets rescued the dysfunctional proinsulin-to-insulin ratio. We show that although CPE is not directly OGlcNAc modified in islets, overexpression of the suspected OGT target eIF4G1, previously shown to regulate CPE translation in -cells, increases islet CPE levels, and fully reverses OGTKO islet-induced hyperproinsulinemia. Furthermore, our results reveal that OGT O-GlcNAc-modifies eIF4G1 at Ser-61 and that this modification is critical for eIF4G1 protein stability. Together, these results indicate a direct link between nutrient-sensitive OGT and insulin processing, underscoring the importance of post-translational O-GlcNAc modification in general cell physiology.
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
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