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Previously we utilized a murine model to demonstrate that Ogt deletion in pancreatic progenitors (OgtKOPanc) causes pancreatic hypoplasia, partly mediated by a reduction in the Pdx1-expressing pancreatic progenitor pool. Here, we continue to explore the role of Ogt in pancreas development by deletion of Ogt in the endocrine progenitors (OgtKOEndo). At birth OgtKOEndo, were normoglycemic and had comparable pancreas weight and α-cell, and β-cell mass to littermate controls. At postnatal day 23, OgtKOEndo displayed wide ranging but generally elevated blood glucose levels, with histological analyses showing aberrant islet architecture with α-cells invading the islet core. By postnatal day 60, these mice were overtly diabetic and showed significant loss of both α-cell and β-cell mass. Together, these results highlight the indispensable role of Ogt in maintenance of β-cell mass and glucose homeostasis.

Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring including increased susceptibility to type 2 diabetes (T2D) in adulthood. In rat Reduced Uteroplacental Perfusion Pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimics aspects of preeclampsia and resulted in female embryonic day (e)19 offspring with reduced β-cell area and increased β-cell apoptosis compared to offspring of Sham pregnancies. Decreased pancreatic β-cell area persists to Postnatal Day (PD)13 in females and could influence whether T2D develops in adulthood. Macrophage changes also occur in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic β-cell area in female offspring following chronic placental ischemia. The macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared to Sham. Postnatal injections of clodronate liposomes into female RUPP and Sham offspring on PD2 and PD9 significantly depleted macrophages compared to animals injected with control liposomes. Depletion of macrophages rescued reduced β-cell area and increased β-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced β-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in offspring. Pancreatic beta cell mass is set very early in life and can influence whether an individual develops Type 2 diabetes. We previously demonstrated that postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension had significantly reduced beta cell area. Previous studies in humans have noted increased circulating C3 in Type 2 diabetics, as well as an important role for intracellular complement C3 in maintaining integrity of the beta cell in the islet. Thus, we hypothesized that decreased C3 in islets is associated with reductions in beta cell area in rat offspring following placental insufficiency. The rat Reduced Uterine Perfusion Pressure (RUPP) model was used to mechanically induce placental insufficiency in the dam by placing silver clips on abdominal aorta and uterine arteries on gestation day 14 of a 21-day gestation, resulting in hypertension in the dam. Pancreatic islets and acinar were isolated by collagenase perfusion and hand picking from PD13 female offspring of RUPP and Sham dams. The concentration of C3 in serum, isolated islets and acinar was determined by ELISA. C3 was significantly increased in serum of female RUPP compared to female Sham offspring. In contrast, C3 in islets was significantly decreased in RUPP female offspring compared to Sham with no significant differences detected in acinar C3 concentrations. Thus, these data suggest that decreased C3 in islets of PD13 offspring may contribute to reduction of beta cell area and long-term susceptibility for Type 2 diabetes.

T2D is associated with reduced pancreatic β-cell mass, in part, by increased apoptosis and identity loss as insulin-producing cells. Nutrient-sensor protein O-GlcNAc Transferase (Ogt) is a regulator of pancreas development and β-cell function. We hypothesized that Ogt also regulates postnatal β-cell identity maintenance, in part, by modulating the activity and localization of key β-cell transcription factors. We genetically ablated Ogt in endocrine progenitors in mice (Ngn3-cre;OgtKOEndo) . While phenotypically normal at birth, at postnatal (p) day 23, normoglycemic OgtKOEndo displayed aberrant islet architecture, along with a significant increase in Ins/Gcg-expressing bihormonal cells. Reduced levels of Pdx1 expression and increased incidence of cytoplasmic Pdx1 localization were observed in islets of OgtKOEndo mice compared to controls. All OgtKOEndo mice were diabetic by p60, with severe reductions in both α and β-cell mass. Next, we generated a β-cell Ogt-deficient mouse model (RIP-cre;OgtKOIns) . Like OgtKOEndo, OgtKOIns mice had increased incidence of bihormonal cells, with a trending increase in α-cell associated gene transcription. α-cell mass and β-cell mass were also reduced at p30 compared to controls. We hypothesized, mechanistically, that deletion of Ogt perturbed the activity of Pdx1, a transcription factor modified by Ogt and a key regulator of β-cell fate and insulin biogenesis. As expected, OgtKOIns islets display reduced Ins1/2 mRNA, total insulin content, and Pdx1 protein. Over-expression of Pdx1 in the OgtKO;PdxOEIns mice rescued islet insulin content, and O-GlcNAc modification of Pdx1 is positively correlated with insulin secretion. However, no rescue in β-cell mass was observed in OgtKO;PdxOEIns mice. Next, we will determine the role of the O-GlcNAc modification of Pdx1 in β-cell identity in vivo using the OgtKO;PdxOEIns mice and in vitro, using a proximity-directed nanobody technology to increase Pdx1 and Ogt interactions. Disclosure A.L.Wong: None. B.Akhaphong: None. A.D.Lockridge: None. E.Alejandro: None. Funding National Institutes of Health (DK115720)

Maternal low-protein diet (LP) throughout gestation affects pancreatic β-cell fraction of the offspring at birth, thus increasing their susceptibility to metabolic dysfunction and type 2 diabetes in adulthood. The present study sought to strictly examine the effects of LP during the last week of gestation (LP12.5) alone as a developmental window for β-cell programming and metabolic dysfunction in adulthood. Islet morphology analysis revealed normal β-cell fraction in LP12.5 newborns. Normal glucose tolerance was observed in 6- to 8-wk-old male and female LP12.5 offspring. However, male LP12.5 offspring displayed glucose intolerance and reduced insulin sensitivity associated with β-cell dysfunction with aging. High-fat diet exposure of metabolically normal 12-wk-old male LP12.5 induced glucose intolerance due to increased body weight, insulin resistance, and insufficient β-cell mass adaptation despite higher insulin secretion. Assessment of epigenetic mechanisms through microRNAs (miRs) by a real-time PCR-based microarray in islets revealed elevation in miRs that regulate insulin secretion (miRs 342, 143), insulin resistance (miR143), and obesity (miR219). In the islets, overexpression of miR143 reduced insulin secretion in response to glucose. In contrast to the model of LP exposure throughout pregnancy, islet protein levels of mTOR and pancreatic and duodenal homeobox 1 were normal in LP12.5 islets. Collectively, these data suggest that LP diet during the last week of pregnancy is critical and sufficient to induce specific and distinct developmental programming effects of tissues that control glucose homeostasis, thus causing permanent changes in specific set of microRNAs that may contribute to the overall vulnerability of the offspring to obesity, insulin resistance, and type 2 diabetes.

Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring, including increased susceptibility to type 2 diabetes (T2D) in adulthood. In a rat reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimicked aspects of preeclampsia and resulted in female embryonic day 19 (e19) offspring with reduced β-cell area and increased β-cell apoptosis compared with offspring of sham pregnancies. Decreased pancreatic β-cell area persisted to postnatal day 13 (PD13) in females and could influence whether T2D developed in adulthood. Macrophage changes also occurred in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic β-cell area in female offspring after chronic placental ischemia. Macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared with sham. Postnatal injections of clodronate liposomes into female RUPP and sham offspring on PD2 and PD9 significantly depleted macrophages compared with injections of control liposomes. Depletion of macrophages rescued reduced β-cell area and increased β-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced β-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

Fetal growth restriction, or low birth weight, is a strong determinant for eventual obesity and type 2 diabetes. Clinical studies suggest placental mechanistic target of rapamycin (mTOR) signaling regulates fetal birth weight and the metabolic health trajectory of the offspring. In the current study, we used a genetic model with loss of placental mTOR function (mTOR-KOPlacenta) to test the direct role of mTOR signaling on birth weight and metabolic health in the adult offspring. mTOR-KOPlacenta animals displayed reduced placental area and total weight, as well as fetal body weight at embryonic day (E) 17.5. Birth weight and serum insulin levels were reduced; however, β cell mass was normal in mTOR-KOPlacenta newborns. Adult mTOR-KOPlacenta offspring, under a metabolic high-fat challenge, displayed exacerbated obesity and metabolic dysfunction compared with 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 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.

Placental insufficiency can result in gestational hypertension and/or intrauterine growth restriction (IUGR) in the offspring. Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in the offspring. Pancreatic β cell mass, set very early in life, can influence whether an individual develops Type 2 diabetes. Our previous studies demonstrated a significant reduction in β cell area in embryonic day 19 and postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension. Previous studies in human islets demonstrated important roles for complement C3 and activation product C3a in determining β cell area, with increased C3a generation associated with prevention of islet cell atrophy. Thus, we hypothesized that increases in self-proteins that limit complement system activation, i.e. complement regulators, are associated with reductions in β cell area in the offspring following placental insufficiency. The Reduced Uterine Perfusion Pressure (RUPP) model was used to induce placental insufficiency in the dam by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation to mechanically reduce placental perfusion. This results in hypertension in the dam and IUGR in the offspring. Islets from the pancreas of (PD)13 female offspring of RUPP and Sham dams were isolated by collagenase perfusion and hand picking. The transcript message for complement regulators CD55 and Crry (regulators of C3 activation) and CD59 (terminal pathway regulator) was determined using qRT-PCR. Complement regulator CD55 was significantly increased in female RUPP offspring (2.01 + 0.29 fold) compared to female Sham offspring (1.00 + 0.13 fold change; p < 0.05; 6-13 pups from 4-7 dams; mixed model ANOVA including litter effect), whereas the Crry increase of 1.38 + 0.20 fold in female RUPP vs Sham offspring was not significant (p = 0.22). No significant change was observed in message for CD59 or C3. Thus, these data suggest increased islet complement regulator CD55 may limit complement activation in islets of PD13 offspring following placental ischemia and thereby contribute to reduction of β cell area and long term susceptibility for Type 2 diabetes.

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational 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.

Maternal hypertension during pregnancy is a major risk factor for intrauterine growth restriction (IUGR), which increases susceptibility to cardiovascular and metabolic disease in adulthood through unclear mechanisms. The aim of this study was to characterize the pancreatic β-cell area and function in the fetal rat offspring of a reduced uterine perfusion pressure (RUPP) model of gestational hypertension. At embryonic day 19.5, RUPP dams exhibited lower body weight, elevated mean blood pressure, reduced litter size, and higher blood glucose compared with sham-operated controls. In RUPP placental lysates, a nonsignificant change in mammalian target of rapamycin (mTOR) activity markers, phosphorylated S6 at serine 240, and phosphorylated AKT (at S473) was observed. RUPP offspring showed significantly reduced β-cell-to-pancreas area and increased β-cell death but normal insulin levels in serum. Isolated islets had normal insulin content and secretory function in response to glucose and palmitate. Fetal pancreatic lysates showed a tendency for reduced insulin levels, with a significant reduction in total mTOR protein with RUPP surgery. In addition, its downstream complex 2 targets phosphorylation of AKT at S473, and pAKT at Thr308 tended to be reduced in the fetal RUPP pancreas. Altogether, these data show that RUPP offspring demonstrated increased β-cell death, reduced β-cell area, and altered nutrient-sensor mTOR protein level in the pancreas. This could represent a mechanistic foundation in IUGR offspring’s risk for enhanced susceptibility to type 2 diabetes and other metabolic vulnerabilities seen in adulthood.

Deletion of the only nutrient-sensor enzyme, O-GlcNAc Transferase (OGT), catalyzing O-GlcNAc addition onto target proteins in pancreatic β-cells induces diabetes through β-cell failure. We hypothesized that OGT, expressed at a very high level in β-cells, has key developmental regulatory properties. In this study, we tested the hypothesis that OGT is required for pancreas and β-cell development by deleting OGT in pancreatic (Pdx1Cre, OGTKOPanc) or endocrine (Ngn3Cre, OGTKOEndo) progenitors. We show that mice lacking OGT in pancreatic progenitors (Pdx1+, OGTKOPanc) show near pancreas agenesis and loss of β-cell mass at birth. Few mice that grow into adulthood develop overt diabetes due to functional β-cell mass failure. OGT ablation in endocrine progenitors (Ngn3+, OGTKOEndo) resulted in normal pancreas mass but reduced β-cell mass and diabetes early in life, strongly revealing the temporal and importance of OGT for β-cell development. We observed loss of Pdx1 staining at birth and on embryonic (e) day 14.5 in OGTKOPanc. Transcriptome analysis of e14.5 progenitor single cells show distinct cell clustering populations in OGTKOPanc compared to littermate controls. Upstream regulator analysis of altered genes show enhanced activation of p53, also known as TP53 or tumor protein. Next we identified p53 to be O-GlcNAc-modified in β-cells. Concomitant deletion of p53 in the OGTKOPanc background (p53,OGTKOPanc) does not rescue pancreas agenesis in OGTKOPanc newborn, and adult p53,OGTKOPanc mice develop severe hyperglycemia and diabetes. These data highlight the novel role O-GlcNAc-modifications in pancreas and β-cell development and provide a new model to characterize the developmental defects associated with pancreatic agenesis. Disclosure E. Alejandro: None. D. Baumann: None. S. Jo: None. B. Akhaphong: None. A.D. Lockridge: None. R. Mohan: None.

NMDA receptors (NMDARs) have recently been discovered as functional regulators of pancreatic β-cell insulin secretion. While these excitatory receptor channels have been extensively studied in the brain for their role in synaptic plasticity and development, little is known about how they work in β-cells. In neuronal cells, NMDAR activation requires the simultaneous binding of glutamate and a rate-limiting co-agonist, such as D-serine. D-serine levels and availability in most of the brain rely on endogenous synthesis by the enzyme serine racemase (Srr). Srr transcripts have been reported in human and mouse islets but it is not clear whether Srr is functionally expressed in β-cells or what its role in the pancreas might be. In this investigation, we reveal that Srr protein is highly expressed in primary human and mouse β-cells. Mice with whole body deletion of Srr (Srr KO) show improved glucose tolerance through enhanced insulin secretory capacity, possibly through Srr-mediated alterations in islet NMDAR expression and function. We observed elevated insulin sensitivity in some animals, suggesting Srr metabolic regulation in other peripheral organs as well. Srr expression in neonatal and embryonic islets, and adult deficits in Srr KO pancreas weight and islet insulin content, point toward a potential role for Srr in pancreatic development. These data reveal the first evidence that Srr may regulate glucose homeostasis in peripheral tissues and provide circumstantial evidence that D-serine may be an endogenous islet NMDAR co-agonist in β-cells.