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Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin’s impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 μM (therapeutic range) and 2000 μM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 μM metformin treated cells, 2000 μM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 μM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impairs trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

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.

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.

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.

The nutrient-sensor O-GlcNAc transferase (Ogt), the sole enzyme that adds an O-GlcNAc-modification onto proteins, plays a critical role for pancreatic β-cell survival and insulin secretion. We hypothesized that β-cell Ogt overexpression would confer protection from β-cell failure in response to metabolic stressors, such as high-fat diet (HFD) and streptozocin (STZ). Here, we generated a β-cell-specific Ogt in overexpressing (βOgtOE) mice, where a significant increase in Ogt protein level and O-GlcNAc-modification of proteins were observed in islets under a normal chow diet. We uncovered that βOgtOE mice show normal peripheral insulin sensitivity and glucose tolerance with a regular chow diet. However, when challenged with an HFD, only female βOgtOE (homozygous) Hz mice developed a mild glucose intolerance, despite increased insulin secretion and normal β-cell mass. While female mice are normally resistant to low-dose STZ treatments, the βOgtOE Hz mice developed hyperglycemia and glucose intolerance post-STZ treatment. Transcriptome analysis between islets with loss or gain of Ogt by RNA sequencing shows common altered pathways involving pro-survival Erk and Akt and inflammatory regulators IL1β and NFkβ. Together, these data show a possible gene dosage effect of Ogt and the importance O-GlcNAc cycling in β-cell survival and function to regulate glucose homeostasis.

Robust evidence of fetal programming of adult disease has surfaced in the last several decades. Human and preclinical investigations of intrauterine insults report perturbations in placental nutrient sensing by the mechanistic target of rapamycin (mTOR). This review focuses on pregnancy complications associated with placental mTOR regulation, such as fetal growth restriction (FGR), fetal overgrowth, gestational diabetes mellitus (GDM), polycystic ovarian syndrome (PCOS), maternal nutrient restriction (MNR), preeclampsia (PE), maternal smoking, and related effects on offspring birthweight. The link between mTOR-associated birthweight outcomes and offspring metabolic health trajectory with a focus on sexual dimorphism are discussed. Both human physiology and animal models are summarized to facilitate in depth understanding. GDM, PCOS and fetal overgrowth are associated with increased placental mTOR, whereas FGR, MNR and maternal smoking are linked to decreased placental mTOR activity. Generally, birth weight is reduced in complications with decreased mTOR (i.e., FGR, MNR, maternal smoking) and higher with increased mTOR (GDM, PCOS). Offspring display obesity or a higher body mass index in childhood and adulthood, impaired glucose and insulin tolerance in adulthood, and deficiencies in pancreatic beta-cell mass and function compared to offspring from uncomplicated pregnancies. Defining causal players in the fetal programming of offspring metabolic health across the lifespan will aid in stopping the vicious cycle of obesity and type II diabetes.

To ensure fetal lipid supply, maternal blood triglyceride (TG) concentrations are robustly elevated during pregnancy. Interestingly, a lower increase in maternal blood TG concentrations has been observed in some obese mothers. We have shown that high-fat (HF) feeding during pregnancy significantly reduces maternal blood TG levels. Therefore, we performed this study to investigate if and how obesity alters maternal blood TG levels. Maternal obesity was established by prepregnant HF (ppHF) feeding, which avoided the dietary effect during pregnancy. We found not only that maternal blood TG concentrations in ppHF dams were remarkably lower than in control dams but also that the TG peak occurred earlier during gestation. Hepatic TG production and intestinal TG absorption were unchanged in ppHF dams, but systemic lipoprotein lipase (LPL) activity was increased, suggesting that increased blood TG clearance contributes to the decreased blood TG concentrations in ppHF dams. Although significantly higher levels of UCP1 protein were observed in interscapular brown adipose tissue (iBAT) of ppHF dams, Ucp1 gene deletion did not restore blood TG concentrations in ppHF dams. Expression of the angiopoietin-like protein 4 (ANGPTL4), a potent endogenous LPL inhibitor, was significantly increased during pregnancy. However, the pregnancy-induced elevation of blood TG was almost abolished in Angptl4(−/−) dams. Compared with control dams, Angptl4 mRNA levels were significantly lower in iBAT, gonadal white adipose tissue, and livers of ppHF dams. Importantly, ectopic overexpression of ANGPTL4 restored maternal blood TG concentrations in ppHF dams. Together, these results indicate that ANGPTL4 plays a vital role in increasing maternal blood TG concentrations during pregnancy. Obesity impairs the rise of maternal blood TG concentrations by reducing ANGPTL4 expression in mice.

An early hallmark of type 2 diabetes is a failure of proinsulin-to-insulin processing in pancreatic β-cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and β-cell–specific deletion of the nutrient-sensing protein modifier OGlcNAc transferase (βOGTKO) causes β-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 4γ1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in βOGTKO mice. We first established that βOGTKO hyperproinsulinemia is independent of age, sex, glucose levels, and endoplasmic reticulum–CCAAT enhancer-binding protein homologous protein (CHOP)–mediated stress status. Of note, OGT loss was associated with a reduction in β-cell–resident CPE, and genetic reconstitution of CPE in βOGTKO 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 β-cells, increases islet CPE levels, and fully reverses βOGTKO 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.

O-GlcNAc transferase (OGT), a nutrient sensor sensitive to glucose flux, is highly expressed in the pancreas. However, the role of OGT in the mitochondria of β-cells is unexplored. In this study, we identified the role of OGT in mitochondrial function in β-cells. Constitutive deletion of OGT (βOGTKO) or inducible ablation in mature β-cells (iβOGTKO) causes distinct effects on mitochondrial morphology and function. Islets from βOGTKO, but not iβOGTKO, mice display swollen mitochondria, reduced glucose-stimulated oxygen consumption rate, ATP production, and glycolysis. Alleviating endoplasmic reticulum stress by genetic deletion of Chop did not rescue the mitochondrial dysfunction in βOGTKO mice. We identified altered islet proteome between βOGTKO and iβOGTKO mice. Pancreatic and duodenal homeobox 1 (Pdx1) was reduced in in βOGTKO islets. Pdx1 overexpression increased insulin content and improved mitochondrial morphology and function in βOGTKO islets. These data underscore the essential role of OGT in regulating β-cell mitochondrial morphology and bioenergetics. In conclusion, OGT couples nutrient signal and mitochondrial function to promote normal β-cell physiology.

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.

An intricate network of regulation between the brain and the pancreas modulates hormone secretion and organ function. Dysfunction of the brain-pancreas axis occurs in disease states such as diabetes and pancreatitis. Given the delicate nature of the mouse brain, procurement for tissue and cellular analysis is facilitated by fixation by perfusion with paraformaldehyde (PFA). The brain is hardened by PFA during the preservation process, but this hardening also occurs in the pancreas, as well as the remainder of the intra-abdominal organs. This hardening makes the pancreas friable and difficult to dissect without damaging and fragmenting the organ. Additionally, this fixation may preclude the ability to perform analytic techniques such as western blot and quantitative PCR (qPCR) simultaneously. Performing a simple cross-clamping of the thoracic aorta allows for differential perfusion of organs and maximal use of limited samples from a single animal. The brain can be perfused with PFA without compromising tissue collection of the pancreas and other intra-abdominal organs. This simple maneuver allows for greater tissue collection and analysis per mouse in studies evaluating the brain-pancreas or brain-gut axis. © 2021 Wiley Periodicals LLC. Basic Protocol: Differential fixation by perfusion using aortic cross-clamp.

Insulin-secreting pancreatic &beta, cells express proteins characteristic of D-serine regulated synapses, but the acute effect of D-serine co-agonism on its presumptive &beta, cell target, N-methyl D-aspartate receptors (NMDARs), is unclear. We used multiple models to evaluate glucose homeostasis and insulin secretion in mice with a systemic increase in D-serine (intraperitoneal injection or DAAO mutants without D-serine catabolism) or tissue-specific loss of Grin1-encoded GluN1, the D-serine binding NMDAR subunit. We also investigated the effects of D-serine &plusmn, NMDA on glucose-stimulated insulin secretion (GSIS) and &beta, cell depolarizing membrane oscillations, using perforated patch electrophysiology, in &beta, cell-containing primary isolated mouse islets. In vivo models of elevated D-serine correlated to improved blood glucose and insulin levels. In vitro, D-serine potentiated GSIS and &beta, cell membrane excitation, dependent on NMDAR activating conditions including GluN1 expression (co-agonist target), simultaneous NMDA (agonist), and elevated glucose (depolarization). Pancreatic GluN1-loss females were glucose intolerant and GSIS was depressed in islets from younger, but not older, &beta, Grin1 KO mice. Thus, D-serine is capable of acute antidiabetic effects in mice and potentiates insulin secretion through excitatory &beta, cell NMDAR co-agonism but strain-dependent shifts in potency and age/sex-specific Grin1-loss phenotypes suggest that context is critical to the interpretation of data on the role of D-serine and NMDARs in &beta, cell function.

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 both humans and mice. In the present 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 (βeIF4G1KO). 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 both 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.

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.

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 (αOGTKO) and an inducible glucagon-Cre (i-αOGTKO), 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. αOGTKO mice demonstrated normal glucose tolerance and insulin sensitivity but displayed significantly lower glucagon levels during both fed and fasted states. αOGTKO mice exhibited significantly lower α-cell glucagon content and α-cell mass at 6 months of age. In fasting, αOGTKO mice showed impaired pyruvate stimulated gluconeogenesis in vivo and reduced glucagon secretion in vitro. i-αOGTKO mice showed similarly reduced blood glucagon levels, defective in vitro glucagon secretion, and normal α-cell mass. Interestingly, both αOGTKO and i-αOGTKO 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.

Gestational diabetes mellitus (GDM), characterized by a transitory form of diabetes induced by insulin resistance and pancreatic β-cell dysfunction during pregnancy, has been identified as one of the major obstacles in achieving improved maternal and child health. Approximately 9–25% of pregnancies worldwide are impacted by the acute, long-term, and transgenerational health complications of this disease. Here, we discuss how GDM affects longstanding maternal and neonatal outcomes, as well as health risks that likely persist into future generations. In addition to the current challenges in the management and diagnosis of and the complications associated with GDM, we discuss current preclinical models of GDM to better understand the underlying pathophysiology of the disease and the timely need to increase our scientific toolbox to identify strategies to prevent and treat GDM, thereby advancing clinical care.

Summary During early obesity, pancreatic β 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 β cell O-GlcNAcylation, a nutrient-responsive post-translational protein modification regulated by O-GlcNAc transferase (OGT), is critical for coupling hyperlipidemia to β 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 β-cell-specific OGT loss abolish hyperinsulinemic responses to lipids, in vivo and in vitro. We identify the endoplasmic reticulum (ER) Ca2+ ATPase SERCA2 as a β 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 β 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.

The prevalence of obesity and type 2 (T2D) diabetes is a major health concern in the United States and around the world. T2D is a complex disease characterized by pancreatic β-cell failure in association with obesity and insulin resistance in peripheral tissues. Although several genes associated with T2D have been identified, it is speculated that genetic variants account for only

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.

Females are more protected against insulin resistance and cardiovascular disease compared with males of the same age or BMI, and this relative protection is diminished in postmenopausal women, attributable to a loss in estrogen signaling (1–4). Evidence from genetic animal models further points to the beneficial effects of estrogen signaling via estrogen receptor-α (ERα) in regard to glucose homeostasis in health and metabolic diseases (5). Mice harboring whole-body deletion of ERα (ERαKO) reveal that estrogen signaling through this receptor regulates glucose homeostasis in part by modulating hepatic insulin sensitivity (6) and glucose uptake in the skeletal muscle and adipose tissue (7). Moreover, ERα signaling has been demonstrated to promote pancreatic insulin-producing β-cell function, survival, and proliferation, as well as protection from development of diabetes in mice of both sexes (8–10). Thus, estrogen is generally thought to positively regulate glucose homeostasis primarily through ERα, which is expressed in both male and female tissues, but the respective importance of nuclear and membrane ERα pools in the regulation of glucose homeostasis is not clear. ERα is predominantly characterized as a nuclear receptor and thus noted for exclusively regulating transcription of target genes. However, extranuclear ERα action or membrane compartment–initiated estrogen signaling is now widely accepted to activate different pathways occurring in the cytoplasm and nucleus. For example, membrane localization of ERα (which accounts for ∼5–10% of the ERα pool depending on cell type) facilitates membrane-initiated signaling events important for reproduction and vascular physiology (11) as well as β-cell function and survival (12). Although it is clear that ERα positively regulates glucose homeostasis, the distinct and overlapping contributions of the extranuclear and nuclear pool of ERα remain unknown. Therefore, mouse models specifically engineered to have only nuclear or membrane ERα action are necessary to delineate specific ERα functions related to …

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.

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.

Obesity, metabolic syndrome, and diabetes continue to be a scourge of modern society, despite much research and developed therapies. Research in these areas is ongoing, and in this review we discuss models of obesity, metabolic syndrome, and diabetes in the rat. There are many experimental and genetic ways to achieve these conditions, and these states can be either experimentally induced or spontaneously developed. Here we cover the various ways in which these disease states are achieved, and experimental uses of these disease models. The goal is to provide a practical and scientific resource for the experimenter who aims to study these disease states. First, we describe dietary methods to induce obesity, as well as sleep deprivation and physical activity models that lead to obesity. Next, aspects and models of metabolic syndrome are presented. Finally, models and caveats to the study of diabetes rat models are described.

An early hallmark of type 2 diabetes is a failure of proinsulin-to-insulin processing in pancreatic β-cells, resulting in hyperproinsulinemia. Proinsulin processing is quite sensitive to nutrient flux, and β-cell–specific deletion of the nutrient-sensing protein modifier OGlcNAc transferase (βOGTKO) causes β-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 4γ1 (eIF4G1) and carboxypeptidase E (CPE)-dependent proinsulin processing in βOGTKO mice. We first established that βOGTKO hyperproinsulinemia is independent of age, sex, glucose levels, and endoplasmic reticulum–CCAAT enhancer-binding protein homologous protein (CHOP)–mediated stress status. Of note, OGT loss was associated with a reduction in β-cell–resident CPE, and genetic reconstitution of CPE in βOGTKO 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 β-cells, increases islet CPE levels, and fully reverses βOGTKO 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.

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.

Fetal growth restriction is one of the most common obstetrical complications resulting in significant perinatal morbidity and mortality. The most frequent etiology of human singleton fetal growth restriction is placental insufficiency, which occurs secondary to reduced utero-placental perfusion, abnormal placentation, impaired trophoblast invasion and spiral artery remodeling, resulting in altered nutrient and oxygen transport. Two nutrient-sensing proteins involved in placental development and glucose and amino acid transport are mechanistic target of rapamycin (mTOR) and O-linked N-acetylglucosamine transferase (OGT), which are both regulated by availability of oxygen. Impairment in either of these pathways is associated with fetal growth restriction and accompanied by cellular stress in the forms of hypoxia, oxidative and endoplasmic reticulum (ER) stress, metabolic dysfunction and nutrient starvation in the placenta. Recent evidence has emerged regarding the potential impact of nutrient sensors on fetal stress response, which occurs in a sexual dysmorphic manner, indicating a potential element of genetic gender susceptibility to fetal growth restriction. In this mini review, we focus on the known role of mTOR and OGT in placental development, nutrient regulation and response to cellular stress in human fetal growth restriction with supporting evidence from rodent models.

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.

β cell loss of OGlcNAc transferase (βOGTKO), the sole enzyme that adds OGlcNAc post-translational modification onto target proteins impacting their function and stability, leads to diabetes and islet failure in mice. βOGTKO mice present hyperproinsulinemia, typified by increased serum proinsulin/insulin ratio. Here, we show that βOGTKO islets have reduced carboxypeptidase E protein but not mRNA (CPE, an exopeptidase that converts proinsulin to insulin), independent of sex, age and glucose levels. We identified that CPE is neither the target nor a binding partner of OGT. Eukaryotic initiation factor 4 gamma 1 (eIF4G1), previously shown to regulate CPE in vitro, is reduced at the protein level, but not mRNA, in βOGTKO islets. We identified that eIF4G1 is OGlcNAc-modified in β cells and mutation of the putative OGlcNAc site at serine 61 to alanine led to decreased protein stability, but not on CPE translation. To assess the role of eIF4G1 on CPE regulation in vivo, we generated mice with β cell specific deletion of eIF4G1 (RIPCre; eIF4G1flox/flox (βeIF4G1KO)), in R1207H eIF4G1 (eIF4G1) background. At 6 weeks of age in normal chow diet, glucose intolerance was evident between βeIF4G1KO and littermate control eIF4G1 mice, without alteration in peripheral insulin sensitivity. Surprisingly, no apparent insulin processing deficit was observed between βeIF4G1KO and eIF4G1 mice, consistent with the observed normal level of CPE protein among islets of βeIF4G1 KO, eIF4G1 or wild type mice via immunofluorescence, and comparable serum proinsulin/insulin ratio in βeIF4G1 KO and eIF4G1 mice. Significant reduction in the average β cell size and increased number of α cells was evident in βeIF4G1KO vs. eIF4G1 mice, hinting at loss in β cell mass. Together, these data unveil the regulation of OGT on eIF4G1, and underscore the novel role of eIF4G1 on glucose homeostasis and modulation of β cell size. Disclosure S. Jo: None. A.D. Lockridge: None. E. Alejandro: None.

The nutrient-sensor O-GlcNAc transferase (OGT), the sole enzyme that adds an O-GlcNAc-modification onto proteins, was shown to regulate β-cell survival and function as β-cell deletion of OGT induces diabetes and islet failure in mice. We hypothesized that β-cell OGT overexpression (Rip-cre;OgtOE) would confer protection from streptozotocin (STZ)-induced β-cell failure and high-fat diet (HFD)-induced diabetes. Female mice are typically resistant from HFD- or STZ-induced diabetes. However, the molecular mechanisms underlying this sexual dimorphism are unclear. Here, we uncovered that Rip-cre;OgtOE mice show normal peripheral insulin sensitivity and glucose tolerance in normal chow but when challenged female transgenic mice alone develop glucose intolerance post 8-weeks of HFD and severe hyperglycemia 1-week post STZ treatment. Surprisingly, male transgenic mice are neither protected nor impaired when compared to controls in both treatments. Molecular interrogation of islets validated efficient overexpression of OGT in both sexes by confirming protein expression of the reporter transgene (GFP). However, significant increase in OGT protein was observed only in islets of female Rip-cre;OgtOE mice, suggesting sex difference in OGT protein regulation. Transcriptome analysis by RNA sequencing shows increased expression levels of genes involved in acute inflammation (Ggt5, Ncf1, Ptgs1, Alox5ap) and immune response via agranulocyte adhesion and diapedesis (Glycam1) in islets of female Rip-cre;OgtOE mice, providing a mechanism of susceptibility to STZ and HFD-induced islet failure. Indeed, pancreas analysis shows increased islet infiltrating lymphocytes in female Rip-cre;OgtOE compared to control post 22 weeks of HFD treatment. Together, these data highlight the novel role of OGT in inflammation induced by STZ and HFD in islets and emphasize the importance of OGT dosage and O-GlcNAc cycling in β-cell survival. Disclosure R. Mohan: None. H. Ruan: None. X. Yang: None. E. Alejandro: None.

Highly expressed in the β-cell, the nutrient sensor O-GlcNAc transferase (OGT) couples metabolic environment to cellular behavior through dynamic protein modifications (O-GlcNAcylation) but the role of OGT in β-cell hyperlipidemia is unknown. Here, we observed changes in islet O-GlcNAcylation in obese humans and high-fat diet (HFD, 60%) fed mice vs. lean controls, leading us to hypothesize that OGT mediates β-cell adaptations to diet-induced obesity. In a mouse model of partial β-cell OGT loss, we found that Rip-Cre; OGT f/+ (OGT Het) mice failed to develop hyperinsulinemia over 12 weeks of HFD. Only Het islets isolated from HFD mice showed deficits in stimulated insulin secretion (30 mM KCl, vs. HFD Ogt f/+). Nutrient-driven ATP was not impaired, but islets with full β-cell OGT KO (Rip-Cre; OGT f/f) showed no acute secretory potentiation to palmitate (100 μM) implicating the amplification arm of the stimulus-secretion pathway. HFD β-cell mass was similar (OGT Het vs. WT) but increased Ins+/Gcg+ cells and Ngn3 gene expression in OGT Het islets suggested functional β-cell loss through enhanced dedifferentiation. HFD hypoinsulinemia was reiterated in a tamoxifen-induced OGT loss model (Mip-Cre; OGT f/f), which also showed elevated proinsulin/insulin serum ratios (6 weeks HFD). Transcriptome analysis by RNA-sequencing of OGT KO and WT mouse islets revealed differential gene expression of proteins involved in membrane depolarization (Kcnj11, Abcc8), ER calcium (Atp2a2), lipid uptake (Lpl) and β-cell differentiation (Ucn3). An upstream analysis suggested impairments in free radical scavenging, supported by elevated levels of 4-HNE lipid peroxidation staining in post-HFD OGT Het islets. However, antioxidant supplementation of HFD chow (0.2% alpha-lipoic acid) did not rescue OGT Het in vivo deficits. These data reveal OGT as a critical mediator of β-cell adaptation to diet-induced obesity and highlight protein O-GlcNAcylation as a potential therapeutic target to prevent prediabetic progression. Disclosure A.D. Lockridge: None. S. Jo: None. N.E. Damberg: None. R. Mohan: None. E. Alejandro: None.