Pharmacological modulation of prostaglandin E2 (PGE2 ) EP receptors improves cardiomyocyte function under hyperglycemic conditions.

Type 2 diabetes (T2D) affects >30 million Americans and nearly 70% of individuals with T2D will die from cardiovascular disease (CVD). Circulating levels of the inflammatory signaling lipid, prostaglandin E2 (PGE2 ), are elevated in the setting of obesity and T2D and are associated with decreased cardiac function. The EP3 and EP4 PGE2 receptors have opposing actions in several tissues, including the heart: overexpression of EP3 in cardiomyocytes impairs function, while EP4 overexpression improves function. Here we performed complementary studies in vitro with isolated cardiomyocytes and in vivo using db/db mice, a model of T2D, to analyze the effects of EP3 inhibition or EP4 activation on cardiac function. Using echocardiography, we found that 2 weeks of systemic treatment of db/db mice with 20 mg/kg of EP3 antagonist, beginning at 6 weeks of age, improves ejection fraction and fractional shortening (with no effect on heart rate). We further show that either EP3 blockade or EP4 activation enhances contractility and calcium cycling in isolated mouse cardiomyocytes cultured in both normal and high glucose. Thus, peak [Ca2+ ]I transient amplitude was increased, while time to peak [Ca2+ ]I and [Ca2+ ]I decay were decreased. These data suggest that modulation of EP3 and EP4 activity has beneficial effects on cardiomyocyte contractility and overall heart function.

Pharmacological blockade of the EP3 prostaglandin E2 receptor in the setting of type 2 diabetes enhances ß-cell proliferation and identity and relieves oxidative damage.

OBJECTIVE: Type 2 diabetes is characterized by hyperglycemia and inflammation. Prostaglandin E2, which signals through four G protein-coupled receptors (EP1-4), is a mediator of inflammation and is upregulated in diabetes. We have shown previously that EP3 receptor blockade promotes ß-cell proliferation and survival in isolated mouse and human islets ex vivo. Here, we analyzed whether systemic EP3 blockade could enhance ß-cell mass and identity in the setting of type 2 diabetes using mice with a spontaneous mutation in the leptin receptor (Leprdb). METHODS: Four- or six-week-old, db/+, and db/db male mice were treated with an EP3 antagonist daily for two weeks. Pancreata were analyzed for α-cell and ß-cell proliferation and ß-cell mass. Islets were isolated for transcriptomic analysis. Selected gene expression changes were validated by immunolabeling of the pancreatic tissue sections. RESULTS: EP3 blockade increased ß-cell mass in db/db mice through enhanced ß-cell proliferation. Importantly, there were no effects on α-cell proliferation. EP3 blockade reversed the changes in islet gene expression associated with the db/db phenotype and restored the islet architecture. Expression of the GLP-1 receptor was slightly increased by EP3 antagonist treatment in db/db mice. In addition, the transcription factor nuclear factor E2-related factor 2 (Nrf2) and downstream targets were increased in islets from db/db mice in response to treatment with an EP3 antagonist. The markers of oxidative stress were decreased. CONCLUSIONS: The current study suggests that EP3 blockade promotes ß-cell mass expansion in db/db mice. The beneficial effects of EP3 blockade may be mediated through Nrf2, which has recently emerged as a key mediator in the protection against cellular oxidative damage.

Maternal Western-style diet affects offspring islet composition and function in a non-human primate model of maternal over-nutrition.

OBJECTIVE: In humans, offspring of women who are overweight or obese are more likely to develop metabolic disease later in life. Studies in lower animal species reveal that a calorically-dense maternal diet is associated with alterations in islet cell mass and function. The long-term effects of maternal diet on the structure and function of offspring islets with characteristics similar to humans are unknown. We used a well-established non-human primate (NHP) model to determine the consequences of exposure to Western-Style Diet (WSD) in utero and during lactation on islet cell mass and function in the offspring. METHODS: Female Japanese Macaques (Macaca fuscata) were fed either control (CTR) or WSD before and throughout pregnancy and lactation. Offspring were weaned onto CTR or WSD to generate four different groups based on maternal/offspring diets: CTR/CTR, WSD/CTR, CTR/WSD, and WSD/WSD. Offspring were analyzed at three years of age. Pancreatic tissue sections were immunolabelled to measure α- and ß-cell mass and proliferation as well as islet vascularization. Live islets were also isolated to test the effects of WSD-exposure on islet function ex vivo. Offspring glucose tolerance was correlated with various maternal characteristics. RESULTS: α-cell mass was reduced as a result of maternal WSD exposure. α-cell proliferation was reduced in response to offspring WSD. Islet vasculature did not differ among the diet groups. Islets from WSD/CTR offspring secreted a greater amount of insulin in response to glucose ex vivo. We also found that maternal glucose tolerance and parity correlated with offspring glucose tolerance. CONCLUSIONS: Maternal WSD exposure results in persistently decreased α-cell mass in the three-year old offspring. WSD/CTR islets secreted greater amounts of insulin ex vivo, suggesting that these islets are primed to hyper-secrete insulin under certain metabolic stressors. Although WSD did not induce overt impaired glucose tolerance in dams or offspring, offspring born to mothers with higher glucose excursions during a glucose tolerance test were more likely to also show higher glucose excursions.

Cooperative function of Pdx1 and Oc1 in multipotent pancreatic progenitors impacts postnatal islet maturation and adaptability.

The transcription factors pancreatic and duodenal homeobox 1 (Pdx1) and onecut1 (Oc1) are coexpressed in multipotent pancreatic progenitors (MPCs), but their expression patterns diverge in hormone-expressing cells, with Oc1 expression being extinguished in the endocrine lineage and Pdx1 being maintained at high levels in ß-cells. We previously demonstrated that cooperative function of these two factors in MPCs is necessary for proper specification and differentiation of pancreatic endocrine cells. In those studies, we observed a persistent decrease in expression of the ß-cell maturity factor MafA. We therefore hypothesized that Pdx1 and Oc1 cooperativity in MPCs impacts postnatal ß-cell maturation and function. Here our model of Pdx1-Oc1 double heterozygosity was used to investigate the impact of haploinsufficiency for both of these factors on postnatal ß-cell maturation, function, and adaptability. Examining mice at postnatal day (P) 14, we observed alterations in pancreatic insulin content in both Pdx1 heterozygotes and double heterozygotes. Gene expression analysis at this age revealed significantly decreased expression of many genes important for glucose-stimulated insulin secretion (e.g., Glut2, Pcsk1/2, Abcc8) exclusively in double heterozygotes. Analysis of P14 islets revealed an increase in the number of mixed islets in double heterozygotes. We predicted that double-heterozygous ß-cells would have an impaired ability to respond to stress. Indeed, we observed that ß-cell proliferation fails to increase in double heterozygotes in response to either high-fat diet or placental lactogen. We thus report here the importance of cooperation between regulatory factors early in development for postnatal islet maturation and adaptability.

Vascular-derived connective tissue growth factor (Ctgf) is critical for pregnancy-induced ß cell hyperplasia in adult mice.

During pregnancy, maternal ß cells undergo compensatory changes including hypertrophy, hyperplasia, and increased glucose-stimulated insulin secretion (GSIS). Failure of these adaptations to occur can result in gestational diabetes mellitus. The secreted protein, Connective tissue growth factor (Ctgf), is critical for normal ß cell development and promotes regeneration after partial ß cell ablation. During embryogenesis, Ctgf is expressed in pancreatic ducts, vasculature, and ß cells. In the adult pancreas, Ctgf is expressed only in the vasculature. Here, we report that pregnant mice with global Ctgf haploinsufficiency (CtgfLacZ/+) have an impairment in maternal ß cell proliferation, while ß cell proliferation in virgin CtgfLacZ/+ females is unaffected. Additionally, α-cell proliferation, ß cell size, and GSIS were unaffected in CtgfLacZ/+ mice, suggesting that vascular-derived Ctgf has a specific role in islet compensation during pregnancy.

Unexpected effects of the MIP-CreER transgene and tamoxifen on ß-cell growth in C57Bl6/J male mice.

Transgenic mouse models have been fundamental in the discovery of factors that regulate ß-cell development, mass, and function. Several groups have recently shown that some of these models display previously uncharacterized phenotypes due to the transgenic system itself. These include impaired islet function and increased ß-cell mass due to the presence of a human growth hormone (hGH) minigene as well as impaired ß-cell proliferation in response to tamoxifen (TM) administration. We aimed to determine how these systems impact ß-cell mass and proliferation during high fat diet (HFD). To this end, we utilized C57Bl6/J male MIP-CreER mice, which are known to express hGH, or wild-type (WT) mice treated with vehicle corn oil or TM In the absence of TM, MIP-CreER mice fed a chow diet have increased ß-cell mass due to hypertrophy, whereas replication is unchanged. Similarly, after 1 week on HFD, MIP-CreER mice have increased ß-cell mass compared to WT, and this is due to hypertrophy rather than increased proliferation. To assess the impact of TM on ß-cell proliferation and mass, WT mice were treated with vehicle corn oil or TM and then fed a chow diet or HFD for 3 days. We observed that TM-treated mice have improved glucose homeostasis on chow diet but impaired ß-cell proliferation in response to 3 days HFD feeding. These results unveil additional complications associated with commonly used pancreas-specific mouse models.

Connective tissue growth factor is critical for proper ß-cell function and pregnancy-induced ß-cell hyperplasia in adult mice.

During pregnancy, maternal ß-cells undergo compensatory changes, including increased ß-cell mass and enhanced glucose-stimulated insulin secretion. Failure of these adaptations to occur results in gestational diabetes mellitus. The secreted protein connective tissue growth factor (CTGF) is critical for normal ß-cell development and promotes regeneration after partial ß-cell ablation. During embryogenesis, CTGF is expressed in pancreatic ducts, vasculature, and ß-cells. In adult pancreas, CTGF is expressed only in the vasculature. Here we show that pregnant mice with global Ctgf haploinsufficiency (Ctgf(LacZ/+)) have an impairment in maternal ß-cell proliferation; no difference was observed in virgin Ctgf(LacZ/+) females. Using a conditional CTGF allele, we found that mice with a specific inactivation of CTGF in endocrine cells (Ctgf(ΔEndo)) develop gestational diabetes during pregnancy, but this is due to a reduction in glucose-stimulated insulin secretion rather than impaired maternal ß-cell proliferation. Moreover, virgin Ctgf(ΔEndo) females also display impaired GSIS with glucose intolerance, indicating that underlying ß-cell dysfunction precedes the development of gestational diabetes in this animal model. This is the first time a role for CTGF in ß-cell function has been reported.

Macrophages are essential for CTGF-mediated adult ß-cell proliferation after injury.

OBJECTIVE: Promotion of endogenous ß-cell mass expansion could facilitate regeneration in patients with diabetes. We discovered that the secreted protein CTGF (aka CCN2) promotes adult ß-cell replication and mass regeneration after injury via increasing ß-cell immaturity and shortening the replicative refractory period. However, the mechanism of CTGF-mediated ß-cell proliferation is unknown. Here we focused on whether CTGF alters cells of the immune system to enhance ß-cell replication. METHODS: Using mouse models for 50% ß-cell ablation and conditional, ß-cell-specific CTGF induction, we assessed changes in immune cell populations by performing immunolabeling and gene expression analyses. We tested the requirement for macrophages in CTGF-mediated ß-cell proliferation via clodronate-based macrophage depletion. RESULTS: CTGF induction after 50% ß-cell ablation increased both macrophages and T-cells in islets. An upregulation in the expression of several macrophage and T-cell chemoattractant genes was also observed in islets. Gene expression analyses suggest an increase in M1 and a decrease in M2 macrophage markers. Depletion of macrophages (without changes in T cell number) blocked CTGF-mediated ß-cell proliferation and prevented the increase in ß-cell immaturity. CONCLUSIONS: Our data show that macrophages are critical for CTGF-mediated adult ß-cell proliferation in the setting of partial ß-cell ablation. This is the first study to link a specific ß-cell proliferative factor with immune-mediated ß-cell proliferation in a ß-cell injury model.

Activation of FoxM1 Revitalizes the Replicative Potential of Aged ß-Cells in Male Mice and Enhances Insulin Secretion.

Type 2 diabetes incidence increases with age, while ß-cell replication declines. The transcription factor FoxM1 is required for ß-cell replication in various situations, and its expression declines with age. We hypothesized that increased FoxM1 activity in aged ß-cells would rejuvenate proliferation. Induction of an activated form of FoxM1 was sufficient to increase ß-cell mass and proliferation in 12-month-old male mice after just 2 weeks. Unexpectedly, at 2 months of age, induction of activated FoxM1 in male mice improved glucose homeostasis with unchanged ß-cell mass. Cells expressing activated FoxM1 demonstrated enhanced glucose-stimulated Ca2+ influx, which resulted in improved glucose tolerance through enhanced ß-cell function. Conversely, our laboratory has previously demonstrated that mice lacking FoxM1 in the pancreas display glucose intolerance or diabetes with only a 60% reduction in ß-cell mass, suggesting that the loss of FoxM1 is detrimental to ß-cell function. Ex vivo insulin secretion was therefore examined in size-matched islets from young mice lacking FoxM1 in ß-cells. Foxm1-deficient islets indeed displayed reduced insulin secretion. Our studies reveal that activated FoxM1 increases ß-cell replication while simultaneously enhancing insulin secretion and improving glucose homeostasis, making FoxM1 an attractive therapeutic target for diabetes.

High-fat diet-induced ß-cell proliferation occurs prior to insulin resistance in C57Bl/6J male mice.

Both short- (1 wk) and long-term (2-12 mo) high-fat diet (HFD) studies reveal enhanced ß-cell mass due to increased ß-cell proliferation. ß-Cell proliferation following HFD has been postulated to occur in response to insulin resistance; however, whether HFD can induce ß-cell proliferation independent of insulin resistance has been controversial. To examine the kinetics of HFD-induced ß-cell proliferation and its correlation with insulin resistance, we placed 8-wk-old male C57Bl/6J mice on HFD for different lengths of time and assayed the following: glucose tolerance, insulin secretion in response to glucose, insulin tolerance, ß-cell mass, and ß-cell proliferation. We found that ß-cell proliferation was significantly increased after only 3 days of HFD feeding, weeks before an increase in ß-cell mass or peripheral insulin resistance was detected. These results were confirmed by hyperinsulinemic euglycemic clamps and measurements of α-hydroxybutyrate, a plasma biomarker of insulin resistance in humans. An increase in expression of key islet-proliferative genes was found in isolated islets from 1-wk HFD-fed mice compared with chow diet (CD)-fed mice. These data indicate that short-term HFD feeding enhances ß-cell proliferation before insulin resistance becomes apparent.

Activated FoxM1 attenuates streptozotocin-mediated ß-cell death.

The forkhead box transcription factor FoxM1, a positive regulator of the cell cycle, is required for ß-cell mass expansion postnatally, during pregnancy, and after partial pancreatectomy. Up-regulation of full-length FoxM1, however, is unable to stimulate increases in ß-cell mass in unstressed mice or after partial pancreatectomy, probably due to the lack of posttranslational activation. We hypothesized that expression of an activated form of FoxM1 could aid in recovery after ß-cell injury. We therefore derived transgenic mice that inducibly express an activated version of FoxM1 in ß-cells (RIP-rtTA;TetO-hemagglutinin (HA)-Foxm1(Δ)(NRD) mice). This N-terminally truncated form of FoxM1 bypasses 2 posttranslational controls: exposure of the forkhead DNA binding domain and targeted proteasomal degradation. Transgenic mice were subjected to streptozotocin (STZ)-induced ß-cell ablation to test whether activated FoxM1 can promote ß-cell regeneration. Mice expressing HA-FoxM1(ΔNRD) displayed decreased ad libitum-fed blood glucose and increased ß-cell mass. ß-Cell proliferation was actually decreased in RIP-rtTA:TetO-HA-Foxm1(NRD) mice compared with that in RIP-rtTA mice 7 days after STZ treatment. Unexpectedly, ß-cell death was decreased 2 days after STZ treatment. RNA sequencing analysis indicated that activated FoxM1 alters the expression of extracellular matrix and immune cell gene profiles, which may protect against STZ-mediated death. These studies highlight a previously underappreciated role for FoxM1 in promoting ß-cell survival.