The PGE2 EP3 Receptor Regulates Diet-Induced Adiposity in Male Mice.

Mice carrying a targeted disruption of the prostaglandin E2 (PGE2) E-prostanoid receptor 3 (EP3) gene, Ptger3, were fed a high-fat diet (HFD), or a micronutrient matched control diet, to investigate the effects of disrupted PGE2-EP3 signaling on diabetes in a setting of diet-induced obesity. Although no differences in body weight were seen in mice fed the control diet, when fed a HFD, EP3(-/-) mice gained more weight relative to EP3(+/+) mice. Overall, EP3(-/-) mice had increased epididymal fat mass and adipocyte size; paradoxically, a relative decrease in both epididymal fat pad mass and adipocyte size was observed in the heaviest EP3(-/-) mice. The EP3(-/-) mice had increased macrophage infiltration, TNF-α, monocyte chemoattractant protein-1, IL-6 expression, and necrosis in their epididymal fat pads as compared with EP3(+/+) animals. Adipocytes isolated from EP3(+/+) or EP3(-/-) mice were assayed for the effect of PGE2-evoked inhibition of lipolysis. Adipocytes isolated from EP3(-/-) mice lacked PGE2-evoked inhibition of isoproterenol stimulated lipolysis compared with EP3(+/+). EP3(-/-) mice fed HFD had exaggerated ectopic lipid accumulation in skeletal muscle and liver, with evidence of hepatic steatosis. Both blood glucose and plasma insulin levels were similar between genotypes on a control diet, but when fed HFD, EP3(-/-) mice became hyperglycemic and hyperinsulinemic when compared with EP3(+/+) fed HFD, demonstrating a more severe insulin resistance phenotype in EP3(-/-). These results demonstrate that when fed a HFD, EP3(-/-) mice have abnormal lipid distribution, developing excessive ectopic lipid accumulation and associated insulin resistance.

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.

Connective tissue growth factor modulates adult ß-cell maturity and proliferation to promote ß-cell regeneration in mice.

Stimulation of endogenous ß-cell expansion could facilitate regeneration in patients with diabetes. In mice, connective tissue growth factor (CTGF) is expressed in embryonic ß-cells and in adult ß-cells during periods of expansion. We discovered that in embryos CTGF is necessary for ß-cell proliferation, and increased CTGF in ß-cells promotes proliferation of immature (MafA(-)) insulin-positive cells. CTGF overexpression, under nonstimulatory conditions, does not increase adult ß-cell proliferation. In this study, we tested the ability of CTGF to promote ß-cell proliferation and regeneration after partial ß-cell destruction. ß-Cell mass reaches 50% recovery after 4 weeks of CTGF treatment, primarily via increased ß-cell proliferation, which is enhanced as early as 2 days of treatment. CTGF treatment increases the number of immature ß-cells but promotes proliferation of both mature and immature ß-cells. A shortened ß-cell replication refractory period is also observed. CTGF treatment upregulates positive cell-cycle regulators and factors involved in ß-cell proliferation, including hepatocyte growth factor, serotonin synthesis, and integrin ß1. Ex vivo treatment of whole islets with recombinant human CTGF induces ß-cell replication and gene expression changes consistent with those observed in vivo, demonstrating that CTGF acts directly on islets to promote ß-cell replication. Thus, CTGF can induce replication of adult mouse ß-cells given a permissive microenvironment.

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.

Differential regulation of embryonic and adult ß cell replication.

Diabetes results from an inadequate functional ß cell mass, either due to autoimmune destruction (Type 1 diabetes) or insulin resistance combined with ß cell failure (Type 2 diabetes). Strategies to enhance ß cell regeneration or increase cell proliferation could improve outcomes for patients with diabetes. Research conducted over the past several years has revealed that factors regulating embryonic ß cell mass expansion differ from those regulating replication of ß cells post-weaning. This article aims to compare and contrast factors known to control embryonic and postnatal ß cell replication. In addition, we explore the possibility that connective tissue growth factor (CTGF) could increase adult ß cell replication. We have already shown that CTGF is required for embryonic ß cell proliferation and is sufficient to induce replication of embryonic ß cells. Here we examine whether adult ß cell replication and expansion of ß cell mass can be enhanced by increased CTGF expression in mature ß cells.