The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in ß-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the ß-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the ß cell. We hypothesized ß-cell-specific EP3 knockout (EP3 ßKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 ßKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 ßKO mice as compared with wild-type controls, with no effect of ß-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 ßKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of ß-cell replication and survival, revealing severe ß-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating ß-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of ß-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed ß-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.

Prostaglandin EP3 receptor signaling is required to prevent insulin hypersecretion and metabolic dysfunction in a non-obese mouse model of insulin resistance.

When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 wk of age, frankly diabetic. Previous work has shown prostaglandin EP3 receptor (EP3) expression and activity is upregulated in islets from BTBR-Ob mice as compared with lean controls, actively contributing to their ß-cell dysfunction. In this work, we aimed to test the impact of ß-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse-an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain-generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in ß-cell fractional area or markers of ß-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective upregulation of constitutively active EP3 splice variants in islets from young, lean BTBR mice as compared with C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females versus Balb/c males was fully consistent with their sexually dimorphic metabolic phenotype after loss of EP3-coupled Gαz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion.NEW & NOTEWORTHY Islet prostaglandin EP3 receptor (EP3) signaling is well known as upregulated in the pathophysiological conditions of type 2 diabetes, contributing to ß-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously unknown relationship between mouse insulin resistance and the penetrance of rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating ß-cell-specific knockouts.

Short-term methionine deprivation improves metabolic health via sexually dimorphic, mTORC1-independent mechanisms.

Obesity and diabetes are major challenges to global health, and there is an urgent need for interventions that promote weight loss. Dietary restriction of methionine promotes leanness and improves metabolic health in mice and humans. However, poor long-term adherence to this diet limits its translational potential. In this study, we develop a short-term methionine deprivation (MD) regimen that preferentially reduces fat mass, restoring normal body weight and glycemic control to diet-induced obese mice of both sexes. The benefits of MD do not accrue from calorie restriction, but instead result from increased energy expenditure. MD promotes increased energy expenditure in a sex-specific manner, inducing the fibroblast growth factor (Fgf)-21-uncoupling protein (Ucp)-1 axis only in males. Methionine is an agonist of the protein kinase mechanistic target of rapamycin complex (mTORC)-1, which has been proposed to play a key role in the metabolic response to amino acid-restricted diets. In our study, we used a mouse model of constitutive hepatic mTORC1 activity and demonstrate that suppression of hepatic mTORC1 signaling is not required for the metabolic effects of MD. Our study sheds new light on the mechanisms by which dietary methionine regulates metabolic health and demonstrates the translational potential of MD for the treatment of obesity and type 2 diabetes.-Yu, D., Yang, S. E., Miller, B. R., Wisinski, J. A., Sherman, D. S., Brinkman, J. A., Tomasiewicz, J. L., Cummings, N. E., Kimple, M. E., Cryns, V. L., Lamming, D. W. Short-term methionine deprivation improves metabolic health via sexually dimorphic, mTORC1-independent mechanisms.

Restoration of metabolic health by decreased consumption of branched-chain amino acids.

KEY POINTS: We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, modestly improves glucose tolerance and slows fat mass gain. In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice. Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high-fat, high-sugar diet. A low BCAA diet transiently induces FGF21 (fibroblast growth factor 21) and increases energy expenditure. We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets. ABSTRACT: Obesity and diabetes are increasing problems around the world, and although even moderate weight loss can improve metabolic health, reduced calorie diets are notoriously difficult to sustain. Branched-chain amino acids (BCAAs; leucine, isoleucine and valine) are elevated in the blood of obese, insulin-resistant humans and rodents. We recently demonstrated that specifically reducing dietary levels of BCAAs has beneficial effects on the metabolic health of young, growing mice, improving glucose tolerance and modestly slowing fat mass gain. In the present study, we examine the hypothesis that reducing dietary BCAAs will promote weight loss, reduce adiposity, and improve blood glucose control in diet-induced obese mice with pre-existing metabolic syndrome. We find that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucoregulatory control in diet-induced obese mice. Most dramatically, mice eating an otherwise unhealthy high-calorie, high-sugar Western diet with reduced levels of BCAAs lost weight and fat mass rapidly until regaining a normal weight. Importantly, this normalization of weight was mediated not by caloric restriction or increased activity, but by increased energy expenditure, and was accompanied by a transient induction of the energy balance regulating hormone FGF21 (fibroblast growth factor 21). Consumption of a Western diet reduced in BCAAs was also accompanied by a dramatic improvement in glucose tolerance and insulin resistance. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance.

EPAC-RAP1 Axis-Mediated Switch in the Response of Primary and Metastatic Melanoma to Cyclic AMP.

Cyclic AMP (cAMP) is an important second messenger that regulates a wide range of physiologic processes. In mammalian cutaneous melanocytes, cAMP-mediated signaling pathways activated by G-protein-coupled receptors (GPCR), like melanocortin 1 receptor (MC1R), play critical roles in melanocyte homeostasis including cell survival, proliferation, and pigment synthesis. Impaired cAMP signaling is associated with increased risk of cutaneous melanoma. Although mutations in MAPK pathway components are the most frequent oncogenic drivers of melanoma, the role of cAMP in melanoma is not well understood. Here, using the Braf(V600E)/Pten-null mouse model of melanoma, topical application of an adenylate cyclase agonist, forskolin (a cAMP inducer), accelerated melanoma tumor development in vivo and stimulated the proliferation of mouse and human primary melanoma cells, but not human metastatic melanoma cells in vitro The differential response of primary and metastatic melanoma cells was also evident upon pharmacologic inhibition of the cAMP effector protein kinase A. Pharmacologic inhibition and siRNA-mediated knockdown of other cAMP signaling pathway components showed that EPAC-RAP1 axis, an alternative cAMP signaling pathway, mediates the switch in response of primary and metastatic melanoma cells to cAMP. Evaluation of pERK levels revealed that this phenotypic switch was not correlated with changes in MAPK pathway activity. Although cAMP elevation did not alter the sensitivity of metastatic melanoma cells to BRAF(V600E) and MEK inhibitors, the EPAC-RAP1 axis appears to contribute to resistance to MAPK pathway inhibition. These data reveal a MAPK pathway-independent switch in response to cAMP signaling during melanoma progression.Implications: The prosurvival mechanism involving the cAMP-EPAC-RAP1 signaling pathway suggest the potential for new targeted therapies in melanoma. Mol Cancer Res; 15(12); 1792-802. ©2017 AACR.

The EP3 Receptor/Gz Signaling Axis as a Therapeutic Target for Diabetes and Cardiovascular Disease.

Cardiovascular disease is a common co-morbidity found with obesity-linked type 2 diabetes. Current pharmaceuticals for these two diseases treat each of them separately. Yet, diabetes and cardiovascular disease share molecular signaling pathways that are increasingly being understood to contribute to disease pathophysiology, particularly in pre-clinical models. This review will focus on one such signaling pathway: that mediated by the G protein-coupled receptor, Prostaglandin E2 Receptor 3 (EP3), and its associated G protein in the insulin-secreting beta-cell and potentially the platelet, Gz. The EP3/Gz signaling axis may hold promise as a dual target for type 2 diabetes and cardiovascular disease.

The Inhibitory G Protein α-Subunit, Gαz, Promotes Type 1 Diabetes-Like Pathophysiology in NOD Mice.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes ß-cell death and inhibits ß-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional ß-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive ß-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive ß-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, ß-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a ß-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in ß-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of ß-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

Synergy Between Gαz Deficiency and GLP-1 Analog Treatment in Preserving Functional ß-Cell Mass in Experimental Diabetes.

A defining characteristic of type 1 diabetes mellitus (T1DM) pathophysiology is pancreatic ß-cell death and dysfunction, resulting in insufficient insulin secretion to properly control blood glucose levels. Treatments that promote ß-cell replication and survival, thus reversing the loss of ß-cell mass, while also preserving ß-cell function, could lead to a real cure for T1DM. The α-subunit of the heterotrimeric Gz protein, Gαz, is a tonic negative regulator of adenylate cyclase and downstream cAMP production. cAMP is one of a few identified signaling molecules that can simultaneously have a positive impact on pancreatic islet ß-cell proliferation, survival, and function. The purpose of our study was to determine whether mice lacking Gαz might be protected, at least partially, from ß-cell loss and dysfunction after streptozotocin treatment. We also aimed to determine whether Gαz might act in concert with an activator of the cAMP-stimulatory glucagon-like peptide 1 receptor, exendin-4 (Ex4). Without Ex4 treatment, Gαz-null mice still developed hyperglycemia, albeit delayed. The same finding held true for wild-type mice treated with Ex4. With Ex4 treatment, Gαz-null mice were protected from developing severe hyperglycemia. Immunohistological studies performed on pancreas sections and in vitro apoptosis, cytotoxicity, and survival assays demonstrated a clear effect of Gαz signaling on pancreatic ß-cell replication and death; ß-cell function was also improved in Gαz-null islets. These data support our hypothesis that a combination of therapies targeting both stimulatory and inhibitory pathways will be more effective than either alone at protecting, preserving, and possibly regenerating ß-cell mass and function in T1DM.

Glucagon-Like Peptide-1 Regulates Cholecystokinin Production in ß-Cells to Protect From Apoptosis.

Cholecystokinin (CCK) is a classic gut hormone that is also expressed in the pancreatic islet, where it is highly up-regulated with obesity. Loss of CCK results in increased ß-cell apoptosis in obese mice. Similarly, islet α-cells produce increased amounts of another gut peptide, glucagon-like peptide 1 (GLP-1), in response to cytokine and nutrient stimulation. GLP-1 also protects ß-cells from apoptosis via cAMP-mediated mechanisms. Therefore, we hypothesized that the activation of islet-derived CCK and GLP-1 may be linked. We show here that both human and mouse islets secrete active GLP-1 as a function of body mass index/obesity. Furthermore, GLP-1 can rapidly stimulate ß-cell CCK production and secretion through direct targeting by the cAMP-modulated transcription factor, cAMP response element binding protein (CREB). We find that cAMP-mediated signaling is required for Cck expression, but CCK regulation by cAMP does not require stimulatory levels of glucose or insulin secretion. We also show that CREB directly targets the Cck promoter in islets from obese (Leptin(ob/ob)) mice. Finally, we demonstrate that the ability of GLP-1 to protect ß-cells from cytokine-induced apoptosis is partially dependent on CCK receptor signaling. Taken together, our work suggests that in obesity, active GLP-1 produced in the islet stimulates CCK production and secretion in a paracrine manner via cAMP and CREB. This intraislet incretin loop may be one mechanism whereby GLP-1 protects ß-cells from apoptosis.