Efficacy of glucagon-like peptide-1 and estrogen dual agonist in pancreatic islets protection and pre-clinical models of insulin-deficient diabetes.

We study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 ß cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.

Estrogen receptor α protects pancreatic ß-cells from apoptosis by preserving mitochondrial function and suppressing endoplasmic reticulum stress.

Estrogen receptor α (ERα) action plays an important role in pancreatic ß-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote ß-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 ß-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 ß-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes ß-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

The Role of Estrogens in Pancreatic Islet Physiopathology.

In rodent models of insulin-deficient diabetes, 17ß-estradiol (E2) protects pancreatic insulin-producing ß-cells against oxidative stress, amyloid polypeptide toxicity, gluco-lipotoxicity, and apoptosis. Three estrogen receptors (ERs)-ERα, ERß, and the G protein-coupled ER (GPER)-have been identified in rodent and human ß-cells. This chapter describes recent advances in our understanding of the role of ERs in islet ß-cell function, nutrient homeostasis, survival from pro-apoptotic stimuli, and proliferation. We discuss why and how ERs represent potential therapeutic targets for the maintenance of functional ß-cell mass.

Effect of targeted estrogen delivery using glucagon-like peptide-1 on insulin secretion, insulin sensitivity and glucose homeostasis.

The female estrogen 17ß-estradiol (E2) enhances pancreatic ß-cell function via estrogen receptors (ERs). However, the risk of hormone dependent cancer precludes the use of general estrogen therapy as a chronic treatment for diabetes. To target E2 to ß-cells without the undesirable effects of general estrogen therapy, we created fusion peptides combining active or inactive glucagon-like peptide-1 (GLP-1) and E2 in a single molecule (aGLP1-E2 and iGLP1-E2 respectively). By combining the activities of GLP-1 and E2, we envisioned synergistic insulinotropic activities of these molecules on ß-cells. In cultured human islets and in C57BL/6 mice, both aGLP1 and aGLP1-E2 enhanced glucose-stimulated insulin secretion (GSIS) compared to vehicle and iGLP1-E2 without superior efficacy of aGLP1-E2 compared to GLP-1 alone. However, aGLP1-E2 decreased fasting and fed blood glucose to a greater extent than aGLP1 and iGLP1-E2 alone. Further, aGLP1-E2 exhibited improved insulin sensitivity compared to aGLP1 and iGLP1-E2 alone (HOMA-IR and insulin tolerance test). In conclusion, targeted estrogen delivery to non-diabetic islets in the presence of GLP-1 does not enhance GSIS. However, combining GLP-1 to estrogen delivers additional efficacy relative to GLP-1 alone on insulin sensitivity and glucose homeostasis in non-diabetic mice.

SMAD3 negatively regulates serum irisin and skeletal muscle FNDC5 and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) during exercise.

Beige adipose cells are a distinct and inducible type of thermogenic fat cell that express the mitochondrial uncoupling protein-1 and thus represent a powerful target for treating obesity. Mice lacking the TGF-ß effector protein SMAD3 are protected against diet-induced obesity because of browning of their white adipose tissue (WAT), leading to increased whole body energy expenditure. However, the role SMAD3 plays in WAT browning is not clearly understood. Irisin is an exercise-induced skeletal muscle hormone that induces WAT browning similar to that observed in SMAD3-deficient mice. Together, these observations suggested that SMAD3 may negatively regulate irisin production and/or secretion from skeletal muscle. To address this question, we used wild-type and SMAD3 knock-out (Smad3(-/-)) mice subjected to an exercise regime and C2C12 myotubes treated with TGF-ß, a TGF-ß receptor 1 pharmacological inhibitor, adenovirus expressing constitutively active SMAD3, or siRNA against SMAD3. We find that in Smad3(-/-) mice, exercise increases serum irisin and skeletal muscle FNDC5 (irisin precursor) and its upstream activator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) to a greater extent than in wild-type mice. In C2C12 myotubes, TGF-ß suppresses FNDC5 and PGC-1α mRNA and protein levels via SMAD3 and promotes SMAD3 binding to the FNDC5 and PGC-1α promoters. These data establish that SMAD3 suppresses FNDC5 and PGC-1α in skeletal muscle cells. These findings shed light on the poorly understood regulation of irisin/FNDC5 by demonstrating a novel association between irisin and SMAD3 signaling in skeletal muscle.

Targeted estrogen delivery reverses the metabolic syndrome.

We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1-targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1-estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

Molecular mechanisms of estrogen receptors' suppression of lipogenesis in pancreatic ß-cells.

The gonadal steroid, 17ß-estradiol (E2), suppresses pancreatic islet fatty acid and glycerolipid synthesis and prevents ß-cell failure in rodent models of type 2 diabetes. ß-Cell estrogen receptors (ER) mediate these actions by suppressing the expression and enzymatic activity of fatty acid synthase (FAS). Here, we explored the mechanism of FAS suppression. We show that E2, and pharmacological agonists for ERα, ERß, and the G protein-coupled ER, suppress mRNA and protein expression of the transcriptional regulators of FAS, namely, sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP) in insulin-secreting INS-1 cells. ER suppress SREBP1c and ChREBP mRNA and protein expression via an extranuclear localization. Using two mouse lines with pancreas-specific null deletion of either ERα or the signal transducer and activator of transcription 3 (STAT3), we show that ERα activation in vivo reduces SREBP1c and ChREBP mRNA expression via a direct islet action involving STAT3 activation. The master regulators of lipogenesis, liver X receptor (LXR) α and ß, transcriptionally up-regulate SREBP1c and ChREBP. We find that activation of ERα, ERß, and G protein-coupled ER suppresses LXR's mRNA expression in INS-1 cells. We also observe that activation of ERα in mouse islets in vivo suppresses LXR mRNA in a STAT3-dependent manner. Finally, we show that E2 also activates and uses AMP-activated protein kinase in INS-1 cells to suppress SREBP1c protein expression. This study identifies extranuclear ER pathways involving STAT3 and AMP-activated protein kinase in the genetic control of lipogenesis with therapeutic implications to protect ß-cells in type 2 diabetes.

Importance of oestrogen receptors to preserve functional ß-cell mass in diabetes.

Protecting the functional mass of insulin-producing ß cells of the pancreas is a major therapeutic challenge in patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). The gonadal hormone 17ß-oestradiol (E2) is involved in reproductive, bone, cardiovascular and neuronal physiology. In rodent models of T1DM and T2DM, treatment with E2 protects pancreatic ß cells against oxidative stress, amyloid polypeptide toxicity, lipotoxicity and apoptosis. Three oestrogen receptors (ERs)--ERα, ERß and the G protein-coupled ER (GPER)--have been identified in rodent and human ß cells. Whereas activation of ERα enhances glucose-stimulated insulin biosynthesis, reduces islet toxic lipid accumulation and promotes ß-cell survival from proapoptotic stimuli, activation of ERß increases glucose-stimulated insulin secretion. However, activation of GPER protects ß cells from apoptosis, raises glucose-stimulated insulin secretion and lipid homeostasis without affecting insulin biosynthesis. Oestrogens are also improving islet engraftment in rodent models of pancreatic islet transplantation. This Review describes developments in the role of ERs in islet insulin biosynthesis and secretion, lipid homeostasis and survival. Moreover, we discuss why and how enhancing ER action in ß cells without the undesirable effect of general oestrogen therapy is a therapeutic avenue to preserve functional ß-cell mass in patients with diabetes mellitus.

Estrogen receptor activation reduces lipid synthesis in pancreatic islets and prevents ß cell failure in rodent models of type 2 diabetes.

The failure of pancreatic ß cells to adapt to an increasing demand for insulin is the major mechanism by which patients progress from insulin resistance to type 2 diabetes (T2D) and is thought to be related to dysfunctional lipid homeostasis within those cells. In multiple animal models of diabetes, females demonstrate relative protection from ß cell failure. We previously found that the hormone 17ß-estradiol (E2) in part mediates this benefit. Here, we show that treating male Zucker diabetic fatty (ZDF) rats with E2 suppressed synthesis and accumulation of fatty acids and glycerolipids in islets and protected against ß cell failure. The antilipogenic actions of E2 were recapitulated by pharmacological activation of estrogen receptor α (ERα) or ERß in a rat ß cell line and in cultured ZDF rat, mouse, and human islets. Pancreas-specific null deletion of ERα in mice (PERα-/-) prevented reduction of lipid synthesis by E2 via a direct action in islets, and PERα-/- mice were predisposed to islet lipid accumulation and ß cell dysfunction in response to feeding with a high-fat diet. ER activation inhibited ß cell lipid synthesis by suppressing the expression (and activity) of fatty acid synthase via a nonclassical pathway dependent on activated Stat3. Accordingly, pancreas-specific deletion of Stat3 in mice curtailed ER-mediated suppression of lipid synthesis. These data suggest that extranuclear ERs may be promising therapeutic targets to prevent ß cell failure in T2D.

Early-life exposure to testosterone programs the hypothalamic melanocortin system.

In mammals, males consume more food, which is considered a masculinized behavior, but the underlying mechanism of this sex-specific feeding behavior is unknown. In mice, neonatal testosterone (NT) is critical to masculinize the developing brain, leading to sex differences in reproductive physiology. The proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus (ARC) are critical to suppress energy intake and POMC innervation of hypothalamic feeding circuits develops to a large extent neonatally. We hypothesized that NT programs the masculinization of energy intake by programming POMC neurons. We tested this hypothesis by comparing control females and control males (CMs) with female mice neonatally androgenized with testosterone (NTFs). We show that increased food intake in CMs is associated with reduced POMC expression and decreased intensity of neuronal projections from POMC neurons within the ARC compared with control females. We found that NTFs display a masculinized energy intake and ARC POMC expression and innervation as observed in CMs, which can be mimicked by neonatal exposure to the androgen receptor agonist dihydrotestosterone (DHT). NTFs also exhibit hyperleptinemia and a decreased ability of leptin to up-regulate POMC, suppress food intake, and prevent adipose tissue accumulation, independent of signal transducer and activator of transcription 3. However, this leptin resistance is specific to NTFs, is not a consequence of masculinization, and is reproduced by neonatal exposure to estrogen but not DHT. Thus, NT programs a sexual differentiation of POMC neurons in female mice via DHT but also predisposes to leptin resistance and obesity in an estrogen-dependent manner.

Extranuclear estrogen receptor-alpha stimulates NeuroD1 binding to the insulin promoter and favors insulin synthesis.

Estrogen receptors (ERs) protect pancreatic islet survival in mice through rapid extranuclear actions. ERalpha also enhances insulin synthesis in cultured islets. Whether ERalpha stimulates insulin synthesis in vivo and, if so, through which mechanism(s) remain largely unknown. To address these issues, we generated a pancreas-specific ERalpha knockout mouse (PERalpha KO(-/-)) using the Cre-loxP strategy and used a combination of genetic and pharmacologic tools in cultured islets and beta cells. Whereas 17beta-estradiol (E2) treatment up-regulates pancreatic insulin gene and protein content in control ERalpha lox/lox mice, these E2 effects are abolished in PERalpha KO(-/-) mice. We find that E2-activated ERalpha increases insulin synthesis by enhancing glucose stimulation of the insulin promoter activity. Using a knock-in mouse with a mutated ERalpha eliminating binding to the estrogen response elements (EREs), we show that E2 stimulation of insulin synthesis is independent of the ERE. We find that the extranuclear ERalpha interacts with the tyrosine kinase Src, which activates extracellular signal-regulated kinases(1/2), to increase nuclear localization and binding to the insulin promoter of the transcription factor NeuroD1. This study supports the importance of ERalpha in beta cells as a regulator of insulin synthesis in vivo.

Mitogen-associated protein kinase- and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells.

Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.