GPR30 Research Articles

Current progress and prospects for G protein-coupled estrogen receptor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease. The G protein-coupled estrogen receptor (GPER) plays a crucial role in mediating the effect of estrogen and estrogen-like compounds in TNBC cells. Compared with other subtypes, GPER has a higher expression in TNBC. The GPER mechanisms have been thoroughly characterized and analyzed in estrogen receptor α (ERα) positive breast cancer, but not in TNBC. Our previous work revealed that a higher expression of GPER mRNA indicates a better prognosis for ERα-positive breast cancer; however, its effects in TNBC differ. Whether GPER could serve as a predictive prognostic marker or therapeutic target for TNBC remains unclear. In this review, we provide a detailed introduction to the subcellular localization of GPER, the different effects of various ligands, and the interactions between GPER and closely associated factors in TNBC. We focused on the internal molecular mechanisms specific to TNBC and thoroughly explored the role of GPER in promoting tumor development. We also discussed the interaction of GPER with specific cytokines and chemokines, and the relationship between GPER and immune evasion. Additionally, we discussed the feasibility of using GPER as a therapeutic target in the context of existing studies. This comprehensive review highlights the effects of GPER on TNBC, providing a framework and directions for future research.

Lactate/GPR81 recruits regulatory T cells by modulating CX3CL1 to promote immune resistance in a highly glycolytic gastric cancer

ABSTRACT Lactate plays an important role in shaping immune tolerance in tumor microenvironment (TME) and correlates with poor prognosis in various solid tumors. Overcoming the immune resistance in an acidic TME may improve the anti-tumor immunity. Here, this study elucidated that via G-protein-coupled receptor 81 (GPR81), lactate could modulate immune tolerance in TME by recruiting regulatory T cells (Tregs) in vitro and in vivo. A high concentration of lactate was detected in cell supernatant and tissues of gastric cancer (GC), which was modulated by lactic dehydrogenase A (LDHA). GPR81 was the natural receptor of lactate and was overexpressed in different GC cell lines and samples, which correlated with poor outcomes in GC patients. Lactate/GPR81 signaling could promote the infiltration of Tregs into TME by inducing the expression of chemokine CX3CL1. GPR81 deficiency could decrease the infiltration of Tregs into TME, thereby inhibiting GC progression by weakening the inhibition of CD8+T cell function in a humanized mouse model. In conclusion, targeting the lactate/GPR81 signaling may potentially serve as a critical process to overcome immune resistance in highly glycolytic GC.

Modulation of GPER1 alleviates early brain injury via inhibition of A1 reactive astrocytes activation after intracerebral hemorrhage in mice

BackgroundEarly brain injury (EBI) caused by inflammatory responses in acute phase of Intracerebral hemorrhage (ICH) plays a vital role in the pathological progression of ICH. Increasing evidences demonstrate A1 reactive astrocytes are associated with the severity of EBI. G-protein coupled estrogen receptor 1 (GPER1) has been proved mediating the neuroprotective effects of estrogen in central nervous system (CNS) disease. However, whether GPER1 plays a protective effect on ICH and A1 reactive astrocytes activation is not well studied. MethodsICH model was established by infused the autologous whole blood into the right basal ganglia in wild type and GPER1 knockout mice. GPER1 specific agonist G1 and antagonist G15 were administered by intraperitoneal injection at 1 h or 0.5 h after ICH. Neurological function was detected on day 1 and day 3 by open field test and corner turn test following ICH. Besides, A1 reactive astrocytes were determined by immunofluorescence staining after ICH on day 3. To further identify the possible mechanism of GPER1 mediated neuroprotective effect, Western blot assays was performed after ICH on day 3. ResultsAfter ICH, G1 treatment alleviated mice neurobehavior deficits on day 1 and day 3. Meanwhile, G1 treatment also significantly reduced the GFAP positive astrocytes and the C3 positive cells after ICH. Interestingly, G15 reversed the protective effect of G1 on the neurobehavior of ICH mice. Meanwhile, the expression of GFAP+C3+ A1 reactive astrocytes were also reduced by activation of GPER1. Mechanistic studies indicated TLR4 and NF-κB mediated the neuroprotective effect of GPER1. ConclusionGenerally, activation of GPER1 alleviated the EBI through inhibiting A1 reactive astrocytes activation via TLR4/NF-κB pathway after ICH in mice. Additionally, GPER1may be a promising target for ICH treatment.

Estrogen receptor GPR30 in the anterior cingulate cortex mediates exacerbated neuropathic pain in ovariectomized mice

Menopausal women experience neuropathic pain 63% more frequently than men do, which may attribute to the estrogen withdrawal. However, the underlying mechanisms remain unclear. Here, the role of estrogen receptors (ERs) in ovariectomized (OVX) female mice following chronic constriction injury (CCI) was investigated. With 17β-estradiol (E2) supplemented, aggravated mechanical allodynia in OVX mice could be significantly alleviated, particularly after intra-anterior cingulate cortex (ACC) E2 delivery. Pharmacological interventions further demonstrated that the agonist of G-protein-coupled estrogen receptor 30 (GPR30), rather than ERα or ERβ in the ACC, exhibited the similar analgesic effect as E2, whereas antagonist of GPR30 exacerbated allodynia. Furthermore, OVX surgery reduced GPR30 expression in the ACC, which could be restored with estrogen supplementation. Selective downregulation of GPR30 in the ACC of naïve female mice induces mechanical allodynia, whereas GPR30 overexpression in the ACC remarkedly alleviated OVX-exacerbated allodynia. Collectively, estrogen withdrawal could downregulate the ACC GPR30 expression, resulting in exacerbated neuropathic pain. Our findings highlight the importance of GPR30 in the ACC in aggravated neuropathic pain during menopause, and offer a potential therapeutic candidate for neuropathic pain management in menopausal women.

Growth differentiation factor 9 regulates the expression of estrogen receptors via Smad2/3 signaling in goat cumulus cells

Both oocyte secretory factors (OSFs) and estrogen are essential for the development and function of mammalian ovarian follicles, playing synergistic role in regulating oocyte growth. OSFs can significantly affect the biological processes regulated by estrogen in cumulus cells (CCs). It is a scientific question worth investigating whether oocyte secretory factors can influence the expression of estrogen receptors in CCs. In our study, we observed a significant increase in the mRNA and protein expressions of estrogen receptor β (Esr2/ERβ) and G-protein-coupled estrogen receptor (GPER) in cumulus cells of goat cumulus-oocyte complexes (COCs) cultured in vitro for 6 h. Furthermore, the addition of 10 ng/mL growth-differentiation factor 9 (GDF9) and 5 ng/mL bone morphogenetic protein 15 (BMP15) to the culture medium of goat COCs resulted in a significant increase in the expressions of ERβ and GPER in cumulus cells. To explore the mechanism further, we performed micromanipulation to remove oocyte contents and co-cultured the oocytectomized complexes (OOXs) with denuded oocytes (DOs) or GDF9/BMP15. The expressions of ERβ and GPER in the co-culture groups were significantly higher than those in the OOXs group, but there was no difference compared to the COCs group. Mechanistically, we found that SB431542 (inhibitor of GDF9 bioactivity), but not LDN193189 (inhibitor of BMP15 bioactivity), abolished the upregulation of ERβ and GPER in cumulus cells and the activation of Smad2/3 signaling. In conclusion, our results demonstrate that the oocyte secretory factor GDF9 promotes the activation of Smad2/3 signaling in cumulus cells during goat COCs culture in vitro, and the phosphorylation of Smad2/3 induces the expression of estrogen receptors ERβ and GPER in cumulus cells.

Lactate Protects Intestinal Epithelial Barrier Function from Dextran Sulfate Sodium-Induced Damage by GPR81 Signaling.

The dysregulation of the intestinal epithelial barrier significantly contributes to the inflammatory progression of ulcerative colitis. Recent studies have indicated that lactate, produced by gut bacteria or derived from fermented foods, plays a key role in modulating inflammation via G-protein-coupled receptor 81 (GPR81). In this study, we aimed to investigate the potential role of GPR81 in the progression of colitis and to assess the impact of lactate/GPR81 signaling on intestinal epithelial barrier function. Our findings demonstrated a downregulation of GPR81 protein expression in patients with colitis. Functional verification experiments showed that Gpr81-deficient mice exhibited more severe damage to the intestinal epithelial barrier and increased susceptibility to DSS-induced colitis, characterized by exacerbated oxidative stress, elevated inflammatory cytokine secretion, and impaired expression of tight-junction proteins. Mechanistically, we found that lactate could suppress TNF-α-induced MMP-9 expression and prevent the disruption of tight-junction proteins by inhibiting NF-κB activation through GPR81 in vitro. Furthermore, our study showed that dietary lactate could preserve intestinal epithelial barrier function against DSS-induced damage in a GPR81-dependent manner in vivo. Collectively, these results underscore the crucial involvement of the lactate/GPR81 signaling pathway in maintaining intestinal epithelial barrier function, providing a potential therapeutic strategy for ulcerative colitis.

The role of estrogen receptor gene polymorphisms in menopausal symptoms and estradiol levels in perimenopausal women – Findings from the Swiss Perimenopause Study

ObjectivesFluctuating estradiol (E2) levels seem to be associated with menopausal symptoms, though not all women suffer from these symptoms to the same extent despite experiencing these hormonal changes. This suggests underlying, interindividual mechanisms, such as single-nucleotide polymorphisms (SNPs) influencing estrogen receptors α and β, and the g-protein-coupled estrogen receptor (GPER). As research is scarce, we aimed to address this research gap by assessing genetic traits, E2 levels, and menopausal symptoms longitudinally. Study design129 perimenopausal women (aged 40–56 years) participated in the 13-month longitudinal Swiss Perimenopause Study. Main outcome measuresMenopausal symptoms were assessed fortnightly using the Menopause Rating Scale (MRS II). Salivary E2 levels were assessed 14 times over two non-consecutive months. Blood samples were collected using the dried blood spot (DBS) technique to analyze ESR1 rs2234693, ESR1 rs9340799, ESR2 rs1256049, ESR2 rs4906938, and GPER rs3808350. Group-based trajectory modeling was performed to identify interindividual trajectories of menopausal symptoms. Multinomial logistic regression models were employed to identify factors associated with these trajectories. ResultsFour distinct trajectory groups of menopausal symptoms were identified (increase, moderate, rebound, decrease). ER gene polymorphisms and E2 fluctuation were significantly associated with group membership. Furthermore, ER gene polymorphisms modulated the effect of E2 fluctuations on menopausal symptom trajectory. ConclusionsThis study illuminates the multifaceted factors contributing to the individuality of the perimenopausal experience. ER gene polymorphisms emerged as integral factors by modulating the effect of E2 fluctuations on menopausal symptom trajectory. This underscores the intricate interplay of genetic factors, E2 fluctuations, and menopausal symptoms during perimenopause.

GPR55 is expressed in glutamate neurons and functionally modulates drug taking and seeking in rats and mice

G protein-coupled receptor 55 (GPR55) has been thought to be a putative cannabinoid receptor. However, little is known about its functional role in cannabinoid action and substance use disorders. Here we report that GPR55 is predominantly found in glutamate neurons in the brain, and its activation reduces self-administration of cocaine and nicotine in rats and mice. Using RNAscope in situ hybridization, GPR55 mRNA was identified in cortical vesicular glutamate transporter 1 (VgluT1)-positive and subcortical VgluT2-positive glutamate neurons, with no detection in midbrain dopamine (DA) neurons. Immunohistochemistry detected a GPR55-like signal in both wildtype and GPR55-knockout mice, suggesting non-specific staining. However, analysis using a fluorescent CB1/GPR55 ligand (T1117) in CB1-knockout mice confirmed GPR55 binding in glutamate neurons, not in midbrain DA neurons. Systemic administration of the GPR55 agonist O-1602 didnt impact ∆9-THC-induced analgesia, hypothermia and catalepsy, but significantly mitigated cocaine-enhanced brain-stimulation reward caused by optogenetic activation of midbrain DA neurons. O-1602 alone failed to alter extracellar DA, but elevated extracellular glutamate, in the nucleus accumbens. In addition, O-1602 also demonstrated inhibitory effects on cocaine or nicotine self-administration under low fixed-ratio and/or progressive-ratio reinforcement schedules in rats and wildtype mice, with no such effects observed in GPR55-knockout mice. Together, these findings suggest that GPR55 activation may functionally modulate drug-taking and drug-seeking behavior possibly via a glutamate-dependent mechanism, and therefore, GPR55 deserves further study as a new therapeutic target for treating substance use disorders.

Targeting metabolic sensing switch GPR84 on macrophages for cancer immunotherapy

IntroductionAs one of the major components of the tumor microenvironment, tumor-associated macrophages (TAMs) possess profound inhibitory activity against T cells and facilitate tumor escape from immune checkpoint blockade therapy. Converting this pro-tumorigenic toward the anti-tumorigenic phenotype thus is an important strategy for enhancing adaptive immunity against cancer. However, a plethora of mechanisms have been described for pro-tumorigenic differentiation in cancer, metabolic switches to program the anti-tumorigenic property of TAMs are elusive. Materials and methodsFrom an unbiased analysis of single-cell transcriptome data from multiple tumor models, we discovered that anti-tumorigenic TAMs uniquely express elevated levels of a specific fatty acid receptor, G-protein-coupled receptor 84 (GPR84). Genetic ablation of GPR84 in mice leads to impaired pro-inflammatory polarization of macrophages, while enhancing their anti-inflammatory phenotype. By contrast, GPR84 activation by its agonist, 6-n-octylaminouracil (6-OAU), potentiates pro-inflammatory phenotype via the enhanced STAT1 pathway. Moreover, 6-OAU treatment significantly retards tumor growth and increases the anti-tumor efficacy of anti-PD-1 therapy. ConclusionOverall, we report a previously unappreciated fatty acid receptor, GPR84, that serves as an important metabolic sensing switch for orchestrating anti-tumorigenic macrophage polarization. Pharmacological agonists of GPR84 hold promise to reshape and reverse the immunosuppressive TME, and thereby restore responsiveness of cancer to overcome resistance to immune checkpoint blockade.

Maternal Phlorizin Intake Protects Offspring from Maternal Obesity-Induced Metabolic Disorders in Mice via Targeting Gut Microbiota to Activate the SCFA-GPR43 Pathway.

Maternal obesity increases the risk of obesity and metabolic disorders (MDs) in offspring, which can be mediated by the gut microbiota. Phlorizin (PHZ) can improve gut dysbiosis and positively affect host health; however, its transgenerational metabolic benefits remain largely unclear. This study aimed to investigate the potential of maternal PHZ intake in attenuating the adverse impacts of a maternal high-fat diet on obesity-related MDs in dams and offspring. The results showed that maternal PHZ reduced HFD-induced body weight gain and fat accumulation and improved glucose intolerance and abnormal lipid profiles in both dams and offspring. PHZ improved gut dysbiosis by promoting expansion of SCFA-producing bacteria, Akkermansia and Blautia, while inhibiting LPS-producing and pro-inflammatory bacteria, resulting in significantly increased fecal SCFAs, especially butyric acid, and reduced serum lipopolysaccharide levels and intestinal inflammation. PHZ also promoted intestinal GLP-1/2 secretion and intestinal development and enhanced gut barrier function by activating G protein-coupled receptor 43 (GPR43) in the offspring. Antibiotic-treated mice receiving FMT from PHZ-regulated offspring could attenuate MDs induced by receiving FMT from HFD offspring through the gut microbiota to activate the GPR43 pathway. It can be regarded as a promising functional food ingredient for preventing intergenerational transmission of MDs and breaking the obesity cycle.

The role of GPR81–cAMP–PKA pathway in endurance training-induced intramuscular triglyceride accumulation and mitochondrial content changes in rats

The athlete's paradox phenomenon involves the accumulation of intramuscular triglycerides (IMTG) in both insulin-resistant and insulin-sensitive endurance athletes. Nevertheless, a complete understanding of this phenomenon is yet to be achieved. Recent research indicates that lactate, a common byproduct of physical activity, may increase the accumulation of IMTG in skeletal muscle. This is achieved through the activation of G protein-coupled receptor 81 (GPR81) leads to the suppression of the cyclic adenosine monophosphate–protein kinase A (cAMP–PKA) pathway. The mechanism accountable for the increase in mitochondrial content in skeletal muscle triggered by lactate remains incomprehensible. Based on current research, our objective is to explore the role of the GPR81-inhibited cAMP–PKA pathway in the aggregation of IMTG and the increase in mitochondrial content as a result of prolonged exercise. The GPR81–cAMP–PKA-signaling pathway regulates the buildup of IMTG caused by extended periods of endurance training (ET). This is likely due to a decrease in proteins related to fat breakdown and an increase in proteins responsible for fat production. It is possible that the GPR81–cAMP–PKA pathway does not contribute to the long-term increase in mitochondrial biogenesis and content, which is induced by chronic ET. Additional investigation is required to explore the possible hindrance of the mitochondrial biogenesis and content process during physical activity by the GPR81–cAMP–PKA signal.

Allosteric modulation and G-protein selectivity of the Ca2+-sensing receptor.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor1 (GPCR) that has a central role in regulating systemic calcium homeostasis2,3. Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional Gi versus Gq proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both Gi and Gq drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein-lipid interactions. Selective Gi and Gq coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR-G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid L-tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands.

GPR55 Inactivation Diminishes Splenic Responses and Improves Neurological Outcomes in the Mouse Ischemia/Reperfusion Stroke Model.

Although strokes are frequent and severe, treatment options are scarce. Plasminogen activators, the only FDA-approved agents for clot treatment (tissue plasminogen activators (tPAs)), are used in a limited patient group. Moreover, there are few approaches for handling the brain's inflammatory reactions to a stroke. The orphan G protein-coupled receptor 55 (GPR55)'s connection to inflammatory processes has been recently reported; however, its role in stroke remains to be discovered. Post-stroke neuroinflammation involves the central nervous system (CNS)'s resident microglia activation and the infiltration of leukocytes from circulation into the brain. Additionally, splenic responses have been shown to be detrimental to stroke recovery. While lymphocytes enter the brain in small numbers, they regularly emerge as a very influential leukocyte subset that causes secondary inflammatory cerebral damage. However, an understanding of how this limited lymphocyte presence profoundly impacts stroke outcomes remains largely unclear. In this study, a mouse model for transient middle cerebral artery occlusion (tMCAO) was used to mimic ischemia followed by a reperfusion (IS/R) stroke. GPR55 inactivation, with a potent GPR55-specific antagonist, ML-193, starting 6 h after tMCAO or the absence of the GPR55 in mice (GPR55 knock out (GPR55ko)) resulted in a reduced infarction volume, improved neurological outcomes, and decreased splenic responses. The inhibition of GPR55 with ML-193 diminished CD4+T-cell spleen egress and attenuated CD4+T-cell brain infiltration. Additionally, ML-193 treatment resulted in an augmented number of regulatory T cells (Tregs) in the brain post-tMCAO. Our report offers documentation and the functional evaluation of GPR55 in the brain-spleen axis and lays the foundation for refining therapeutics for patients after ischemic attacks.

GPR39 regulated spinal glycinergic inhibition and mechanical inflammatory pain.

G protein-coupled receptor 39 (GPR39) senses the change of extracellular divalent zinc ion and signals through multiple G proteins to a broad spectrum of downstream effectors. Here, we found that GPR39 was prevalent at inhibitory synapses of spinal cord somatostatin-positive (SOM+) interneurons, a mechanosensitive subpopulation that is critical for the conveyance of mechanical pain. GPR39 complexed specifically with inhibitory glycine receptors (GlyRs) and helped maintain glycinergic transmission in a manner independent of G protein signalings. Targeted knockdown of GPR39 in SOM+ interneurons reduced the glycinergic inhibition and facilitated the excitatory output from SOM+ interneurons to spinoparabrachial neurons that engaged superspinal neural circuits encoding both the sensory discriminative and affective motivational domains of pain experience. Our data showed that pharmacological activation of GPR39 or augmenting GPR39 interaction with GlyRs at the spinal level effectively alleviated the sensory and affective pain induced by complete Freund's adjuvant and implicated GPR39 as a promising therapeutic target for the treatment of inflammatory mechanical pain.

Positive correlation between the nuclear expression of GPER and pGLI3 in prostate cancer tissues from patients with different Gleason scores.

Prostate cancer (PCa) is the most prevalent cause of death in the male population worldwide. The G Protein-Coupled Estrogen Receptor (GPER) has been gaining relevance in the development of PCa. Hedgehog (Hh) pathway activation is associated with aggressiveness, metastasis, and relapse in PCa patients. To date, no studies have evaluated the crosstalk between the GPER and the Hh pathway along different group grades in PCa. We conducted an analysis of paraffin-embedded tissues derived from patients with different prognostic grade of PCa using immunohistochemistry. Expression and correlation between GPER and glioma associated oncogene homologue (GLI) transcriptional factors in the parenchyma and stroma of PCa tumors were evaluated. Our results indicate that GPER is highly expressed in the nucleus and increases with higher grade groups. Additionally, GPER's expression correlates with pGLI3 nuclear expression across different grade groups in PCa tissues; however, whether the receptor induces the activation of GLI transcriptional factors, or the latter modulate the expression of GPER is yet to be discovered, as well as the functional consequence of this correlation.

G Protein Coupled Receptor 87 (GPR87) in End Stage Kidney and Associated Tumors.

End-stage renal disease (ESRD) and acquired cystic renal disease (ACRD) are characterized by progressive inflammation, structural remodeling and by development of unique cancer types. Eosinophilic-vacuolated and chromophobe-like renal cell carcinoma develop exclusively in ACRD kidney. The aim of the study was to investigate the molecular mechanism of ESRD/ACRD carcinogenesis. Our previous Affymetrix array analysis detected GPR87 as one of the highly and specifically expressed genes in ESRD/ACRD kidneys. In this study we analyzed normal and ESRD/ACRD kidneys and related tumors for GPR87 expression by PCR, RT-PCR, and immunohistochemistry. Immunohistochemistry revealed a strong GPR87 expression in proliferating epithelial cells in ESRD/ACRD kidneys and in cells of eosinophilic-vacuolated and chromophobe-like renal cell carcinoma. GPR87 signaling plays an important role in the structural remodeling of ESRD/ACRD kidney and development of ACRD-associated tumors with unique histology.

Outcome of stress on G protein-coupled receptors and hypoxia inducible factor-1α.

Stress drives neuroendocrine signals with detrimental effects to the intestinal homeostasis. The aim of this study was to evaluate the effect of stress on intestinal hypoxia response elements, including G protein-coupled receptor 41 (GPR41), GPR43, and hypoxia inducible factor (HIF)-1α. Groups of five BALB/c mice were subjected to acute (2 h per day) and chronic (2 h per day for 4 days) stress induced by restraint, and the results were compared to those of an unstressed control group. Whole mucosal samples from the colon were collected to evaluate the expression of GPR41, GPR43 and HIF-1α using Western blot chemiluminescent analysis. Compared to the control group, in the chronic stress group the expression of GPR43 (P = 0.0092) and HIF-1α (P < 0.0001) were significantly lower and the expression of GPR41 was similar (P = 0.9184); acute stress significantly increased HIF-1α expression (P = 0.0030) and increased GPR41 expression (P = 0.0937), without affecting GPR43 (P = 0.9184). These findings offer insights into the modulation of hypoxia response elements under stress conditions and their pharmacological implications for developing drugs that mitigate the effects of stress on intestinal homeostasis.

Dihydrotestosterone induces arterial stiffening in female mice

BackgroundAndrogens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER.MethodsThe impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15–16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8–9/group).ResultsIn cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson’s trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003).ConclusionsThese findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

Docosahexanoic Acid Attenuates Palmitate-Induced Apoptosis by Autophagy Upregulation via GPR120/mTOR Axis in Insulin-Secreting Cells.

Polyunsaturated fatty acids (PUFAs) reportedly have protective effects on pancreatic β-cells; however, the underlying mechanisms are unknown. To investigate the cellular mechanism of PUFA-induced cell protection, mouse insulinoma 6 (MIN6) cells were cultured with palmitic acid (PA) and/or docosahexaenoic acid (DHA), and alterations in cellular signaling and apoptosis were examined. DHA treatment remarkably repressed caspase-3 cleavage and terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive red dot signals in PA-treated MIN6 cells, with upregulation of autophagy, an increase in microtubule- associated protein 1-light chain 3 (LC3)-II, autophagy-related 5 (Atg5), and decreased p62. Upstream factors involved in autophagy regulation (Beclin-1, unc51 like autophagy activating kinase 1 [ULK1], phosphorylated mammalian target of rapamycin [mTOR], and protein kinase B) were also altered by DHA treatment. DHA specifically induced phosphorylation on S2448 in mTOR; however, phosphorylation on S2481 decreased. The role of G protein-coupled receptor 120 (GPR120) in the effect of DHA was demonstrated using a GPR120 agonist and antagonist. Additional treatment with AH7614, a GPR120 antagonist, significantly attenuated DHA-induced autophagy and protection. Taken together, DHA-induced autophagy activation with protection against PA-induced apoptosis mediated by the GPR120/mTOR axis. These findings indicate that DHA has therapeutic effects on PA-induced pancreatic β-cells, and that the cellular mechanism of β-cell protection by DHA may be a new research target with potential pharmacotherapeutic implications in β-cell protection.

PGC-1α activation boosts exercise-dependent cellular response in the skeletal muscle

The role of Peroxisome proliferator-activated receptor-gamma coactivator alpha (PGC-1α) in fat metabolism is not well known. In this study, we compared the mechanisms of muscle-specific PGC-1α overexpression and exercise-related adaptation-dependent fat metabolism. PGC-1α trained (PGC-1α Ex) and wild-trained (wt-ex) mice were trained for 10 weeks, five times a week at 30 min per day with 60 percent of their maximal running capacity. The PGC-1α overexpressed animals exhibited higher levels of Fibronectin type III domain-containing protein 5 (FNDC5), 5' adenosine monophosphate-activated protein kinase alpha (AMPK-α), the mammalian target of rapamycin (mTOR), Sirtuin 1 (SIRT1), Lon protease homolog 1 (LONP1), citrate synthase (CS), succinate dehydrogenase complex flavoprotein subunit A (SDHA), Mitofusin-1 (Mfn1), endothelial nitric oxide synthase (eNOS), Hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), G protein-coupled receptor 41 (GPR41), and Phosphatidylcholine Cytidylyltransferase 2 (PCYT2), and lower levels of Sirtuin 3 (SIRT3) compared to wild-type animals. Exercise training increased the protein content levels of SIRT1, HSL, and ATGL in both the wt-ex and PGC-1α trained groups. PGC-1α has a complex role in cellular signaling, including the upregulation of lipid metabolism-associated proteins. Our data reveals that although exercise training mimics the effects of PGC-1α overexpression, it incorporates some PGC-1α-independent adaptive mechanisms in fat uptake and cell signaling.