Fecal microbiota as a predictor of acupuncture responses in patients with postpartum depressive disorder.

Background: There are several clinical and molecular predictors of responses to antidepressant therapy. However, these markers are either too subjective or complex for clinical use. The gut microbiota could provide an easily accessible set of biomarkers to predict therapeutic efficacy, but its value in predicting therapy responses to acupuncture in patients with depression is unknown. Here we analyzed the predictive value of the gut microbiota in patients with postpartum depressive disorder (PPD) treated with acupuncture. Methods: Seventy-nine PPD patients were enrolled: 55 were treated with acupuncture and 24 did not received any treatment. The 17-item Hamilton depression rating scale (HAMD-17) was used to assess patients at baseline and after eight weeks. Patients receiving acupuncture treatment were divided into an acupuncture-responsive group or non-responsive group according to HAMD-17 scores changes. Baseline fecal samples were obtained from the patients receiving acupuncture and were analyzed by high-throughput 16S ribosomal RNA sequencing to characterize the gut microbiome. Results: 47.27% patients responded to acupuncture treatment and 12.5% patients with no treatment recovered after 8-week follow-up. There was no significant difference in α-diversity between responders and non-responders. The ß-diversity of non-responders was significantly higher than responders. Paraprevotella and Desulfovibrio spp. were significantly enriched in acupuncture responders, and these organisms had an area under the curve of 0.76 and 0.66 for predicting responder patients, respectively. Conclusions: Paraprevotella and Desulfovibrioare may be useful predictive biomarkers to predict PPD patients likely to respond to acupuncture. Larger studies and validation in independent cohorts are now needed to validate our findings.

Bifidobacterium bifidum relieved DSS-induced colitis in mice potentially by activating the aryl hydrocarbon receptor.

Inflammatory bowel disease (IBD) characterized by relapsed intestinal inflammation and barrier function disruption is still a great therapeutic challenge. This study aimed to screen probiotics that have the potential to help alleviate IBD and further elucidate their mechanism of action. Caco-2 cell differentiated monolayers and RAW264.7 cells stimulated by lipopolysaccharide (LPS) were used for probiotic screening in vitro, and then the efficacies of the obtained six bacterial strains were evaluated in mice with dextran sulfate sodium (DSS)-induced colitis. The results showed that all of the strains at varying degrees could increase the transepithelial electrical resistance (TEER) value, decrease the influx of FITC-dextran in Caco-2 cell monolayers and attenuate the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-stimulated RAW264.7 cells. In vivo experiments indicated that Bifidobacterium bifidum FL-276.1 (FL-276.1) and Bifidobacterium bifidum FL-228.1 (FL-228.1) showed the best efficacies to ameliorate body weight loss, colon shortening, and intestinal barrier disruption. Accordingly, in FL-276.1 and FL-228.1 groups, the genes of zonula occludens-1 (ZO-1), claudin-4, occludin and mucin 2 (Muc2) in mouse colonic tissues were significantly upregulated, while TNF-α, IL-1ß and IL-6 were downregulated. Further results showed that strains FL-276.1 and FL-228.1 could activate the aryl hydrocarbon receptor (AhR) in the intestine. Our study showed that the two Bifidobacterium bifidum strains, FL-276.1 and FL-228.1, ameliorated DSS-induced colitis by enhancing the intestinal barrier and anti-inflammation potentially via the AhR pathway.

Correlation between TCM Syndromes and Type 2 Diabetic Comorbidities Based on Fully Connected Neural Network Prediction Model.

OBJECTIVE: To predict the major comorbidities of type 2 diabetes based on the distribution characteristics of syndromes, and to explore the relationship between TCM syndromes and comorbidities of type 2 diabetes. METHODS: Based on the electronic medical record data of 3413 outpatient visits from 995 type 2 diabetes patients with comorbidities, descriptive statistical methods were used to analyze the basic characteristics of the population, the distribution characteristics of comorbidities, and TCM syndromes. A neural network model for the prediction of type 2 diabetic comorbidities based on TCM syndromes was constructed. RESULTS: Patients with TCM syndrome of blood amassment in the lower jiao were diagnosed with renal insufficiency with 95% test sensitivity. The patients with spleen deficiency combined with ascending counterflow of stomach qi and cold-damp patterns were diagnosed with gastrointestinal lesions with 92% sensitivity. The patients with TCM syndrome group of spleen heat and exuberance of heart fire were diagnosed as type 2 diabetes complicated with hypertension with a sensitivity of 91%. In addition, the prediction accuracy of combined neuropathy, heart disease, liver disease, and lipid metabolism disorder reached 70∼90% in TCM syndrome groups. CONCLUSION: The fully connected neural network model study showed that syndrome characteristics are highly correlated with type 2 diabetes comorbidities. Syndrome location is commonly in the heart, spleen, stomach, lower jiao, meridians, etc., while syndrome pattern manifests in states of deficiency, heat, phlegm, and blood stasis. The different combinations of disease location and disease pattern reflect the syndrome characteristics of different comorbidities forming the characteristic syndrome group of each comorbidity. Major comorbidities could be predicted with a high degree of accuracy through TCM syndromes. Findings from this study may have further implementations to assist with the diagnosis, treatment, and prevention of diabetic comorbidities at an early stage.

Study on the TCM Syndromes Evolution and Chinese Herbal Characteristics of Type 2 Diabetes Patients with Different Courses of Disease in TCM "Heat Stage": A Real-World Study.

OBJECTIVE: The purpose of this study is to analyze and summarize the syndrome distribution, syndrome evolution, and Chinese herb medicine characteristics of T2D in heat stage. METHOD: In this study, 228 heat-stage T2D patients were divided into three groups based on the course of disease. Group 1 (the course of disease ≤5 years) included 118 patients. Group 2 (5< the course of disease ≤10 years) had 73 patients. Group 3 (the course of disease >10 years) consisted of 37 patients. The main methods used in our study were complex network community partitioning algorithms and Sankey diagram visualization, based on the clinical electronic medical record data we collected. RESULT: In the three groups, the nodes with the highest node degree are all "heat syndrome." Edge weight between "heat" and "dampness," "qi stagnation," "phlegm," "liver," and "stomach" is the largest. During the whole course of treatment, 60.17%, 63.01%, and 62.16% of the patients' syndromes in groups 1, 2, and 3, respectively, were ascribed to the heat stage all the time. The patients' syndromes in groups 1 and 2 easily transformed to the syndrome of deficiency of both qi and yin of the spleen and stomach. In group 3, 27% of the patients' syndromes were easily transformed into kidney yin deficiency and qi deficiency and blood stasis syndrome. The largest Chinese herb communities of the patients whose syndromes did not change after treatment in the three groups were all heat-clearing drugs. The proportion of blood-activating drugs in patients with syndrome changes increased significantly after treatment. CONCLUSION: (1) The basic syndrome of T2D patients in the heat stage is liver-stomach heat syndrome. (2) T2D patients in the heat stage tend to deteriorate towards the direction of qi and yin deficiency syndrome. However, the longer the course of the disease is, the more likely it is to deteriorate to the direction of kidney yin deficiency syndrome and blood stasis syndrome. (3) Drugs that can help T2D patients in the heat stage to maintain their condition stably are heat-clearing drugs represented by Coptis chinensis, which usually need to be combined with warming interior drugs such as Zingiberis Rhizoma and Pinelliae Rhizoma.

Ligilactobacillus Salivarius LCK11 Prevents Obesity by Promoting PYY Secretion to Inhibit Appetite and Regulating Gut Microbiota in C57BL/6J Mice.

SCOPE: Obesity is a common disease worldwide and there is an urgent need for strategies to preventing obesity. METHODS AND RESULTS: The anti-obesity effect and mechanism of Ligilactobacillus salivarius LCK11 (LCK11) is studied using a C57BL/6J male mouse model in which obesity is induced by a high-fat diet (HFD). Results show that LCK11 can prevent HFD-induced obesity, reflected as inhibited body weight gain, abdominal and liver fat accumulation and dyslipidemia. Analysis of its mechanism shows that on the one hand, LCK11 can inhibit food intake through significantly improving the transcriptional and translational levels of peptide YY (PYY) in the rectum, in addition to the eventual serum PYY level; this is attributed to the activation of the toll-like receptor 2/nuclear factor-κB signaling pathway in enteroendocrine L cells by the peptidoglycan of LCK11. On the other hand, LCK11 supplementation effectively reduces the Firmicutes/Bacteroidetes ratio and shifts the overall structure of the HFD-disrupted gut microbiota toward that of mice fed on a low-fat diet; this also contributes to preventing obesity. CONCLUSION: LCK11 shows the potential to be used as a novel probiotic for preventing obesity by both promoting PYY secretion to inhibit food intake and regulating gut microbiota.

Lactiplantibacillus plantarum H-87 prevents high-fat diet-induced obesity by regulating bile acid metabolism in C57BL/6J mice.

Bile salt hydrolase (BSH)-producing bacteria are negatively related to the body weight gain and energy storage of the host. We aimed to obtain a novel BSH-producing strain with excellent anti-obesity effect and explained its mechanism. Here, we selected a strain named Lactiplantibacillus plantarum H-87 (H-87) with excellent ability to hydrolyze glycochenodeoxycholic acid (GCDCA) and tauroursodeoxycholic acid (TUDCA) in vitro from 12 lactobacilli, and evaluated its anti-obesity effect in high-fat diet (HFD)-fed C57BL/6J mice. The results suggested that H-87 could inhibit HFD-induced body weight gain, fat accumulation, liver lipogenesis and injury, insulin resistance and dyslipidemia. In addition, H-87 could colonize in the ileum and hydrolyze GCDCA and TUDCA, reflected as changes in the concentrations of GCDCA, TUDCA, CDCA and UDCA in the ileum or liver. Furthermore, the study identified that H-87 reduced TUDCA and GCDCA levels in the ileum, which decreased the GLP-1 secretion by L cells to alleviate insulin resistance in HFD-fed mice. Furthermore, H-87 increased the CDCA level in the ileum and liver to activate FXR signaling pathways to inhibit liver lipogenesis in HFD-fed mice. In addition, the decrease of intestinal conjugated bile acids (TUDCA and GCDCA) also increased fecal lipid content and decreased intestinal lipid digestibility. In conclusion, H-87 could inhibit liver fat deposition, insulin resistance and lipid digestion by changing bile acid enterohepatic circulation, and eventually alleviate HFD-induced obesity.

Explaining Divergent Observations Regarding Osteocalcin/GPRC6A Endocrine Signaling.

A new schema proposes that the bone-derived osteocalcin (Ocn) peptide hormone activates the G-protein-coupled receptor GPRC6A to directly regulate glucose and fat metabolism in liver, muscle, and fat, and to stimulate the release of metabolism-regulating hormones, including insulin, fibroblast growth factor 21, glucagon-like peptide 1, testosterone, and interleukin 6. Ocn/GPRC6A activation has also been implicated in cancer progression. GPRC6A is activated by cations, amino acids, and testosterone. The multiligand specificity, the regulation of energy metabolism in diverse tissues, and the coordinated release of metabolically active hormones make the GPRC6A endocrine networks unique. Recently, the significance of Ocn/GPRCA has been questioned. There is a lack of metabolic abnormalities in newly created genetically engineered Ocn- and Gprc6a-deficient mouse models. There are also paradoxical observations that GPRC6A may function as a tumor suppressor. In addition, discordant published studies have cast doubt on the function of the most prevalent uniquely human GPRC6A-KGKY polymorphism. Explanations for these divergent findings are elusive. We provide evidence that the metabolic susceptibility of genetically engineered Ocn- and Gprc6a-deficient mice is influenced by environmental challenges and genetic differences in mouse strains. In addition, the GPRC6A-KGKY polymorphism appears to be a gain-of-function variant. Finally, alternatively spliced isoforms of GPRC6A may alter ligand specificity and signaling that modulate oncogenic effects. Thus, genetic, post-translational and environmental factors likely account for the variable results regarding the functions of GPRC6A in animal models. Pending additional information, GPRC6A should remain a potential therapeutic target for regulating energy and fat metabolism, hormone production, and cancer progression.

The carboxylation status of osteocalcin has important consequences for its structure and dynamics.

BACKGROUND: The carboxylation status of Osteocalcin (Ocn) not only influences formation and structure in bones but also has important endocrine functions affecting energy metabolism and expenditure. In this study, the role of γ-carboxylation of the glutamate residues in the structure-dynamics-function relationship in Ocn is investigated. METHODS: Three forms of Ocn, differentially carboxylated at the Glu-17, 21 and 24 residues, along with a mutated form of Ocn carrying Glu/Ala mutations, are modeled and simulated using molecular dynamics (MD) simulation in the presence of calcium ions. RESULTS: Characterization of the global conformational dynamics of Ocn, described in terms of the orientational variations within its 3-helical domain, highlights large structural variations in the non-carboxylated osteocalcin (nOcn). The bi-carboxylated Ocn (bOcn) and tri-carboxylated (tOcn) species, in contrast, display relatively rigid tertiary structures, with the dynamics of most regions strongly correlated. Radial distribution functions calculated for both bOcn and tOcn show long-range ordering of the calcium ion distribution around the carboxylated glutamate (γGlu) residues, likely playing an important role in promoting stability of these Ocns. Additionally, the same calcium ions are observed to coordinate with neighboring γGlu, better shielding their negative charges and in turn stabilizing these systems more than do the singly coordinating calcium ions observed in the case of nOcn. bOcn is also found to exhibit a more helical C-terminal structure, that has been shown to activate its cellular receptor GPRC6A, highlighting the allosteric role of Ocn carboxylation in modulating the stability and binding potential of the active C-terminal. CONCLUSIONS: The carboxylation status of Ocn as well and its calcium coordination appear to have a direct influence on Ocn structure and dynamics, possibly leading to the known differences in Ocn biological function. GENERAL SIGNIFICANCE: Modification of Ocn sequence or its carboxylation state may provide the blueprint for developing high-affinity peptides targeting its cellular receptor GPRC6A, with therapeutic potential for treatment of metabolic disorders.

Humanized GPRC6AKGKY is a gain-of-function polymorphism in mice.

GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.

Role of GPRC6A in Regulating Hepatic Energy Metabolism in Mice.

GPRC6A is a widely expressed G-protein coupled receptor that regulates energy metabolism. Global deletion of Gprc6a in mice is reported to result in a metabolic syndrome-like phenotype and conditional deletion of Gprc6a in pancreatic ß-cell and skeletal muscle respectively impair insulin secretion and glucose uptake. In the current study, we explore the hepatic functions of GPRC6A by conditionally deleting Gprc6a in hepatocytes by cross breeding Alb-Cre and Gprc6aflox/flox mice to obtain Gprc6aLiver-cko mice. Gprc6aLiver-cko mice on a normal diet showed excessive hepatic fat accumulation and glycogen depletion. These mice also exhibit impaired glucose and pyruvate tolerance, but normal insulin sensitivity. Decreased circulating FGF-21 levels and FGF-21 message expression in the liver were found in Gprc6aLiver-cko mice. Hepatic transcriptome analysis identified alterations in multiple pathways regulating glucose, fat and glycogen metabolism in Gprc6aLiver-cko mice. Taken together, our studies suggest that GPRC6A directly regulates hepatic metabolism as well as regulates the production and release of FGF-21 to control systemic energy homeostasis. GPRC6A's unique regulation of ß-cell, skeletal muscle and hepatic function may represent a new therapeutic target for treating disordered energy metabolism metabolic syndrome and type 2 diabetes.

Human GPRC6A Mediates Testosterone-Induced Mitogen-Activated Protein Kinases and mTORC1 Signaling in Prostate Cancer Cells.

G protein-coupled receptor family C group 6 member A (GPRC6A) is activated by testosterone and modulates prostate cancer progression. Most humans have a GPRC6A variant that contains a recently evolved KGKY insertion/deletion in the third intracellular loop (ICL3) (designated as GPRC6AICL3_KGKY) that replaces the ancestral KGRKLP sequence (GPRC6AICL3_RKLP) present in all other species. In vitro assays purport that human GPRC6AICL3_KGKY is retained intracellularly and lacks function. These findings contrast with ligand-dependent activation and coupling to mammalian target of rapamycin complex 1 (mTORC1) signaling of endogenous human GPRC6AICL3_KGKY in PC-3 cells. To understand these discrepant results, we expressed mouse (mGPRC6AICL3_KGRKLP), human (hGPRC6AICL3_KGKY), and humanized mouse (mGPRC6AICL3_KGKY) GPRC6A into human embryonic kidney 293 cells. Our results demonstrate that mGPRC6AICL3_KGRKLP acts as a classic G protein-coupled receptor, which is expressed at the cell membrane and internalizes in response to ligand activation by testosterone. In contrast, hGPRC6AICL3_KGKY and humanized mouse mGPRC6AICL3_KGKY are retained intracellularly in ligand naive cells, yet exhibit ß-arrestin-dependent signaling responses, mitogen-activated protein kinase [i.e., extracellular signal-regulated kinase (ERK)], and p70S6 kinase phosphorylation in response to testosterone, indicating that hGPRC6AICL3_KGKY is functional. Indeed, testosterone stimulates time- and dose-dependent activation of ERK, protein kinase B, and mTORC1 signaling in wild-type PC-3 cells that express endogenous GPRC6AICL3_KGKY In addition, testosterone stimulates GPRC6A-dependent cell proliferation in wild-type PC-3 cells and inhibits autophagy by activating mTORC1 effectors eukaryotic translation initiation factor 4E binding protein 1 and Unc-51 like autophagy activating kinase 1. Testosterone activation of GPRC6A has the obligate requirement for calcium in the incubation media. In contrast, in GPRC6A-deficient cells, the effect of testosterone to activate downstream signaling is abolished, indicating that human GPRC6A is required for mediating the effects of testosterone on cell proliferation and autophagy.

Cardiovascular Interactions between Fibroblast Growth Factor-23 and Angiotensin II.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5-1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

Computationally identified novel agonists for GPRC6A.

New insights into G protein coupled receptor regulation of glucose metabolism by ß-cells, skeletal muscle and liver hepatocytes identify GPRC6A as a potential therapeutic target for treating type 2 diabetes mellitus (T2D). Activating GPRC6A with a small molecule drug represents a potential paradigm-shifting opportunity to make significant strides in regulating glucose homeostasis by simultaneously correcting multiple metabolic derangements that underlie T2D, including abnormalities in ß-cell proliferation and insulin secretion and peripheral insulin resistance. Using a computational, structure-based high-throughput screening approach, we identified novel tri-phenyl compounds predicted to bind to the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A. Experimental testing found that these compounds dose-dependently stimulated GPRC6A signaling in a heterologous cell expression system. Additional chemical modifications and functional analysis identified one tri-phenyl lead compound, DJ-V-159 that demonstrated the greatest potency in stimulating insulin secretion in ß-cells and lowering serum glucose in wild-type mice. Collectively, these studies show that GPRC6A is a "druggable" target for developing chemical probes to treat T2DM.

Yak milk casein as potential precursor of angiotensin I-converting enzyme inhibitory peptides based on in silico proteolysis.

Yak milk casein was selected as a potential precursor of bioactive peptides based on in silico analysis. Most notable among these are the angiotensin I-converting enzyme (ACE) inhibitory peptides. First, yak milk casein has high homology with cow milk casein by homologous analysis. The potential of yak milk casein for the releasing bioactive peptides was evaluated by determining the frequency of occurrence of fragments with a given activity. Through the BIOPEP database analysis, there are many bioactive peptides in yak milk casein sequences. Then, an in silico proteolysis using single or combined enzymes to obtained ACE inhibitory peptides was investigated. Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all in silico proteolysis derived ACE inhibitory peptides are non-cytotoxic. Overall, the present study highlights a in silico proteolysis approach to assist the yak milk casein releasing ACE inhibitory peptides and provides a guidance for the actual hydrolysis of proteins for the production of bioactive peptides.

GPCR6A Is a Molecular Target for the Natural Products Gallate and EGCG in Green Tea.

SCOPE: The molecular mechanisms whereby gallates in green tea exert metabolic effects are poorly understood. METHODS AND RESULTS: We found that GPRC6A, a multi-ligand-sensing G-protein-coupled receptor that regulates energy metabolism, sex hormone production, and prostate cancer progression, is a target for gallates. Sodium gallate (SG), gallic acid (GA) > ethyl gallate (EG) > octyl gallate (OG) dose dependently activated ERK in HEK-293 cells transfected with GPRC6A but not in non-transfected controls. SG also stimulated insulin secretion in ß-cells isolated from wild-type mice similar to the endogenous GPRC6A ligands, osteocalcin (Ocn) and testosterone (T). Side-chain additions to create OG resulted in loss of GPRC6A agonist activity. Another component of green tea, epigallocatechin 3-gallate (EGCG), dose-dependently inhibited Ocn activation of GPRC6A in HEK-293 cells transfected with GPRC6A and blocked the effect of Ocn in stimulating glucose production in CH10T1/2 cells. Using structural models of the venus fly trap (VFT) and 7-transmembrane (7-TM) domains of GPRC6A, calculations suggest that l-amino acids and GA bind to the VFT, whereas EGCG is calculated to bind to sites in both the VFT and 7-TM. CONCLUSION: GA and EGCG have offsetting agonist and antagonist effects on GPRC6A that may account for the variable metabolic effect of green tea consumption.

Cardiovascular Effects of Renal Distal Tubule Deletion of the FGF Receptor 1 Gene.

The bone-derived hormone fibroblast growth factor-23 (FGF-23) activates complexes composed of FGF receptors (FGFRs), including FGFR1, and α-Klotho in the kidney distal tubule (DT), leading to increased sodium retention and hypertension. However, the role of FGFR1 in regulating renal processes linked to hypertension is unclear. Here, we investigated the effects of selective FGFR1 loss in the DT. Conditional knockout (cKO) of FGFR1 in the DT (FGFR1DT-cKO mice) resulted in left ventricular hypertrophy (LVH) and decreased kidney expression of α-Klotho in association with enhanced BP, decreased expression of angiotensin converting enzyme 2, and increased expression of the Na+-K+-2Cl- cotransporter. Notably, recombinant FGF-23 administration similarly decreased the kidney expression of α-Klotho and induced LVH in mice. Pharmacologic activation of FGFR1 with a monoclonal anti-FGFR1 antibody (R1MAb1) normalized BP and significantly attenuated LVH in the Hyp mouse model of excess FGF-23, but did not induce a response in FGFR1DT-cKO mice. The hearts of FGFR1DT-cKO mice showed increased expression of the transient receptor potential cation channel, subfamily C, member 6 (TRPC6), consistent with cardiac effects of soluble Klotho deficiency. Moreover, administration of recombinant soluble Klotho lowered BP in the Hyp mice. Thus, FGFR1 in the DT regulates systemic hemodynamic responses opposite to those predicted by the actions of FGF-23. These cardiovascular effects appear to be mediated by paracrine FGF control of kidney FGFR1 and subsequent regulation of soluble Klotho and TRPC6. FGFR1 in the kidney may provide a new molecular target for treating hypertension.

CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model.

BACKGROUND: GPRC6A is implicated in the pathogenesis of prostate cancer, but its role remains uncertain because of a purported tolerant gene variant created by substitution of a K..Y polymorphism in the 3rd intracellular loop (IL) that evolved in the majority of humans and replaces the ancestral RKLP present in 40% of humans of African descent and all other species. METHODS: We determined whether the K..Y polymorphism is present in human-derived prostate cancer cell lines by sequencing the region of the 3rd IL and assessed the cellular localization of a "humanized" mouse GPRC6A containing the K..Y sequence by immunofluorescence. We assessed functions of GPRC6A in PC-3 cells expressing endogenous GPRC6A and in GPRC6A-deficient PC-3 cells created using CRISPR/Cas9 technology. The effect of GPRC6A on basal and ligand stimulated cell proliferation and migration was evaluated in vitro in wild-type and PC-3-deficient cell lines. The effect of editing GPRC6A on prostate cancer growth and progression in vivo was assessed in a Xenograft mouse model implanted with wild-type and PC-3 deficient cells and treated with the GPRC6A ligand osteocalcin. RESULTS: We found that all of the human prostate cancer cell lines tested endogenously express the "K..Y" polymorphism in the 3rd IL. Comparison of mouse wild-type GPRC6A with a "humanized" mouse GPRC6A construct created by replacing the "RKLP" with the "K..Y" sequence, found that both receptors were predominantly expressed on the cell surface. The transfected "humanized" GPRC6A receptor, however, preferentially activated mTOR compared to ERK signaling in HEK-293 cells. In contrast, in PC-3 cells expressing the endogenous GPRC6A with the "K..Y" polymorphism, the ligand osteocalcin stimulated ERK, AKT and mTOR phosphorylation, promoted cell proliferation and migration, and upregulated genes regulating testosterone biosynthesis. Targeting GPRC6A in PC-3 cells by CRISPR/Cas9 significantly blocked these responses in vitro. In addition, GPRC6A deficient PC-3 xenografts exhibited significantly less growth and were resistant to osteocalcin-induced prostate cancer progression compared to control PC-3 cells expressing GPRC6A. CONCLUSIONS: Human GPRC6A is a functional osteocalcin and testosterone sensing receptor that promotes prostate cancer progression. GPRC6A may contribute to racial disparities in prostate cancer, and is a potential therapeutic target to develop antagonists to treat prostate cancer.

GPRC6A: Jack of all metabolism (or master of none).

BACKGROUND: GPRC6A, a widely expressed G-protein coupled receptor, is proposed to be a master regulator of complex endocrine networks and metabolic processes. GPRC6A is activated by multiple ligands, including osteocalcin (Ocn), testosterone (T), basic amino acids, and various cations. SCOPE OF REVIEW: We review the controversy surrounding GPRC6A functions. In mice, GPRC6A is proposed to integrate metabolic functions through the coordinated secretion of hormones, including insulin, GLP-1, T, and IL-6, and direct effects of this receptor to control glucose and fat metabolism in the liver, skeletal muscle, and fat. Loss-of-GPRC6A results in metabolic syndrome (MetS), and activation of GPRC6A stimulates proliferation of ß-cells, increases peripheral insulin sensitivity, and protects against high fat diet (HFD) induced metabolic abnormalities in most mouse models. Bone, cardiovascular, immune, and skin functions of GPRC6A have also been identified in mice. Expression of GPRC6A is increased in prostate cancer (PCa) cells, and inhibition of GPRC6A attenuates PCa progression in mouse models. The function of GPRC6A in humans, however, is not clear. During evolution, a unique polymorphism of GPRC6A emerged mainly in humans of Asian and European decent that has been proposed to alter membrane trafficking and function. In contrast, the ancestral allele found in all other species is retained in 1%, 15%, and 40% of people of Asian, European and African descent, respectively, suggesting GPRC6A gene variants may contribute to the racial disparities in the risk of developing MetS and PCa. MAJOR CONCLUSIONS: If the regulatory functions of GPRC6A identified in mice translate to humans, and polymorphisms in GPRC6A are found to predict racial disparities in human diseases, GPRC6A may be a new gene target to predict, prevent, and treat MetS, PCa, and other disorders impacted by GPRC6A.

Differential Regulatory Role of Soluble Klothos on Cardiac Fibrogenesis in Hypertension.

BACKGROUND: Soluble Klotho functions as an endocrine factor that plays important roles in a variety of pathophysiological processes. Soluble Klotho contains 130 KDa and 65 KDa isoforms. However, their distinct individual functional heterogeneity remains uncertain. Herein, we investigated the regulatory role of two soluble Klothos on cardiac fibrogenic responses. METHODS AND RESULTS: The effect of soluble Klothos on myofibroblast differentiation, proliferation, and collagen synthesis/degradation were examined in cultured mouse cardiac myofibroblasts. The role of 130 KDa Klotho on fibrosis in hypertensive heart disease were examined in wild type (WT) and Klotho transgenic (Tg/+) mice receiving chronic angiotensin (Ang)II infusion. Our in vitro studies revealed that addition of 130 KDa soluble Klotho isoform increased collagen synthesis in a dose dependent manner. Furthermore, 130 KDa Klotho significantly stimulated myofibroblast differentiation, proliferation, and ERK phosphorylation, which were abolished by fibroblast growth factor (FGF) receptor antagonist (SU5402). In contrast, 65 KDa soluble Klotho treatment significantly suppressed myofibroblast proliferation and collagen synthesis. In vivo study further demonstrated that chronic AngII infusion lead to cardiac fibrosis in both WT and Tg/+ mice. However, cardiac collagen, TGF-ß1, TIMP-2, and α-smooth muscle actin (SMA) levels were markedly upregulated in Tg/+ mice compared to WT cohort. CONCLUSION: Taken together, these findings implicate that 130 KDa soluble Klotho plays a stimulatory role in cardiac myofibroblast growth and activity through FGF pathway, whereas 65 KDa soluble Klotho exerts an anti-fibrotic effect in cardiac myofibroblasts. Thus, two distinct isoforms of soluble Klotho appear to play the counter-regulatory roles in cardiac fibrogenic responses.

Evidence for Osteocalcin Binding and Activation of GPRC6A in ß-Cells.

The possibility that G protein-coupled receptor family C member A (GPRC6A) is the osteocalcin (Ocn)-sensing G protein-coupled receptor that directly regulates pancreatic ß-cell functions is controversial. In the current study, we found that Ocn and an Ocn-derived C-terminal hexapeptide directly activate GPRC6A-dependent ERK signaling in vitro. Computational models probe the structural basis of Ocn binding to GPRC6A and predict that the C-terminal hexapeptide docks to the extracellular side of the transmembrane domain of GPRC6A. Consistent with the modeling, mutations in the computationally identified binding pocket of GPRC6A reduced Ocn and C-terminal hexapeptide activation of this receptor. In addition, selective deletion of Gprc6a in ß-cells (Gprc6a(ß)(-cell-cko)) by crossing Gprc6a(flox/flox) mice with Ins2-Cre mice resulted in reduced pancreatic weight, islet number, insulin protein content, and insulin message expression. Both islet size and ß-cell proliferation were reduced in Gprc6a(ß)(-cell-cko) compared with control mice. Gprc6a(ß)(-cell-cko) exhibited abnormal glucose tolerance, but normal insulin sensitivity. Islets isolated from Gprc6a(ß)(-cell-cko) mice showed reduced insulin simulation index in response to Ocn. These data establish the structural basis for Ocn direct activation of GPRC6A and confirm a role for GPRC6A in regulating ß-cell proliferation and insulin secretion.