Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes.

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.

miR-582-5p targets Skp1 and regulates NF-κB signaling-mediated inflammation.

A well-tuned inflammatory response is crucial for an effective immune process. Nuclear factor-kappa B (NF-κB) is a key mediator of inflammatory and innate immunity responses, and its dysregulation is closely associated with immune-related diseases. MicroRNAs (miRNAs) are important inflammation modulators. However, miRNA-regulated mechanisms that implicate NF-κB activity are not fully understood. This study aimed to identify a potential miRNA that could modulate the dysregulated NF-κB signaling during inflammation. We identified miR-582-5p that was significantly downregulated in inflamed murine adipose tissues and RAW264.7 cells. S-phase kinase-associated protein 1 (SKP1), a core component of an E3 ubiquitin ligase that regulates the NF-κB pathway, was proposed as a biological target of miR-582-5p by using TargetScan. The binding of miR-582-5p to a 3'-untranslated region site on Skp1 was confirmed using a dual-luciferase reporter assay; in addition, transfection with a miR-582-5p mimic suppressed SKP1 expression in RAW264.7 cells. Importantly, exogenous miR-582-5p attenuated the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 through suppressing the degradation of the NF-κB inhibitor alpha, followed by the nuclear translocation of NF-κB. Therefore, exogenously applied miR-582-5p can attenuate the NF-κB signaling pathway via targeting Skp1; this provides a prospective therapeutic strategy for treating inflammatory and immune diseases.

Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.

Inhibition of the ATG4-LC3 pathway suppressed osteoclast maturation.

Autophagy is a non-selective action in which cells degrade parts of themselves, reusing degraded cellular components. Among autophagy-related gene (ATG) family members, ATG4 proteins play crucial roles in the microtubule-associated protein 1 light chain 3 (LC3) phosphatidylethanolamine (PE) system which is essential for autophagosome maturation. Although autophagy has been shown to be involved in osteoclastic bone resorption, the role of ATG4/LC3 in bone resorption remains unclear. When mouse bone marrow cells were treated with various concentrations of NSC185058 (NSC), a specific inhibitor of ATG4B, 1 h prior to treatment with receptor activator of NF-κB ligand (RANKL) in the presence of macrophage colony stimulating factor (M-CSF), NSC inhibited osteoclastogenesis in a dose-dependent manner. Addition of NSC in the late stages of osteoclast differentiation suppressed multinucleation and reduced the expression of markers for mature osteoclasts such as Dc-stamp, Mmp9, and Ctsk. NSC also suppressed actin ring formation and pit formation in mature osteoclasts. When a periodontitis model involving eight-week-old male mice in which the right maxillary second molar had been ligated with silk thread was injected with or without NSC, alveolar bone resorption was suppressed by a decrease in the number of osteoclasts in the NSC-treated group. These results suggest that LC3 is important for the maturation of osteoclasts and that LC3 inhibition is a new therapeutic strategy for periodontal disease.

Osteocalcin promotes proliferation, differentiation, and survival of PC12 cells.

Involvement of the bone matrix protein osteocalcin (OC) in the development of learning and memory, and the prevention of anxiety-like behaviors in mice. However, the direct effects of OC on neurons are still unknown comparing to the mechanism how OC affects systemic energy expenditure and glucose homeostasis. In this study, we investigated the effect of OC on proliferation, differentiation, and survival of neurons using the rat pheochromocytoma cell line PC12. RT-PCR analysis for OC receptor candidates revealed that Gpr158, but not Gprc6a, mRNA was expressed in PC12 cells. The growth of PC12 cells cultured in the presence of 5-50 ng/mL of either uncarboxylated (GluOC) or carboxylated (GlaOC) OC was increased compared to cells cultured in the absence of OC. In addition, NGF-induced neurite outgrowth was enhanced by OC, and H2O2-induced cell death was suppressed by pretreatment with OC. All of these results were observed for both GluOC and GlaOC at comparable levels, suggesting that OC may directly affect cell proliferation, differentiation, and survival by binding to its candidate receptor, GPR158.

Expression of PRIP, a phosphatidylinositol 4,5-bisphosphate binding protein, attenuates PI3K/AKT signaling and suppresses tumor growth in a xenograft mouse model.

Cancer is characterized by uncontrolled proliferation resulting from aberrant cell cycle progression. The activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling, a regulatory pathway for the cell cycle, stabilizes cyclin D1 in the G1 phase by inhibiting the activity of glycogen synthase kinase 3ß (GSK3ß) via phosphorylation. We previously reported that phospholipase C-related catalytically inactive protein (PRIP), a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding protein, regulates PI3K/AKT signaling by competitively inhibiting substrate recognition by PI3K. Therefore, in this study, we investigated whether PRIP is involved in cell cycle progression. PRIP silencing in MCF-7 cells, a human breast cancer cell line, demonstrated PI(3,4,5)P3 signals accumulated at the cell periphery compared to that of the control. This suggests that PRIP reduction enhances PI(3,4,5)P3-mediated signaling. Consistently, PRIP silencing in MCF-7 cells exhibited increased phosphorylation of AKT and GSK3ß which resulted in cyclin D1 accumulation. In contrast, the exogenous expression of PRIP in MCF-7 cells evidenced stronger downregulation of AKT and GSK3ß phosphorylation, reduced accumulation of cyclin D1, and diminished cell proliferation in comparison to control cells. Flow cytometry analysis indicated that MCF-7 cells stably expressing PRIP attenuate cell cycle progression. Importantly, tumor growth of MCF-7 cells stably expressing PRIP was considerably prevented in an in vivo xenograft mouse model. In conclusion, PRIP expression downregulates PI3K/AKT/GSK3ß-mediated cell cycle progression and suppresses tumor growth. Therefore, we propose that PRIP is a new therapeutic target for anticancer therapy.

Kif1c regulates osteoclastic bone resorption as a downstream molecule of p130Cas.

Podosome formation in osteoclasts is an important initial step in osteoclastic bone resorption. Mice lacking c-Src (c-Src-/- ) exhibited osteopetrosis due to a lack of podosome formation in osteoclasts. We previously identified p130Cas (Crk-associated substrate [Cas]) as one of c-Src downstream molecule and osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) mice also exhibited a similar phenotype to c-Src-/- mice, indicating that the c-Src/p130Cas plays an important role for bone resorption by osteoclasts. In this study, we performed a cDNA microarray and compared the gene profiles of osteoclasts from c-Src-/- or p130CasΔOCL-/- mice with wild-type (WT) osteoclasts to identify downstream molecules of c-Src/p130Cas involved in bone resorption. Among several genes that were commonly downregulated in both c-Src-/- and p130CasΔOCL-/- osteoclasts, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization. Reduced Kif1c expression was observed in both c-Src-/- and p130CasΔOCL-/- osteoclasts compared with WT osteoclasts. Kif1c exhibited a broad tissue distribution, including osteoclasts. Knockdown of Kif1c expression using shRNAs in WT osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in p130CasΔOCL-/- osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas (191 words). SIGNIFICANCE OF THE STUDY: We previously showed that the c-Src/p130Cas (Cas) plays an important role for bone resorption by osteoclasts. In this study, we identified kinesin family protein 1c (Kif1c), which regulates the cytoskeletal organization, as a downstream molecule of c-Src/p130Cas axis, using cDNA microarray. Knockdown of Kif1c expression using shRNAs in wild-type osteoclasts suppressed actin ring formation. Kif1c overexpression restored bone resorption subsequent to actin ring formation in osteoclast-specific p130Cas-deficient (p130CasΔOCL-/- ) osteoclasts but not c-Src-/- osteoclasts, suggesting that Kif1c regulates osteoclastic bone resorption in the downstream of p130Cas.

Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic ß-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic ß-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.

An extract from pork bones containing osteocalcin improves glucose metabolism in mice by oral administration.

Osteocalcin (OC) is a bone-derived hormone that regulates energy metabolism. OC exists in two forms, carboxylated (GlaOC) and uncaboxylated (GluOC), but only the latter appears to have an endocrine function. In this study, we prepared an extract containing both Gla- and GluOC from boiled pork bone using 0.2 M carbonate buffer at pH 9.5, and tested whether the extract had beneficial effects on improving metabolic parameters in obese mice. The extract equivalent of 1.2 µg of GluOC/mouse was orally administrated to C57BL/6 female mice fed a high-fat, high-sucrose diet. Daily oral administration of the extract for four weeks decreased blood glucose levels and promoted glucose tolerance as well as insulin sensitivity. Our study shows for the first time that boiled pork bones are a source material for osteocalcin in the large-scale production of supplements designed to improve glucose metabolism.

Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin.

A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca(2+) levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.

The role of adhesion molecules in osteocalcin-induced effects on glucose and lipid metabolism in adipocytes.

Recent findings suggest that uncarboxylated osteocalcin (GluOC) promotes glucose and lipid metabolism via its putative receptor GPRC6A; however, its direct effect on adipocytes remains elusive. In this study, we elucidated the effects of GluOC on adipocytes, with an emphasis on the role of cell adhesion molecules. We determined that GluOC promoted the expression of adipocyte adhesion molecule (ACAM) and its transcription factor Krüppel-like factor 4 and enhanced the cortical actin filament assembly, which ameliorated lipid droplet hypertrophy. Additionally, GluOC upregulated the expression of integrin αVß3 and activation of focal adhesion kinase (FAK) and prevented insulin receptor substrate 1 (IRS1) degradation by inhibiting the ubiquitin-proteasome system via the FAK-PLC-PKC axis, which activated IRS1-Akt-mediated glucose transporter 4 (GLUT4) transport. Furthermore, we showed that GluOC elevated the expression of the insulin-independent glucose transporters GLUT1 and GLUT8, which facilitated insulin stimulation-independent glucose transport. The GluOC-induced activation of integrin αVß3 signaling promoted microtubule assembly, which improved glucose and lipid metabolism via its involvement in intracellular vesicular transport. GluOC treatment also suppressed collagen type 1 formation, which might prevent adipose tissue fibrosis in obese individuals. Overall, our results imply that GluOC promotes glucose and lipid metabolism via ACAM, integrin αVß3, and GLUT1 and 8 expression, directly affecting adipocytes.

Activation of NF-κB signaling regulates ovariectomy-induced bone loss and weight gain.

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss.

The role of microRNAs in understanding sex-based differences in Alzheimer's disease.

BACKGROUND: The incidence of Alzheimer's disease (AD)-the most frequent cause of dementia-is expected to increase as life expectancies rise across the globe. While sex-based differences in AD have previously been described, there remain uncertainties regarding any association between sex and disease-associated molecular mechanisms. Studying sex-specific expression profiles of regulatory factors such as microRNAs (miRNAs) could contribute to more accurate disease diagnosis and treatment. METHODS: A systematic review identified six studies of microRNA expression in AD patients that incorporated information regarding the biological sex of samples in the Gene Expression Omnibus repository. A differential microRNA expression analysis was performed, considering disease status and patient sex. Subsequently, results were integrated within a meta-analysis methodology, with a functional enrichment of meta-analysis results establishing an association between altered miRNA expression and relevant Gene Ontology terms. RESULTS: Meta-analyses of miRNA expression profiles in blood samples revealed the alteration of sixteen miRNAs in female and 22 miRNAs in male AD patients. We discovered nine miRNAs commonly overexpressed in both sexes, suggesting a shared miRNA dysregulation profile. Functional enrichment results based on miRNA profiles revealed sex-based differences in biological processes; most affected processes related to ubiquitination, regulation of different kinase activities, and apoptotic processes in males, but RNA splicing and translation in females. Meta-analyses of miRNA expression profiles in brain samples revealed the alteration of six miRNAs in female and four miRNAs in male AD patients. We observed a single underexpressed miRNA in female and male AD patients (hsa-miR-767-5p); however, the functional enrichment analysis for brain samples did not reveal any specifically affected biological process. CONCLUSIONS: Sex-specific meta-analyses supported the detection of differentially expressed miRNAs in female and male AD patients, highlighting the relevance of sex-based information in biomedical data. Further studies on miRNA regulation in AD patients should meet the criteria for comparability and standardization of information.

Alzheimer's disease (AD)­a neurodegenerative disease mainly affecting older patients­is characterized by cognitive deterioration, memory loss, and progressive incapacitation in daily activities. While AD affects almost twice as many females as males, and cognitive deterioration and brain atrophy develop more rapidly in females, the biological causes of these differences remain poorly understood. MicroRNAs (miRNAs) regulate gene expression and impact a wide variety of biological processes; therefore, studying the differential expression of miRNAs in female and male AD patients could contribute to a better understanding of the disease. We reviewed studies of miRNA expression in female and male AD patients and integrated results using a meta-analysis methodology and then identified those genes regulated by the altered miRNAs to establish an association with biological processes. We found 16 (females) and 22 (males) miRNAs altered in the blood of AD patients. Functional enrichment revealed sex-based differences in the affected altered biological processes­protein modification and degradation and cell death in male AD patients and RNA processing in female AD patients. A similar analysis in the brains of AD patients revealed six (females) and four (males) miRNAs with altered expression; however, our analysis failed to highlight any specifically altered biological processes. Overall, we highlight the sex-based differential expression of miRNAs (and biological processes affected) in the blood and brain of AD patients.

Phospholipase C-related catalytically inactive protein, a novel microtubule-associated protein 1 light chain 3-binding protein, negatively regulates autophagosome formation.

Upon starvation, cells undergo autophagy, an intracellular bulk-degradation process, to provide the required nutrients. Here, we observed that phospholipase C-related catalytically inactive protein (PRIP) binds to microtubule-associated protein 1 light chain 3 (LC3), a mammalian autophagy-related initiator that regulates the autophagy pathway. Then, we examined the involvement of PRIP in the nutrient depletion-induced autophagy pathway. Enhanced colocalization of PRIP with LC3 was clearly seen in nutrient-starved mouse embryonic fibroblasts under a fluorescent microscope, and interaction of the proteins was revealed by immunoprecipitation experiments with an anti-LC3 antibody. Under starvation conditions, there were more green fluorescent protein fused-LC3 dots in mouse embryonic fibroblasts from PRIP-deficient mice than in fibroblasts from wild type cells. The formation of new dots in a single cell increased, as assessed by time-lapse microscopy. Furthermore, the increase in autophagosome formation in PRIP-deficient cells was notably inhibited by exogenously overexpressed PRIP. Taken together, PRIP is a novel LC3-binding protein that acts as a negative modulator of autophagosome formation.

Involvement of PRIP, phospholipase C-related, but catalytically inactive protein, in bone formation.

PRIP (phospholipase C-related, but catalytically inactive protein) is a novel protein isolated in this laboratory. PRIP-deficient mice showed increased serum gonadotropins, but decreased gonadal steroid hormones. This imbalance was similar to that for the cause of bone disease, such as osteoporosis. In the present study, therefore, we analyzed mutant mice with special reference to the bone property. We first performed three-dimensional analysis of the femur of female mice. The bone mineral density and trabecular bone volume were higher in mutant mice. We further performed histomorphometrical assay of bone formation parameters: bone formation rate, mineral apposition rate, osteoid thickness, and osteoblast number were up-regulated in the mutant, indicating that increased bone mass is caused by the enhancement of bone formation ability. We then cultured primary cells isolated from calvaria prepared from both genotypes. In mutant mice, osteoblast differentiation, as assessed by alkaline phosphatase activity and the expression of osteoblast differentiation marker genes, was enhanced. Moreover, we analyzed the phosphorylation of Smad1/5/8 in response to bone morphogenetic protein, with longer phosphorylation in the mutant. These results indicate that PRIP is implicated in the negative regulation of bone formation.

RANKL elevation activates the NIK/NF-κB pathway, inducing obesity in ovariectomized mice.

Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.

Hepatic glycogenolysis is determined by maternal high-calorie diet via methylation of Pygl and it is modified by oteocalcin administration in mice.

OBJECTIVE: Accumulating evidence indicates that an adverse perinatal environment contributes to a higher risk of metabolic disorders in the later life of the offspring. However, the underlying molecular mechanisms remain largely unknown. Thus, we investigated the contribution of maternal high-calorie diet and osteocalcin to metabolic homeostasis in the offspring. METHODS: Eight-week-old C57Bl/6N female mice were mated with age-matched males and allocated randomly to three groups: a normal-diet (ND) or a high-fat, high-sucrose diet group, which was administered either saline (control) or GluOC (10 ng/g body mass) from the day of mating to that of delivery, and the dams were fed a ND after the delivery. Pups weaned at 24 days after birth were analyzed. RESULTS: A maternal high-fat, high-sucrose diet during pregnancy causes metabolic disorders in the liver of the offspring via hypermethylation of the Pygl gene, encoding glycogen phosphorylase L, which mediates hepatic glycogenolysis. The reduced expression of Pygl induced by the maternal diet causes the hepatic accumulation of glycogen and triglyceride in the offspring, which remains in adulthood. In addition, the administration of uncarboxylated osteocalcin during pregnancy upregulates Pygl expression via both direct CREBH and ATF4 and indirect epigenomic pathways, mitigating the maternal diet-induced obesity and abnormal glucose and lipid metabolism in adulthood. CONCLUSIONS: We propose that maternal energy status is reflected in the hepatic glycogenolysis capacity of the offspring via epigenetic modification of Pygl and uncarboxylated osteocalcin regulates glycogenolysis.

GABA(A) receptor subunit alteration-dependent diazepam insensitivity in the cerebellum of phospholipase C-related inactive protein knockout mice.

The GABA(A) receptor, a pentamer composed predominantly of alpha, beta, and gamma subunits, mediates fast inhibitory synaptic transmission. We have previously reported that phospholipase C-related inactive protein (PRIP) is a modulator of GABA(A) receptor trafficking and that knockout (KO) mice exhibit a diazepam-insensitive phenotype in the hippocampus. The alpha subunit affects diazepam sensitivity; alpha1, 2, 3, and 5 subunits assemble with any form of beta and the gamma2 subunits to produce diazepam-sensitive receptors, whereas alpha4 or alpha6/beta/gamma2 receptors are diazepam-insensitive. Here, we investigated how PRIP is implicated in the diazepam-insensitive phenotype using cerebellar granule cells in animals expressing predominantly the alpha6 subunit. The expression of alpha1/beta/gamma2 diazepam-sensitive receptors was decreased in the PRIP-1 and 2 double KO cerebellum without any change in the total number of benzodiazepine-binding sites as assessed by radioligand-binding assay. Since levels of the alpha6 subunit were increased, the alpha1/beta/gamma2 receptors might be replaced with alpha6 subunit-containing receptors. Then, we further performed autoradiographic and electrophysiologic analyses. These results suggest that the expression of alpha6/delta receptors was decreased in cerebellar granule neurons, while that of alpha6/gamma2 receptors was increased. PRIP-1 and 2 double KO mice exhibit a diazepam-insensitive phenotype because of a decrease in diazepam-sensitive (alpha1/gamma2) and increase in diazepam-insensitive (alpha6/gamma2) GABA(A) receptors in the cerebellar granule cells.

The roles of osteocalcin in lipid metabolism in adipose tissue and liver.

Bone provides skeletal support and functions as an endocrine organ by producing osteocalcin, whose uncarboxylated form (GluOC) increases the metabolism of glucose and lipid by activating its putative G protein-coupled receptor (family C group 6 subtype A). Low doses (≤10 ng/ml) of GluOC induce the expression of adiponectin, adipose triglyceride lipase and peroxisome proliferator-activated receptor γ, and promote active phosphorylation of lipolytic enzymes such as perilipin and hormone-sensitive lipase via the cAMP-PKA-Src-Rap1-ERK-CREB signaling axis in 3T3-L1 adipocytes. Administration of high-dose (≥20 ng/ml) GluOC induces programmed necrosis (necroptosis) through a juxtacrine mechanism triggered by the binding of Fas ligand, whose expression is induced by forkhead box O1, to Fas that is expressed in adjacent adipocytes. Furthermore, expression of adiponectin and adipose triglyceride lipase in adipocytes is triggered in the same manner as following low-dose GluOC stimulation; these effects protect mice from diet-induced accumulation of triglycerides in hepatocytes and consequent liver injury through the upregulation of nuclear translocation of nuclear factor-E2-related factor-2, expression of antioxidant enzymes, and inhibition of the c-Jun N-terminal kinase pathway. Evaluation of these molecular mechanisms leads us to consider that GluOC might have potential as a treatment for lipid metabolism disorders. Indeed, there have been many reports demonstrating the negative correlation between serum osteocalcin levels and obesity or non-alcoholic fatty liver disease, a common risk factor for which is dyslipidemia in humans. The present review summarizes the effects of GluOC on lipid metabolism as well as its possible therapeutic application for metabolic diseases including obesity and dyslipidemia.

GLP-1 signaling is required for improvement of glucose tolerance by osteocalcin.

Osteocalcin is a bone-derived hormone that in its uncarboxylated form (GluOC) plays an important role in glucose and energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation through its putative receptor GPRC6A. We previously showed that the effect of GluOC on insulin secretion is mediated predominantly by glucagon-like peptide-1 (GLP-1) released from intestinal endocrine cells in response to GluOC stimulation. Moreover, oral administration of GluOC was found to reduce the fasting blood glucose level, to improve glucose tolerance, and to increase the fasting serum insulin concentration and ß-cell area in the pancreas in wild-type mice. We have now examined the effects of oral GluOC administration for at least 4 weeks in GLP-1 receptor-knockout mice. Such administration of GluOC in the mutant mice triggered glucose intolerance, enhanced gluconeogenesis and promoted both lipid accumulation in the liver as well as adipocyte hypertrophy and inflammation in adipose tissue. Furthermore, inactivation of GLP-1 receptor signaling in association with GluOC administration induced activation of the transcription factor FoxO1 and expression of its transcriptional coactivator PGC1α in the liver, likely accounting for the observed upregulation of gluconeogenic gene expression. Our results thus indicate that the beneficial metabolic effects of GluOC are dependent on GLP-1 receptor signaling.