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.

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.

NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment.

Epidermal growth factor receptor (EGFR) is highly expressed in several types of cancer cells including oral squamous cell carcinoma (OSCC). EGF/EGFR signaling is recognized as an important molecular target in cancer therapy. However, cancer cells often become tolerant to EGF/EGFR signaling-targeted therapies. In the tumor microenvironment, the tumor incites inflammation and the inflammation-derived cytokines make a considerable impact on cancer development. In addition, hyperosmolarity is also induced, but the role of osmotic stress in cancer development has not been fully understood. This study demonstrates molecular insights into hyperosmolarity effect on OSCC development and shows that NFAT5 transcription factor plays an important functional role in enhancing the oral cancer cell proliferation by inducing the EGFR translocation from the endoplasmic reticulum to the plasma membrane through increase the expression of DPAGT1, an essential enzyme for catalyzing the first committed step of N-linked protein glycosylation. These results suggest that hyperosmolarity-induced intra-nuclear translocation of NFAT5 essential for DPAGT1 activation and EGFR subcellular translocation responsible for OSCC tumor progression.

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.

Phospholipase C-related inactive protein type-1 deficiency affects anesthetic electroencephalogram activity induced by propofol and etomidate in mice.

PURPOSE: The general anesthetics propofol and etomidate mainly exert their anesthetic actions via GABA A receptor (GABAA-R). The GABAA-R activity is influenced by phospholipase C-related inactive protein type-1 (PRIP-1), which is related to trafficking and subcellular localization of GABAA-R. PRIP-1 deficiency attenuates the behavioral reactions to propofol but not etomidate. However, the effect of these anesthetics and of PRIP-1 deficiency on brain activity of CNS are still unclear. In this study, we examined the effects of propofol and etomidate on the electroencephalogram (EEG). METHODS: The cortical EEG activity was recorded in wild-type (WT) and PRIP-1 knockout (PRIP-1 KO) mice. All recorded EEG data were offline analyzed, and the power spectral density and 95% spectral edge frequency of EEG signals were compared between genotypes before and after injections of anesthetics. RESULTS: PRIP-1 deficiency induced increases in EEG absolute powers, but did not markedly change the relative spectral powers during waking and sleep states in the absence of anesthesia. Propofol administration induced increases in low-frequency relative EEG activity and decreases in SEF95 values in WT but not in PRIP-1 KO mice. Following etomidate injection, low-frequency EEG power was increased in both genotype groups. At high frequency, the relative power in PRIP-1 KO mice was smaller than that in WT mice. CONCLUSIONS: The lack of PRIP-1 disrupted the EEG power distribution, but did not affect the depth of anesthesia after etomidate administration. Our analyses suggest that PRIP-1 is differentially involved in anesthetic EEG activity with the regulation of GABAA-R activity.

Phospholipase C-related but catalytically inactive proteins regulate ovarian follicle development.

Phospholipase C-related but catalytically inactive proteins PRIP-1 and -2 are inositol-1,4,5-trisphosphate binding proteins that are encoded by independent genes. Ablation of the Prip genes in mice impairs female fertility, which is manifested by fewer pregnancies, a decreased number of pups, and the decreased and increased secretion of gonadal steroids and gonadotropins, respectively. We investigated the involvement of the PRIPs in fertility, focusing on the ovaries of Prip-1 and -2 double-knock-out (DKO) mice. Multiple cystic follicles were observed in DKO ovaries, and a superovulation assay showed a markedly decreased number of ovulated oocytes. Cumulus-oocyte complexes showed normal expansion, and artificial gonadotropin stimulation regulated the ovulation-related genes in a normal fashion, suggesting that the ovulation itself was probably normal. A histological analysis showed atresia in fewer follicles of the DKO ovaries, particularly in the secondary follicle stages. The expression of luteinizing hormone receptor (LHR) was aberrantly higher in developing follicles, and the phosphorylation of extracellular signal-regulated protein kinase, a downstream target of LH-LHR signaling, was higher in DKO granulosa cells. This suggests that the up-regulation of LH-LHR signaling is the cause of impaired follicle development. The serum estradiol level was lower, but estradiol production was unchanged in the DKO ovaries. These results suggest that PRIPs are positively involved in the development of follicles via their regulation of LH-LHR signaling and estradiol secretion. Female DKO mice had higher serum levels of insulin, testosterone, and uncarboxylated osteocalcin, which, together with reduced fertility, are reminiscent of polycystic ovary syndrome in humans.

Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling.

Phospholipase C-related, but catalytically inactive protein (PRIP) was previously identified as a novel inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-δ but lacking phospholipase activity. We recently showed that PRIP gene knock-out (KO) in mice increases bone formation and concomitantly decreases bone resorption, resulting in increased bone mineral density and trabecular bone volume. However, the role of PRIP in osteoclastogenesis has not yet been fully elucidated. Here, we investigated the effects of PRIP on bone remodeling by investigating dynamic tooth movement in mice fitted with orthodontic devices. Morphological analysis indicated that the extent of tooth movement was smaller in the PRIP-KO mice than in wild-type mice. Histological analysis revealed fewer osteoclasts on the bone-resorption side in maxillary bones of PRIP-KO mice, and osteoclast formation assays and flow cytometry indicated lower osteoclast differentiation in bone marrow cells isolated from these mice. The expression of genes implicated in bone resorption was lower in differentiated PRIP-KO cells, and genes involved in osteoclast differentiation, such as the transcription factor NFATc1, exhibited lower expression in immature PRIP-KO cells initiated by M-CSF. Moreover, calcineurin expression and activity were also lower in the PRIP-KO cells. The PRIP-KO cells also displayed fewer M-CSF-induced changes in intracellular Ca2+ and exhibited reduced nuclear localization of NFATc1. Up-regulation of intracellular Ca2+ restored osteoclastogenesis of the PRIP-KO cells. These results indicate that PRIP deficiency impairs osteoclast differentiation, particularly at the early stages, and that PRIP stimulates osteoclast differentiation through calcium-calcineurin-NFATc1 signaling via regulating intracellular Ca2.

Phospholipase C-related Catalytically Inactive Protein Is a New Modulator of Thermogenesis Promoted by ß-Adrenergic Receptors in Brown Adipocytes.

Phospholipase C-related catalytically inactive protein (PRIP) was first identified as an inositol 1,4,5-trisphosphate-binding protein, and was later found to be involved in a variety of cellular events, particularly those related to protein phosphatases. We previously reported that Prip knock-out (KO) mice exhibit a lean phenotype with a small amount of white adipose tissue. In the present study, we examined whether PRIP is involved in energy metabolism, which could explain the lean phenotype, using high-fat diet (HFD)-fed mice. Prip-KO mice showed resistance to HFD-induced obesity, resulting in protection from glucose metabolism dysfunction and insulin resistance. Energy expenditure and body temperature at night were significantly higher in Prip-KO mice than in wild-type mice. Gene and protein expression of uncoupling protein 1 (UCP1), a thermogenic protein, was up-regulated in Prip-KO brown adipocytes in thermoneutral or cold environments. These phenotypes were caused by the promotion of lipolysis in Prip-KO brown adipocytes, which is triggered by up-regulation of phosphorylation of the lipolysis-related proteins hormone-sensitive lipase and perilipin, followed by activation of UCP1 and/or up-regulation of thermogenesis-related genes (e.g. peroxisome proliferator-activated receptor-γ coactivator-1α). The results indicate that PRIP negatively regulates UCP1-mediated thermogenesis in brown adipocytes.

Propofol Anesthesia Is Reduced in Phospholipase C-Related Inactive Protein Type-1 Knockout Mice.

The GABA type A receptor (GABAA-R) is a major target of intravenous anesthetics. Phospholipase C-related inactive protein type-1 (PRIP-1) is important in GABAA-R phosphorylation and membrane trafficking. In this study, we investigated the role of PRIP-1 in general anesthetic action. The anesthetic effects of propofol, etomidate, and pentobarbital were evaluated in wild-type and PRIP-1 knockout (PRIP-1 KO) mice by measuring the latency and duration of loss of righting reflex (LORR) and loss of tail-pinch withdrawal response (LTWR). The effect of pretreatment with okadaic acid (OA), a protein phosphatase 1/2A inhibitor, on propofol- and etomidate-induced LORR was also examined. PRIP-1 deficiency provided the reduction of LORR and LTWR induced by propofol but not by etomidate or pentobarbital, indicating that PRIP-1 could determine the potency of the anesthetic action of propofol. Pretreatment with OA recovered the anesthetic potency induced by propofol in PRIP-1 KO mice. OA injection enhanced phosphorylation of cortical the GABAA-R ß3 subunit in PRIP-1 KO mice. These results suggest that PRIP-1-mediated GABAA-R ß3 subunit phosphorylation might be involved in the general anesthetic action induced by propofol but not by etomidate or pentobarbital.

General anesthetic actions on GABAA receptors in vivo are reduced in phospholipase C-related catalytically inactive protein knockout mice.

PURPOSE: The aim of this study was to investigate the action of general anesthetics in phospholipase C-related catalytically inactive protein (PRIP)-knockout (KO) mice that alter GABAA receptor signaling. METHODS: PRIP regulates the intracellular trafficking of ß subunit-containing GABAA receptors in vitro. In this study, we examined the effects of intravenous anesthetics, propofol and etomidate that act via ß subunit-containing GABAA receptors, in wild-type and Prip-KO mice. Mice were intraperitoneally injected with a drug, and a loss of righting reflex (LORR) assay and an electroencephalogram analysis were performed. RESULTS: The cell surface expression of GABAA receptor ß3 subunit detected by immunoblotting was decreased in Prip-knockout brain compared with that in wild-type brain without changing the expression of other GABAA receptor subunits. Propofol-treated Prip-KO mice exhibited significantly shorter duration of LORR and had lower total anesthetic score than wild-type mice in the LORR assay. The average duration of sleep time in an electroencephalogram analysis was shorter in propofol-treated Prip-KO mice than in wild-type mice. The hypnotic action of etomidate was also reduced in Prip-KO mice. However, ketamine, an NMDA receptor antagonist, had similar effects in the two genotypes. CONCLUSION: PRIP regulates the cell surface expression of the GABAA receptor ß3 subunit and modulates general anesthetic action in vivo. Elucidation of the involved regulatory mechanisms of GABAA receptor-dependent signaling would inform the development of safer anesthetic therapies for clinical applications.

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.

Sphingosine-1-phosphate-enhanced Wnt5a promotes osteogenic differentiation in C3H10T1/2 cells.

In this study, we investigated the involvement of Wnt signaling in sphingosine-1-phosphate (S1P)-enhanced osteogenic differentiation of C3H10T1/2 pluripotent stem cells. We found that S1P enhanced the expression of Wnt5a and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) during osteogenic differentiation. Wnt5a-neutralizing antibody inhibited S1P-enhanced expression of LRP5/6 and alkaline phosphatase, which are essential for osteogenic differentiation. Conversely, S1P did not affect endogenous canonical Wnt signaling. Taken together, S1P-enhanced Wnt5a promotes LRP5/6 expression, resulting in the trigger of osteogenic differentiation of C3H10T1/2 cells. These findings suggest a potential beneficial role for S1P in bone regeneration.

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.

Enhanced lateral inhibition in the barrel cortex by deletion of phospholipase C-related catalytically inactive protein-1/2 in mice.

We previously demonstrated that the deletion of phospholipase C-related catalytically inactive protein-1/2 (PRIP-1/2) enhances the desensitization of GABAA receptors (GABAARs), while it facilitates their resensitization at the offset of GABA puff, causing a hump-like tail current (tail-I) in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex. In the present study, we investigated whether inhibitory synaptic transmission in L3 PCs in the barrel cortex is altered in the PRIP-1/2 double-knockout (PRIP-DKO) mice, and if so, how the interaction between excitation and inhibition is subsequently modified. PRIP-1/2 deletion resulted in the prolongation of the decay phase of inhibitory postsynaptic currents/potentials (IPSCs/IPSPs) in L3 PCs evoked by stimulation of L3, leaving the overall features of miniature IPSCs unchanged. An optical imaging revealed that the spatiotemporal profile of a horizontal excitation spread across columns in L2/3 caused by L4 stimulation in the barrel cortex was more restricted in PRIP-DKO mice compared to the wild type, while those obtained in the presence of bicuculline were almost identical between the two genotypes. These findings suggest that PRIP-1/2 deletion enhances the lateral inhibition by prolonging inhibitory synaptic actions to limit the intercolumnar integration in the barrel cortex. Considering the present findings together with our previous study including a mathematical simulation, the prolongation of inhibitory synaptic actions is likely to result from an enhancement of desensitization followed by an enhanced resensitization in GABAARs.

Enhanced desensitization followed by unusual resensitization in GABAA receptors in phospholipase C-related catalytically inactive protein-1/2 double-knockout mice.

Phospholipase C-related catalytically inactive proteins (PRIP-1/2) are previously reported to be involved in the membrane trafficking of GABAA receptor (GABAAR) and the regulation of intracellular Ca(2+) stores. GABAAR-mediated currents can be regulated by the intracellular Ca(2+). However, in PRIP-1/2 double-knockout (PRIP-DKO) mice, it remains unclear whether the kinetic properties of GABAARs are modulated by the altered regulation of intracellular Ca(2+) stores. Here, we investigated whether GABAAR currents (IGABA) evoked by GABA puff in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex are altered in PRIP-DKO mice. The deletion of PRIP-1/2 enhanced the desensitization of IGABA but induced a hump-like tail current (tail-I) at the GABA puff offset. IGABA and the hump-like tail-I were suppressed by GABAAR antagonists. The enhanced desensitization of IGABA and the hump-like tail-I in PRIP-DKO PCs were mediated by increases in the intracellular Ca(2+) concentration and were largely abolished by a calcineurin inhibitor and ruthenium red. Calcium imaging revealed that Ca(2+)-induced Ca(2+) release (CICR) and subsequent store-operated Ca(2+) entry (SOCE) are more potent in PRIP-DKO PCs than in wild-type PCs. A mathematical model revealed that a slowdown of GABA-unbinding rate and an acceleration of fast desensitization rate by enhancing its GABA concentration dependency are involved in the generation of hump-like tail-Is. These results suggest that in L3 PCs of the barrel cortex in PRIP-DKO mice, the increased calcineurin activity due to the potentiated CICR and SOCE enhances the desensitization of GABAARs and slows the GABA-unbinding rate, resulting in their unusual resensitization following removal of GABA.

Involvement of PRIP (Phospholipase C-Related But Catalytically Inactive Protein) in BMP-Induced Smad Signaling in Osteoblast Differentiation.

Phospholipase C-related but catalytically inactive protein (PRIP) was first isolated as an inositol 1,4,5-trisphosphate binding protein. We generated PRIP gene-deficient mice which exhibited the increased bone mineral density and trabecular bone volume, indicating that PRIP is implicated in the regulation of bone properties. In this study, we investigated the possible mechanisms by which PRIP plays a role in bone morphogenetic protein (BMP) signaling, by analyzing the culture of primary cells isolated from calvaria of two genotypes, the wild type and a mutant. In the mutant culture, enhanced osteoblast differentiation was observed by measuring alkaline phosphatase staining and activity. The promoter activity of Id1 gene, responding immediately to BMP, was also more increased. Smad1/5 phosphorylation in response to BMP showed an enhanced peak and was more persistent in mutant cells, but the dephosphorylation process was not different between the two genotypes. The luciferase assay using calvaria cells transfected with the Smad1 mutated as a constitutive active form showed increased transcriptional activity at similar levels between the genotypes. The expression of BMP receptors was not different between the genotypes. BMP-induced phosphorylation of Smad1/5 was robustly decreased in wild type cells, but not in mutant cells, by pretreatment with DB867, an inhibitor of methyltransferase of inhibitory Smad6. Furthermore, BMP-induced translocation of Smad6 from nucleus to cytosol was not much observed in PRIP-deficient cells. These results indicate that PRIP is implicated in BMP-induced osteoblast differentiation by the negative regulation of Smad phosphorylation, through the methylation of inhibitory Smad6.

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.

Sphingosine-1-phosphate inhibits differentiation of C3H10T1/2 cells into adipocyte.

Mesenchymal stem cells (MSCs) can differentiate into a number of cell types, including adipocytes and osteoblasts. MSC differentiation into adipocytes inhibits osteogenic differentiation and vice versa. Therefore, understanding the mechanisms of MSC differentiation at the signaling level can lead to the development of novel therapeutic strategies toward tissue regeneration. Sphingosine-1-phosphate (S1P) is a signaling molecule that regulates many cellular responses, including cellular differentiation. However, the effects of S1P on MSC differentiation are largely unknown. The purpose of study was to investigate whether S1P drives MSCs toward either adipogenic or osteogenic differentiation, and if so, to clarify the underlying signaling mechanisms for such differentiation. We found that S1P inhibited adipogenic differentiation of C3H10T1/2 multipotent stem cells, while promoting their osteogenic differentiation. During adipogenic differentiation, S1P suppressed the cAMP accumulation in a Gi-protein-dependent manner. The Gi-dependent S1P signaling suppressed C/EBPß expression, which is essential for adipogenic differentiation. Furthermore, S1P did not affect cAMP-independent adipogenic differentiation. These findings suggest that S1P suppresses cAMP accumulation, leading to inhibition of C/EBPß expression, thereby resulting in decreased adipogenic differentiation of C3H10T1/2 cells. Thus, our findings provide novel molecular mechanisms as regards how S1P inhibits adipogenic differentiation of C3H10T1/2 cells, indicating a potential beneficial role for regeneration and repair of tissues.

PRIP (phospholipase C-related but catalytically inactive protein) inhibits exocytosis by direct interactions with syntaxin 1 and SNAP-25 through its C2 domain.

Membrane fusion for exocytosis is mediated by SNAREs, forming trans-ternary complexes to bridge vesicle and target membranes. There is an array of accessory proteins that directly interact with and regulate SNARE proteins. PRIP (phospholipase C-related but catalytically inactive protein) is likely one of these proteins; PRIP, consisting of multiple functional modules including pleckstrin homology and C2 domains, inhibited exocytosis, probably via the binding to membrane phosphoinositides through the pleckstrin homology domain. However, the roles of the C2 domain have not yet been investigated. In this study, we found that the C2 domain of PRIP directly interacts with syntaxin 1 and SNAP-25 but not with VAMP2. The C2 domain promoted PRIP to co-localize with syntaxin 1 and SNAP-25 in PC12 cells. The binding profile of the C2 domain to SNAP-25 was comparable with that of synaptotagmin I, and PRIP inhibited synaptotagmin I in binding to SNAP-25 and syntaxin 1. It was also shown that the C2 domain was required for PRIP to suppress SDS-resistant ternary SNARE complex formation and inhibit high K(+)-induced noradrenalin release from PC12 cells. These results suggest that PRIP inhibits regulated exocytosis through the interaction of its C2 domain with syntaxin 1 and SNAP-25, potentially competing with other SNARE-binding, C2 domain-containing accessory proteins such as synaptotagmin I and by directly inhibiting trans-SNARE complex formation.