Glucosylceramide synthase regulates adipo-osteogenic differentiation through synergistic activation of PPARγ with GlcCer.

Dysregulation of the adipo-osteogenic differentiation balance of mesenchymal stem cells (MSCs), which are common progenitor cells of adipocytes and osteoblasts, has been associated with many pathophysiologic diseases, such as obesity, osteopenia, and osteoporosis. Growing evidence suggests that lipid metabolism is crucial for maintaining stem cell homeostasis and cell differentiation; however, the detailed underlying mechanisms are largely unknown. Here, we demonstrate that glucosylceramide (GlcCer) and its synthase, glucosylceramide synthase (GCS), are key determinants of MSC differentiation into adipocytes or osteoblasts. GCS expression was increased during adipogenesis and decreased during osteogenesis. Targeting GCS using RNA interference or a chemical inhibitor enhanced osteogenesis and inhibited adipogenesis by controlling the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ). Treatment with GlcCer sufficiently rescued adipogenesis and inhibited osteogenesis in GCS knockdown MSCs. Mechanistically, GlcCer interacted directly with PPARγ through A/B domain and synergistically enhanced rosiglitazone-induced PPARγ activation without changing PPARγ expression, thereby treatment with exogenous GlcCer increased adipogenesis and inhibited osteogenesis. Animal studies demonstrated that inhibiting GCS reduced adipocyte formation in white adipose tissues under normal chow diet and high-fat diet feeding and accelerated bone repair in a calvarial defect model. Taken together, our findings identify a novel lipid metabolic regulator for the control of MSC differentiation and may have important therapeutic implications.

Propyl gallate inhibits adipogenesis by stimulating extracellular signal-related kinases in human adipose tissue-derived mesenchymal stem cells.

Propyl gallate (PG) used as an additive in various foods has antioxidant and anti-inflammatory effects. Although the functional roles of PG in various cell types are well characterized, it is unknown whether PG has effect on stem cell differentiation. In this study, we demonstrated that PG could inhibit adipogenic differentiation in human adipose tissue-derived mesenchymal stem cells (hAMSCs) by decreasing the accumulation of intracellular lipid droplets. In addition, PG significantly reduced the expression of adipocyte-specific markers including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein 2 (aP2). PG inhibited adipogenesis in hAMSCs through extracellular regulated kinase (ERK) pathway. Decreased adipogenesis following PG treatment was recovered in response to ERK blocking. Taken together, these results suggest a novel effect of PG on adipocyte differentiation in hAMSCs, supporting a negative role of ERK1/2 pathway in adipogenic differentiation.

Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

An activator of the cAMP/PKA/CREB pathway promotes osteogenesis from human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulation of MSCs differentiation may be a useful tool for regenerative medicine and cell-based therapy. The discovery of small molecule that activates the osteogenic differentiation of MSCs could aid in the development of a new anabolic drug for osteoporosis treatment. We identified CW008, a derivative of pyrazole-pyridine, that stimulates osteoblast differentiation of human MSCs and increases bone formation in ovariectomized mice. CW008 promotes osteogenesis by activating cAMP/PKA/CREB signaling pathway and inhibiting leptin secretion. These results suggest that CW008 is an agonist of cAMP/PKA/CREB pathway in osteogenic differentiation and that application of CW008 may be useful for the treatment of bone-related diseases and for the study of bone biology.

DJ-1 promotes angiogenesis and osteogenesis by activating FGF receptor-1 signaling.

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.

Wedelolactone inhibits adipogenesis through the ERK pathway in human adipose tissue-derived mesenchymal stem cells.

Wedelolactone is an herbal medicine that is used to treat septic shock, hepatitis and venom poisoning. Although in differentiated and cancer cells, wedelolactone has been identified as anti-inflammatory, growth inhibitory, and pro-apoptotic, the effects of wedelolactone on stem cell differentiation remain largely unknown. Here, we report that wedelolactone inhibits the adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Wedelolactone reduced the formation of lipid droplets and the expression of adipogenesis-related proteins, such as CCAAT enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein aP2 (aP2). Wedelolactone mediated this process by sustaining ERK activity. In addition, inhibition of ERK activity with PD98059 resulted in reversion of the wedelolactone-mediated inhibition of adipogenic differentiation. Taken together, these results indicate that wedelolactone inhibits adipogenic differentiation through ERK pathway and suggest a novel inhibitory effect of wedelolactone on adipogenic differentiation in hAMSCs.

PDZ domain-containing 1 (PDZK1) protein regulates phospholipase C-ß3 (PLC-ß3)-specific activation of somatostatin by forming a ternary complex with PLC-ß3 and somatostatin receptors.

Phospholipase C-ß (PLC-ß) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-ß subtypes have different physiological functions despite their similar structures. Because the PLC-ß subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-ß3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-ß3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-ß3, but not PLC-ß1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-ß3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-ß3, but not that of PLC-ß1, significantly inhibited SST-induced intracellular Ca(2+) mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-ß3 is essential for the specific activation of PLC-ß3 and the subsequent physiologic responses by SST.

Human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

Activation of AMP-activated protein kinase is essential for lysophosphatidic acid-induced cell migration in ovarian cancer cells.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-ß3 (PLC-ß3) and calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPKα1, PLC-ß3, or (CaMKKß) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPKα1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer.

Phospholipase C-η1 is activated by intracellular Ca(2+) mobilization and enhances GPCRs/PLC/Ca(2+) signaling.

Phospholipase C-η1 (PLC-η1) is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is unclear. In the present study, we report for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC and Ca(2+) signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)-, bradykinin (BK)-, and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, we found that intracellular Ca(2+) source is enough for PLC-η1 activation. Furthermore, the IP(3) receptor inhibitor, 2-APB, inhibited LPA-induced PLC activity in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca(2+) mobilization in PLC-η1 activation. Finally, we found that LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown, and these knockdown effects were abolished by 2-APB. Taken together, our results strongly suggest that PLC-η1 is activated via intracellular Ca(2+) mobilization from the ER, and therefore amplifies GPCR-mediated signaling.

Subtype-specific roles of phospholipase C-ß via differential interactions with PDZ domain proteins.

Since we first identified the PLC-ß isozyme, enormous studies have been conducted to investigate the functional roles of this protein (Min et al., 1993; Suh et al.,1988). It is now well-known that the four PLC-ß subtypes are major effector molecules in GPCR-mediated signaling, especially for intracellular Ca2+ signaling. Nonetheless, it is still poorly understood why multiple PLC-ß subtype exist. Most cells express multiple subtypes of PLC-ß in different combinations, and each subtype is involved in somewhat different signaling pathways. Therefore, studying the differential roles of each PLC-ß subtype is a very interesting issue. In this regard, we focus here on PDZ domain proteins which are novel PLC-ß interacting proteins. As scaffolders, PDZ domain proteins recruit various target proteins ranging from membrane receptors to cytoskeletal proteins to assemble highly organized signaling complexes; this can give rise to efficiency and diversity in cellular signaling. Because PLC-ß subtypes have different PDZ-binding motifs, it is possible that they are engaged with different PDZ domain proteins, and in turn participate in distinct physiological responses. To date, several PDZ domain proteins, such as the NHERF family, Shank2, and Par-3, have been reported to selectively interact with certain PLC-ß subtypes and GPCRs. Systematic predictions of potential binding partners also suggests differential binding properties between PLC-ß subtypes. Furthermore, we elucidated parallel signaling processes for multiple PLC-ß subtypes, which still perform distinct functions resulting from differential interactions with PDZ domain proteins within a single cell. Therefore, these results highlight the novel function of PDZ domain proteins as intermediaries in subtype-specific role of PLC-ß in GPCR-mediated signaling. Future studies will focus on the physiological meanings of this signaling complex formation by different PDZ domain proteins and PLC-ß subtypes. It has been observed for a long time that the expression of certain PLC-ß subtype fluctuates during diverse physiological conditions. For example, the expression of PLC-ß1 is selectively increased during myoblast and adipocyte differentiation (Faenza et al., 2004; O'Carroll et al., 2009). Likewise, PLC-ß2 is highly up-regulated during breast cancer progression and plays a critical role in cell migration and mitosis (Bertagnolo et al., 2007). Although PLC-ß3 is selectively down-regulated in neuroendocrine tumors, the expression of PLC-ß1 is increased in small cell lung carcinoma (Stalberg et al., 2003; Strassheim et al., 2000). In our hypothetical model, it is most likely that up- and down regulation of certain PLC-ß subtypes are due to their selective coupling with specific GPCR-mediated signaling, implicated in these pathophysiologic conditions. Therefore, better understanding of selective coupling between PLC-ß subtypes, PDZ domain proteins, and GPCRs will shed light on new prognosis and therapy of diverse diseases, and provide potential targets for drug development.

Ochratoxin A inhibits adipogenesis through the extracellular signal-related kinases-peroxisome proliferator-activated receptor-γ pathway in human adipose tissue-derived mesenchymal stem cells.

Ochratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic, and apoptotic potential. Although the toxic effects of OTA in various cell types are well characterized, it is not known whether OTA has an effect on stem cell differentiation. In this study, we demonstrate that OTA inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells, as indicated by decreased accumulation of intracellular lipid droplets. Further, OTA significantly reduces expression of adipocyte-specific markers, including peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT enhancer binding protein-α (C/EBP-α), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2). At the molecular level, OTA phosphorylates PPAR-γ2 through extracellular signal-related kinase activation and inhibits PPAR-γ activity. We also found that treatment with the mitogen-activated protein kinase kinase inhibitor, PD98059, significantly blocked the OTA-induced inhibition of adipogenesis. These results indicate that OTA suppresses adipogenesis in an extracellular signal-related kinase-dependent manner. Taken together, our results suggest a novel effect of OTA on adipocyte differentiation in human adipose tissue-derived mesenchymal stem cells and the possibility that OTA might affect the differentiation of other types of stem cells.

Subtype-specific role of phospholipase C-beta in bradykinin and LPA signaling through differential binding of different PDZ scaffold proteins.

Among phospholipase C (PLC) isozymes (beta, gamma, delta, epsilon, zeta and eta), PLC-beta plays a key role in G-protein coupled receptor (GPCR)-mediated signaling. PLC-beta subtypes are often overlapped in their distribution, but have unique knock-out phenotypes in organism, suggesting that each subtype may have the different role even within the same type of cells. In this study, we examined the possibility of the differential coupling of each PLC-beta subtype to GPCRs, and explored the molecular mechanism underlying the specificity. Firstly, we found that PLC-beta1 and PLC-beta 3 are activated by bradykinin (BK) or lysophosphatidic acid (LPA), respectively. BK-triggered phosphoinositides hydrolysis and subsequent Ca(2+) mobilization were abolished specifically by PLC-beta1 silencing, whereas LPA-triggered events were by PLC-beta 3 silencing. Secondly, we showed the evidence that PDZ scaffold proteins is a key mediator for the selective coupling between PLC-beta subtype and GPCR. We found PAR-3 mediates physical interaction between PLC-beta1 and BK receptor, while NHERF2 does between PLC-beta 3 and LPA(2) receptor. Consistently, the silencing of PAR-3 or NHERF2 blunted PLC signaling induced by BK or LPA respectively. Taken together, these data suggest that each subtype of PLC-beta is selectively coupled to GPCR via PDZ scaffold proteins in given cell types and plays differential role in the signaling of various GPCRs.

Comparative analysis of the secretory proteome of human adipose stromal vascular fraction cells during adipogenesis.

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label-free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT-PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor-beta (TGF-beta) signaling changed during adipogenesis. The expressions of secreted frizzled-related proteins, dickkopf-related proteins, and latent TGF-beta-binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF-beta signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity-related metabolic diseases.

Lysophosphatidylserine regulates blood glucose by enhancing glucose transport in myotubes and adipocytes.

Lysophosphatidylserine (LPS) is known to have diverse cellular effects, but although LPS is present in many biological fluids, its in vivo effects have not been elucidated. In the present study, we investigated the effects of LPS on glucose metabolism in vivo, and how skeletal muscle cells respond to LPS stimulation. LPS enhanced glucose uptake in a dose- and time-dependent manner in L6 GLUT4myc myotubes, and this effect of LPS on glucose uptake was mediated by a Galpha(i) and PI 3-kinase dependent signal pathway. LPS increased the level of GLUT4 on the cell surface of L6 GLUT4myc myotubes, and enhanced glucose uptake in 3T3-L1 adipocytes. In line with its cellular functions, LPS lowered blood glucose levels in normal mice, while leaving insulin secretion unaffected. LPS also had a glucose-lowering effect in STZ-treated type 1 diabetic mice and in obese db/db type 2 diabetic mice. This study shows that LPS-stimulated glucose transport both in skeletal muscle cells and adipocytes, and significantly lowered blood glucose levels both in type 1 and 2 diabetic mice. Our results suggest that LPS is involved in the regulation of glucose homeostasis in skeletal muscle and adipose tissue.

Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways.

The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca(2+) mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference-mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca(2+) mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/G(i)/phospholipase C/Ca(2+) signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.

A myristoylated pseudosubstrate peptide of PKC-zeta induces degranulation in HMC-1 cells independently of PKC-zeta activity.

Mast cells play a central role in allergic disease and host defense against several pathogens through the release of various bioactive compounds via degranulation. In this study, we found that a myristoylated pseudosubstrate of PKC-zeta (zeta-PS; myristoyl-SIYRRGARRWRKL, a PKC-zeta inhibitor) regulates mast cell degranulation. zeta-PS increased [Ca+2]i level at nanomolar concentrations in a PKC-zeta activity-independent manner in HMC-1 cells. Moreover, zeta-PS-induced [Ca+2]i generation was completely abrogated by phospholipase C (PLC), IP3 receptor or Galpha i/o inhibitor and zeta-PS potently induced degranulation in HMC-1 cells which was significantly inhibited by pretreating PLC inhibitors or a calcium chelator. Therefore, our results suggest that zeta-PS can induce degranulation in HMC-1 cells by triggering the calcium signal via a PKC-zeta-independent but Galpha i/o, PLC and IP3-dependent pathways.

Lysophosphatidic acid regulates blood glucose by stimulating myotube and adipocyte glucose uptake.

Lysophosphatidic acid (LPA) is known to have diverse cellular effects, but although LPA is present in many biological fluids, including blood, its effects on glucose metabolism have not been elucidated. In this study, we investigated whether LPA stimulation is related to glucose regulation. LPA was found to enhance glucose uptake in a dose-dependent manner both in L6 GLUT4myc myotubes and 3T3-L1 adipocytes by triggering GLUT4 translocation to the plasma membrane. Moreover, the effect of LPA on glucose uptake was completely inhibited by pretreating both cells with LPA receptor antagonist Ki16425 and Gi inhibitor pertussis toxin. In addition, LPA increased the phosphorylation of AKT-1 with no effects on IRS-1, and LPA-induced glucose uptake was abrogated by pretreatment with the PI 3-kinase inhibitor LY294002. When low concentration of insulin and LPA were treated simultaneously, an additive effect on glucose uptake was observed in both cell types. In line with its cellular functions, LPA significantly lowered blood glucose levels in normal mice but did not affect insulin secretion. LPA also had a glucose-lowering effect in streptozotocin-treated type 1 diabetic mice. In combination, these results suggest that LPA is involved in the regulation of glucose homeostasis in muscle and adipose tissues.

O-GlcNAc modification modulates the expression of osteocalcin via OSE2 and Runx2.

O-Linked beta-N-acetylglucosamine (O-GlcNAc) modification, a reversible post-translational modification, has been implicated in the regulation of protein stability, subcellular localization of proteins and protein-protein interaction. Here, we demonstrate that O-GlcNAc modification regulates the expression of osteocalcin, an osteoblast-specific marker, via Runx2 transcriptional activity in osteoblastic differentiation. Protein-associated O-GlcNAc was increased during osteoblastic differentiation in MC3T3-E1 preosteoblasts. In addition, PUGNAc, an inhibitor of O-GlcNAcase, potentiated the expression of osteocalcin caused by ascorbic acid, parathyroid hormone (PTH) and forskolin. By conducting activity assays of the osteocalcin promoter and transcription factor, we found that the OSE2 site in the osteocalcin promoter and Runx2 were important for increased osteocalcin promoter activity by PUGNAc. Furthermore, PUGNAc led to increased O-GlcNAc modification of Runx2, which regulated the transcription of its target gene osteocalcin. Thus, these data provide evidence that O-GlcNAc modification may be a new mode of osteoblastic differentiation regulation.

Inhibition of phospholipase C-beta1-mediated signaling by O-GlcNAc modification.

Here we report inhibition of phospholipase C-beta1 (PLC-beta1)-mediated signaling by post-translational glycosylation with beta-N-acetylglucosamine (O-GlcNAc modification). In C2C12 myoblasts, isoform-specific knock-down experiments using siRNA showed that activation of bradykinin (BK) receptor led to stimulation of PLC-beta1 and subsequent intracellular Ca2+ mobilization. In C2C12 myotubes, O-GlcNAc modification of PLC-beta1 was markedly enhanced in response to treatment with glucosamine (GlcNH2), an inhibitor of O-GlcNAase (PUGNAc) and hyperglycemia. This was associated with more than 50% inhibition of intracellular production of IP3 and Ca2+ mobilization in response to BK. Since the abundance of PLC-beta1 remained unchanged, these data suggest that O-GlcNAc modification of PLC-beta1 led to inhibition of its activity. Moreover, glucose uptake stimulated by BK was significantly blunted by treatment with PUGNAc. These data support the notion that O-GlcNAc modification negatively modulates the activity of PLC-beta1.