Hydroxylation site-specific and production-dependent effects of endogenous oxysterols on cholesterol homeostasis: Implications for SREBP-2 and LXR.

The cholesterol metabolites, oxysterols, play central roles in cholesterol feedback control. They modulate the activity of two master transcription factors that control cholesterol homeostatic responses, sterol regulatory element-binding protein-2 (SREBP-2) and liver X receptor (LXR). Although the role of exogenous oxysterols in regulating these transcription factors has been well established, whether endogenously synthesized oxysterols similarly control both SREBP-2 and LXR remains poorly explored. Here, we carefully validate the role of oxysterols enzymatically synthesized within cells in cholesterol homeostatic responses. We first show that SREBP-2 responds more sensitively to exogenous oxysterols than LXR in Chinese hamster ovary cells and rat primary hepatocytes. We then show that 25-hydroxycholesterol (25-HC), 27-hydroxycholesterol, and 24S-hydroxycholesterol endogenously synthesized by CH25H, CYP27A1, and CYP46A1, respectively, suppress SREBP-2 activity at different degrees by stabilizing Insig (insulin-induced gene) proteins, whereas 7α-hydroxycholesterol has little impact on SREBP-2. These results demonstrate the role of site-specific hydroxylation of endogenous oxysterols. In contrast, the expression of CH25H, CYP46A1, CYP27A1, or CYP7A1 fails to induce LXR target gene expression. We also show the 25-HC production-dependent suppression of SREBP-2 using a tetracycline-inducible CH25H expression system. To induce 25-HC production physiologically, murine macrophages are stimulated with a Toll-like receptor 4 ligand, and its effect on SREBP-2 and LXR is examined. The results also suggest that de novo synthesis of 25-HC preferentially regulates SREBP-2 activity. Finally, we quantitatively determine the specificity of the four cholesterol hydroxylases in living cells. Based on our current findings, we conclude that endogenous side-chain oxysterols primarily regulate the activity of SREBP-2, not LXR.

HNF4α is required for Tkfc promoter activation by ChREBP.

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we show that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of two hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and one carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.

Lactic acid bacteria-derived γ-linolenic acid metabolites are PPARδ ligands that reduce lipid accumulation in human intestinal organoids.

Gut microbiota regulate physiological functions in various hosts, such as energy metabolism and immunity. Lactic acid bacteria, including Lactobacillus plantarum, have a specific polyunsaturated fatty acid saturation metabolism that generates multiple fatty acid species, such as hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and trans-fatty acids. How these bacterial metabolites impact host physiology is not fully understood. Here, we investigated the ligand activity of lactic acid bacteria-produced fatty acids in relation to nuclear hormone receptors expressed in the small intestine. Our reporter assays revealed two bacterial metabolites of γ-linolenic acid (GLA), 13-hydroxy-cis-6,cis-9-octadecadienoic acid (γHYD), and 13-oxo-cis-6,cis-9-octadecadienoic acid (γKetoD) activated peroxisome proliferator-activated receptor delta (PPARδ) more potently than GLA. We demonstrate that both γHYD and γKetoD bound directly to the ligand-binding domain of human PPARδ. A docking simulation indicated that four polar residues (T289, H323, H449, and Y473) of PPARδ donate hydrogen bonds to these fatty acids. Interestingly, T289 does not donate a hydrogen bond to GLA, suggesting that bacterial modification of GLA introducing hydroxy and oxo group determines ligand selectivity. In human intestinal organoids, we determined γHYD and γKetoD increased the expression of PPARδ target genes, enhanced fatty acid ß-oxidation, and reduced intracellular triglyceride accumulation. These findings suggest that γHYD and γKetoD, which gut lactic acid bacteria could generate, are naturally occurring PPARδ ligands in the intestinal tract and may improve lipid metabolism in the human intestine.

Identification of α-ionone, nootkatone, and their derivatives as TGR5 agonists.

TGR5 is a G-protein-coupled receptor that is activated by bile acids. The activation of TGR5 in brown adipose tissue (BAT) increases energy expenditure by increasing the expression level of thermogenesis-related genes, such as peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, uncoupling protein 1, and type II iodothyronine deiodinase. Therefore, TGR5 is a potential drug target in treating obesity and associated metabolic disorders. In this study, we identified the aroma compounds α-ionone and nootkatone as well as their derivatives as TGR5 agonists by using the luciferase reporter assay system. These compounds had little effect on the activity of the farnesoid X receptor, a nuclear receptor activated by bile acids. Mice fed 0.2% α-ionone containing high-fat diet (HFD) increased the thermogenesis-related gene expression level in BAT and suppressed weight gain compared with mice fed a normal HFD. These findings indicate that aromatic compounds with TGR5 agonist activity are promising chemicals to prevent obesity.

A decreased preoperative platelet-to-lymphocyte ratio, systemic immune-inflammation index, and pan-immune-inflammation value are associated with the poorer survival of patients with a stent inserted as a bridge to curative surgery for obstructive colorectal cancer.

PURPOSE: Inflammation is one of the hallmarks of cancer, and inflammation-based markers that are calculated easily from laboratory results have shown predictive abilities. We investigated the prognostic values of the preoperative platelet-to-lymphocyte ratio (PLR), systemic immune-inflammation index (SII), and pan-immune-inflammation value (PIV) in patients with non-metastatic obstructive colorectal cancer (OCRC) and a self-expandable metallic stent inserted as a bridge to curative surgery. METHODS: The subjects of this retrospective study were 86 patients with pathological stage I to III OCRC. We examined the associations of these biomarkers with short- and long-term outcomes. RESULTS: Multivariate analyses revealed that a preoperative PLR < 149, SII < 597, and PIV < 209 were independently associated with poorer relapse-free survival (RFS) (P = 0.007, P < 0.001, and P = 0.002, respectively) and that a PIV < 209 was independently associated with poorer cancer-specific survival (P = 0.030). A platelet count < 240 was significantly associated with worse RFS, whereas the lymphocyte count was not. Pre-stenting PLR < 221 was an independent poor prognostic factor for RFS (P = 0.045). CONCLUSION: This study showed that decreased preoperative PLR, SII, PIV, and pre-stenting PLR were associated with poorer RFS, contrary to the findings of most previous studies. Our results suggest that platelets and obstruction contributed primarily to the opposite relationships, which might provide new insight into the possible pathophysiology of platelet-tumor interactions generated in the OCRC environment.

Prognostic significance of Ishii's sarcopenia screening score for patients undergoing curative surgery for obstructive colorectal cancer after intraluminal decompression.

PURPOSE: Sarcopenia influences the short- and long-term outcomes of various medical conditions including malignancy. Ishii's screening test estimates the probability of sarcopenia based on a score calculated by three simple variables: age, grip strength, and calf circumference. We investigated the clinical significance of Ishii's score for patients with non-metastatic obstructive colorectal cancer (OCRC) who underwent curative surgery after intraluminal decompression. METHODS: Ishii's score was calculated in 79 patients with OCRC. Muscle volume loss and decreased muscle quality were evaluated by computed tomography (CT) images as skeletal muscle index (SMI) and intramuscular adipose tissue content (IMAC), respectively. RESULTS: There were 46 men and 33 women, with a median age of 70 years old. The cutoff value for Ishii's score was 155.1 and 15 patients were in the high-score group. The high-score group was significantly associated with worse time to recurrence (TTR) and overall survival (OS), and a high Ishii's score was an independent negative prognostic factor for TTR (hazard ratio = 2.93, P = 0.015). A high Ishii's score was significantly associated with a low SMI value but not with the IMAC value. CONCLUSION: A high Ishii's score was independently associated with poorer TTR in patients with non-metastatic OCRC.

Pathophysiological levels of GDF11 activate Smad2/Smad3 signaling and induce muscle atrophy in human iPSC-derived myocytes.

Skeletal muscle mass is negatively regulated by several TGF-ß superfamily members. Myostatin (MSTN) is the most prominent negative regulator of muscle mass. Recent studies show that in addition to MSTN, GDF11, which shares a high sequence identity with MSTN, induces muscle atrophy in vitro and in vivo at supraphysiological levels, whereas controversy regarding its roles exists. Furthermore, higher circulating GDF11 levels associate with frailty in humans. On the other hand, little is known about the effect of pathophysiological levels of GDF11 on muscle atrophy. Here we seek to determine whether pathophysiological levels of GDF11 are sufficient to activate Smad2/Smad3 signaling and induce muscle atrophy using human iPSC-derived myocytes (hiPSC myocytes). We first show that incubating hiPSC myocytes with pathophysiological concentrations of GDF11 significantly reduces myocyte diameters. We next demonstrate that pathophysiological levels of GDF11 are sufficient to activate Smad2/3 signaling. Finally, we show that pathophysiological levels of GDF11 are capable of inducing the expression of Atrogin-1, an atrophy-promoting E3 ubiquitin ligase and that FOXO1 blockage reverses the GDF11-induced Atrogin-1 expression and atrophic phenotype. Collectively, our results suggest that GDF11 induces skeletal muscle atrophy at the pathophysiological levels through the GDF11-FOXO1 axis.

Insulin-induced genes INSIG1 and INSIG2 mediate oxysterol-dependent activation of the PERK-eIF2α-ATF4 axis.

Insulin-induced genes (INSIGs) encode endoplasmic reticulum-resident proteins that regulate intracellular cholesterol metabolism. Oxysterols are oxygenated derivatives of cholesterol, some of which orchestrate lipid metabolism via interaction with INSIGs. Recently, it was reported that expression of activating transcription factor-4 (ATF4) was induced by certain oxysterols; the precise of mechanism is unclear. Herein, we show that INSIGs mediate ATF4 upregulation upon interaction with oxysterol. Oxysterols that possess a high affinity for INSIG, such as 27- and 25-hydroxycholesterol (25HC), markedly induced the increase of ATF4 protein when compared with other oxysterols. In addition, ATF4 upregulation by these oxysterols was attenuated in INSIG1/2-deficient Chinese hamster ovary cells and recovered by either INSIG1 or INSIG2 rescue. Mechanistic studies revealed that the binding of 25HC to INSIG is critical for increased ATF4 protein via activation of protein kinase RNA-activated-like ER kinase and eukaryotic translation initiation factor 2α. Knockout of INSIG1 or INSIG2 in human hepatoma Huh7 cells attenuated ATF4 protein upregulation, indicating that only one of the endogenous INSIGs, unlike overexpression of intrinsic INSIG1 or INSIG2, was insufficient for ATF4 induction. Furthermore, ATF4 proactively upregulated the cell death-inducible gene expression, such as Chop, Chac1, and Trb3, thereby markedly reducing cell viability with 25HC. These findings support a model whereby that INSIGs sense an increase in oxysterol in the endoplasmic reticulum and induce an increase of ATF4 protein via the protein kinase RNA-activated-like ER kinase-eukaryotic translation initiation factor 2α pathway, thereby promoting cell death.

Muscle-specific TGR5 overexpression improves glucose clearance in glucose-intolerant mice.

TGR5, a G protein-coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle-specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia.

Prognostic significance of the mean corpuscular volume (MCV) and red cell distribution width (RDW) in obstructive colorectal cancer patients with a stent inserted as a bridge to curative surgery.

PURPOSE: The prognostic significance of the mean corpuscular volume (MCV) and red cell distribution width (RDW) in patients with malignancy have not been intensely investigated and are largely overlooked. We, therefore, investigated the clinical significance of MCV and RDW in non-metastatic obstructive colorectal cancer (OCRC) patients with a self-expandable metallic stent inserted as a bridge to curative surgery. METHODS: Eighty-five pathological stage II and III OCRC patients were retrospectively evaluated. The associations of the preoperative MCV and RDW values with short- and long-term outcomes were examined. RESULTS: There were 50 males and 35 females, and the median age was 71 years old. The median interval between stenting and surgery was 17 days, and the median postoperative hospital stay was 16 days. Fifty-six patients were in the MCV ≥ 87 group, and 47 were in the RDW ≥ 13.8 group. Multivariate analyses revealed the MCV ≥ 87 status to be independently associated with a poor relapse-free survival (hazard ratio [HR] = 4.70, 95% confidence interval [CI] 1.52-14.58, P = 0.007). The RDW ≥ 13.8% was an independent predictor of postoperative infectious complications (HR = 7.28, 95% CI 1.24-42.70, P = 0.028). CONCLUSION: The MCV and RDW are simple but strong predictors of postoperative outcomes in OCRC patients.

A longer interval after stenting compromises the short- and long-term outcomes after curative surgery for obstructive colorectal cancer.

PURPOSE: Intestinal decompression using self-expandable metallic colonic stents (SEMSs) as a bridge to surgery is now considered an attractive alternative to emergency surgery. However, data regarding the optimal timing of surgery after stenting are limited. METHODS: We investigated the impact of the interval between stenting and surgery on short- and long-term outcomes in 92 obstructive colorectal cancer (OCRC) patients who had a SEMS inserted and subsequently received curative surgery. RESULTS: The median age of the patients was 70.5 years, and the median interval between SEMS insertion and the surgery was 17 (range 5-47) days. There were 35 postoperative complications, including seven major postoperative complications. An interval of more than 16 days was an independent predictor of a poor relapse-free survival (hazard ratio [HR] = 3.12, 95% confidence interval [CI] 1.24-7.81, p = 0.015). An interval of more than 35 days was independently associated with major postoperative complications (HR = 16.6, 95% CI 2.21-125, p = 0.006). CONCLUSION: A longer interval between stenting and surgery significantly compromised the short- and long-term outcomes. Surgery within 16 days after stenting might help maximize the benefit of SEMS without interfering with short- and long-term outcomes.

Ring finger protein 5 activates sterol regulatory element-binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP.

Sterol regulatory element-binding protein 2 (SREBP2) is the master transcription factor that regulates cholesterol metabolism. SREBP2 activation is regulated by SREBP chaperone SCAP. Here we show that ring finger protein 5 (RNF5), an endoplasmic reticulum-anchored E3 ubiquitin ligase, mediates the Lys-29-linked polyubiquitination of SCAP and thereby activates SREBP2. RNF5 knockdown inhibited SREBP2 activation and reduced cholesterol biosynthesis in human hepatoma cells, and RNF5 overexpression activated SREBP2. Mechanistic studies revealed that RNF5 binds to the transmembrane domain of SCAP and ubiquitinates the Lys-305 located in cytosolic loop 2 of SCAP. Moreover, the RNF5-mediated ubiquitination enhanced an interaction between SCAP luminal loop 1 and loop 7, a crucial event for SREBP2 activation. Notably, an overexpressed K305R SCAP variant failed to restore the SREBP2 pathway in SCAP-deficient cell lines. These findings define a new mechanism by which an ubiquitination-induced SCAP conformational change regulates cholesterol biosynthesis.

MIG12 is involved in the LXR activation-mediated induction of the polymerization of mammalian acetyl-CoA carboxylase.

Liver X receptors (LXR) α and ß are a family of nuclear receptors that regulate lipogenesis by controlling the expression of the genes involved in the synthesis of fatty acids. MID1IP1, which encodes MIG12, is a target gene of LXR. MIG12 induces fatty acid synthesis by stimulating the polymerization-mediated activation of acetyl-CoA carboxylase (ACC). Here, we show that LXR's activation stimulates ACC polymerization in HepG2 cells by increasing the expression of MIG12. A knockdown of MID1IP1 abrogated the stimulation completely. The mutations of MIG12's leucine-zipper domain reduced the interaction between MIG12 and ACC, thus decreasing the MIG12's capacity to stimulate ACC polymerization. These results indicate that LXR's activation stimulates lipogenesis not only through the induction of the genes encoding lipogenic enzymes but also through MIG12's stimulation of ACC polymerization.

Polymethoxyflavones in orange peel extract prevent skeletal muscle damage induced by eccentric exercise in rats.

Polymethoxyflavones (PMFs) contained in the peel of citrus fruits have anti-inflammatory, anticancer, and antidepressant effects. However, their effects on skeletal muscle are unknown. We investigated whether PMFs could prevent skeletal muscle damage induced by eccentric exercise in rats. Downhill running for 90 min increased the levels of the inflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), and interleukin-1ß (IL-1ß) in skeletal muscles, especially in vastus lateralis, and the plasma creatine kinase levels. These increases were attenuated by a single oral administration of orange peel extract (OPE) 30 min before downhill running. A mixture of nobiletin, sinensetin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeretin, which are the major PMFs of OPE, also showed similar effects on muscle damage. These results suggest that OPE has a protective effect against eccentric exercise-induced skeletal muscle damage, and that the effects may be attributed to the 4 major PMFs.

Orange peel extract reduces the inflammatory state of skeletal muscle after downhill running via an increase in IL-1RA.

It has been reported that orange peel extract (OPE) and the 4 major polymethoxyflavones (PMFs) in OPE have a protective effect against downhill running (DR)-induced skeletal muscle inflammation. However, the mechanism is not well understood. We investigated the potential of OPE and PMF compounds for increasing anti-inflammatory cytokine levels. The plasma interleukin-1 receptor antagonist (IL-1RA) level was increased 1 and 8 h after OPE administration in rats. Nobiletin induced the secretion of IL-1RA from C2C12 myotubes. In the inflammatory state of skeletal muscle after DR, OPE administration reduced nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) expression, NF-κB-DNA binding, and monocyte chemotactic protein-1 mRNA levels, but these effects were all abrogated by the intravenous administration of IL-1RA neutralizing antibody. These results indicated that OPE reduces skeletal muscle inflammatory state after DR via an increase in IL-1RA, and that IL-1 receptor signaling is important for skeletal muscle inflammation after DR.

Maslinic acid activates mTORC1 and human TGR5 and induces skeletal muscle hypertrophy.

Maslinic acid, a naturally occurring pentacyclic triterpene in more than 30 plants (including olives), reportedly increases human muscle mass and muscle strength; however, the underlying molecular mechanism remains unknown. C57BL/6J mice were fed a standard diet or supplemented with 0.27% maslinic acid for 4 weeks, and their skeletal muscle mass was measured. Mice that consumed maslinic acid displayed significant increases in gastrocnemius and soleus muscle mass. Cultured mouse-C2C12 skeletal muscle cells were treated with mammalian target of rapamycin complex 1 (mTORC1) or protein kinase b (Akt) inhibitor, and protein synthesis was quantified. Maslinic acid accelerated protein synthesis via mTORC1 activation independent of Akt. Furthermore, maslinic acid activated human Takeda G protein-coupled receptor 5 (TGR5) more strongly than mouse TGR5, augmenting the expression of several genes related to muscular hypertrophy. Maslinic acid activated mTORC1 and human TGR5, implying its contribution to human muscular hypertrophy through these effects.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids.

Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

The Controlling Nutritional Status (CONUT) Score as a prognostic factor for obstructive colorectal cancer patients received stenting as a bridge to curative surgery.

PURPOSE: The Controlling Nutritional Status (CONUT) Score, originally developed as a nutritional screening tool, is a cumulative score calculated from the serum albumin level, total cholesterol level, and total lymphocyte count. Previous studies have demonstrated that the score has significant prognostic value in various malignancies. We investigated the relationship between the CONUT score and long-term survival in obstructive colorectal cancer (OCRC) patients who underwent self-expandable metallic colonic stent placement and subsequently received curative surgery. METHODS: We retrospectively analyzed 57 pathological stage II and III OCRC patients between 2013 and 2019. The associations between the preoperative CONUT score and clinicopathological factors and patient survival were evaluated. RESULTS: A receiver operating characteristic curve analysis revealed that the optimal cut-off value for the CONUT score was 7. A CONUT score of ≥ 7 was significantly associated with elevated CA19-9 level (p = 0.03). Multivariate analyses revealed that a CONUT score of ≥ 7 was independently associated with cancer-specific survival (hazard ratio [HR] = 10.2, 95% confidence interval [CI] 1.2-85.9, p = 0.03) and disease-free survival (HR = 7.1, 95% CI 2.3-21.7, p = 0.0006). CONCLUSION: The results demonstrated that the CONUT score was a potent prognostic indicator. Evaluating the CONUT score might result in more precise patient assessment and tailored treatment.

[Study of Laparoscopic Surgery for Elderly Patients with Colorectal Cancer].

The number of elderly patients and colorectal cancer patients is increasing, so laparoscopic surgery for colorectal cancer in elderly patients is suspected to increase. In 456 patients who underwent laparoscopic surgery for colorectal cancer, we investigated whether laparoscopic surgery for elderly patients with colon cancer patients could be performed equally compared to non-elderly patients. Preoperative ASA-PS was slightly poorer in elderly patients. There was no significant difference in pStage. The 5-year overall survival rate was lower in the elderly, but there were no significant differences in blood loss, operation time, postoperative hospital stays and incidence of complications of Clavien-Dindo classification grade 3 or higher. It was suggested that laparoscopic surgery for elderly patients with colorectal cancer may be safely performed compared with non-elderly patients.

Bridging molecules are secreted from the skeletal muscle and potentially regulate muscle differentiation.

Muscle myogenesis is an essential step for muscle development and recovery. During muscle fusion, multiple molecules are thought to be necessary for the formation of normal myotubes. Milk fat globule-EGF factor 8 (MFG-E8) and Gas6 are phosphatidylserine-recognizing bridging molecules that are secreted mainly from immune cells. In this study, we confirmed that these molecules are expressed and secreted from C2C12 cells. Mouse muscle and satellite cells also expressed these molecules. MFG-E8 was highly expressed and secreted in both undifferentiated and differentiated C2C12 cells. We observed that MFG-E8 and Gas6 were bound to the surface of differentiated C2C12 cells more compared with undifferentiated cells. Additionally, the treatment of recombinant MFG-E8 upregulated expression of myogenic genes and suppressed apoptosis during myogenesis in C2C12 cells. In this paper, we discuss the presence of novel functional molecules expressed and secreted in the skeletal muscle. The results of this study suggest that bridging molecules are one of the determinants of myogenesis or other muscle responses.