The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism.

Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.

Rantes secreted from macrophages disturbs skeletal muscle regeneration after cardiotoxin injection in Cbl-b-deficient mice.

Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages.

YKL-40 secreted from adipose tissue inhibits degradation of type I collagen.

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.

Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes.

Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.

Reduced carnitine level causes death from hypoglycemia: possible involvement of suppression of hypothalamic orexin expression during weaning period.

The mechanism of onset of hypoglycemia in patients with carnitine deficiency has yet to be determined. Using mice with systemic carnitine deficiency (JVS mice), we examined this mechanism, focusing on the weaning period (days 14-28 postpartum). For normal mice, the survival rate was 100%, and no hypoglycemia was observed at all. Gastric lactose began to decrease on day 17, and cellulose increased sharply in amount thereafter. For JVS mice, the survival rate was 77% on day 14 and 28% on day 28. From day 21 on, hypoglycemia was noted. Gastric lactose had disappeared almost completely by day 17, and cellulose was almost undetectable from days 14 to 28. Expression of orexin mRNA in the hypothalamus did not differ between JVS and normal mice on day 14, but was suppressed in JVS mice on days 21 and 28. When JVS mice were fed a carnitine-rich diet, suppression of expression of orexin mRNA in hypothalamus was eliminated, and on day 28 lactose and cellulose were detected in the stomach without hypoglycemia. In conclusion, the suppression of the expression of orexin in the hypothalamus during the weaning period may be involved in the marked anorexia in JVS mice, which eventually leads to death from hypoglycemia.

Intermedilysin is essential for the invasion of hepatoma HepG2 cells by Streptococcus intermedius.

Streptococcus intermedius causes endogenous infections leading to abscesses. This species produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed ily knockout mutant UNS38 B3 and complementation strain UNS38 B3R1 in order to investigate the role of ILY in deep-seated infections. Strain UNS38 reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A. Isogenic mutant strain UNS38 B3 was not cytotoxic in either cell line. Quantification of S. intermedius revealed that in infected HepG2 cells UNS38 but not UNS38 B3 increased intracellularly concomitantly with increasing cell damage. This difference between UNS38 and UNS38 B3 was not observed with UNS38 B3R1. Invasion and proliferation in BRL3A cells was not observed. Masking UNS38 or UNS38 B3R1 with ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating strain UNS38 B3 with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. At 1 day post-infection, many intact UNS38 were detected in the damaged phagosomes of HepG2 with bacterial proliferation observed in the cytoplasm of dead HepG2 after an additional 2 day incubation. These results indicate that surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium and that secretion of ILY within host cells is essential for subsequent host cell death. These data strongly implicate ILY as an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

Bridge-linked bis-quaternary ammonium anti-microbial agents: relationship between cytotoxicity and anti-bacterial activity of 5,5'-[2,2'-(tetramethylenedicarbonyldioxy)-diethyl]bis(3-alkyl-4-methylthiazonium iodide)s.

We examined the correlation between the anti-bacterial activity against Escherichia coli and the cytotoxicity of five synthesized bridge types of bis-quaternary ammonium compounds (bis-QACs) as follows: thioether type, 4,4'-(p-xylydithio)bis(1-octylpyridinium iodide) (4DTBP-X,8); amide type, N,N'-tetramethylenebis(1-dodecyl-4-carbamoylpyridinium iodide) (4BCAP-4,12), N,N'-(phenylene)bis(1-octyl-4-carbamoylpyridinium iodide) (4BCAP-P,8); anti-amide type, 4,4'-(tetramethylenedicarbonyldiamino)bis(1-octylpyridinium iodide) (4DCABP-4,8), 4,4'-(phenylenedicarbonyldiamino)bis(1-octylpyridinium iodide) (4DCABP-P,8); ester type, 4,4'-(1,6-hexamethylenedioxydicarbonyl)bis(1-dodecylpyridinium iodide) (4DOCBP-6,12); and an anti-ester type, 5,5'-[2,2'-(tetramethylenedicarbonyldioxy)diethyl]bis(3-alkyl-4-methylthiazolium iodide) (5DEBT-4,n, The letter n indicates the carbon number of the alkyl group). 5DEBT-4,8 showed low cytotoxicity (LD(50)) to human erythrocytes (97+/-6microM) and the NB1RGB cell line (111+/-20microM) and remarkable anti-bacterial activity (MIC) toward E. coli K12 W3110 (7.9microM). Moreover, 5DEBT-4,8 indicated 1144 conformers by global minimum analysis and had two minimum dGW (solvation free energy) points as well as 4DTBP-6,8, which had been previously examined and concluded to be a significant useful anti-bacterial compound.

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

Intermedilysin is a pore-forming cytolysin belonging to the streptolysin O gene family known as the 'Cholesterol-binding/dependent cytolysins' and is unique within the family in that it is highly humanspecific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome-embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome-binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human-specific step of intermedilysin action, that the host cell membrane-binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human-specific action step and the cell-binding site of domain 4 lies within the 56 amino acids of the C-terminal, excluding the undecapeptide region.

Analysis of structural features of bis-quaternary ammonium antimicrobial agents 4,4'-(alpha,omega-polymethylenedithio)bis (1-alkylpyridinium iodide)s using computational simulation.

The bis-quaternary ammonium compounds (QACs) consisted of two identical alkylpyridinium rings and a bridge structure linking the rings to each other. The QACs have a methylene bridge except for 4DCABP-P,12 which has a phenyl ring as a bridge. These bis-QACs are as follows; amide type: N,N'-tetramethylenebis(1-dodecyl-4-carbamoylpyridinium iodide) (4BCAP-4,12), N,N'-hexamethylenebis(1-decyl-4-carbamoylpyridinium iodide) (4BCAP-6,10), anti-amide type: 4,4'-(1,4-tetramethylenedicarbonyldiamine)bis(1-decylpyridinium iodide) (4DCABP-4,10), 4,4'-(1,4-tetramethylenedicarbonyldiamine)bis (1-dodecylpyridinium iodide) (4DCABP-4,12), 4,4'-(1,4-phenyldicarbonyldiamine)bis(1-dodecylpyridinium iodide) (4DCABP-P,12), ester type: 4,4'-(1,6-hexamethylenedioxydicarbonyl)bis(1-dodecylpyridinium iodide) (4DOCBP-6,12), thioether type: 4,4'-(1,6-hexamethylenedithio)bis(1-octylpyridinium iodide) (4DTBP-6,8). From the investigation of the relationship between the median lethal dose (LD(50)) and the minimum inhibitory concentration (MIC) of these compounds, 4DTBP-6,8 as a disinfectant, seems to be very safe for human cells. The global minimum of 4DTBP-6,8 were searched and 1125 conformers obtained. The solvation free energy (dGW) of nine samples, which were extracted from these 1125 conformers, was calculated and two minimum points of dGW were observed. In the conformer-energy analysis of four types of model bridge-molecule, the thioether type bridge indicated a gradual energy increment, while the other three (amide, anti-amide, ester) types indicated an energy jump point in their profiles. Then we considered that the delicate balance between hydrophobicity and structural feature in the bridge-region of 4DTBP-6,8 molecule seemed to be related to its safety antibacterial activity.