Regulation of SREBPs by Sphingomyelin in Adipocytes via a Caveolin and Ras-ERK-MAPK-CREB Signaling Pathway.

Sterol response element binding protein (SREBP) is a key transcription factor in insulin and glucose metabolism. We previously demonstrated that elevated levels of membrane sphingomyelin (SM) were related to peroxisome proliferator-activated receptor-γ (PPARγ), which is a known target gene of SREBP-1 in adipocytes. However, the role of SM in SREBP expression in adipocytes remains unknown. In human abdominal adipose tissue from obese women with various concentrations of fasting plasma insulin, SREBP-1 proteins decreased in parallel with increases in membrane SM levels. An inverse correlation was found between the membrane SM content and the levels of SREBP-1c/ERK/Ras/PPARγ/CREB proteins. For the first time, we demonstrate the effects of SM and its signaling pathway in 3T3-F442A adipocytes. These cells were enriched or unenriched with SM in a range of concentrations similar to those observed in obese subjects by adding exogenous natural SMs (having different acyl chain lengths) or by inhibiting neutral sphingomyelinase. SM accumulated in caveolae of the plasma membrane within 24 h and then in the intracellular space. SM enrichment decreased SREBP-1 through the inhibition of extracellular signal-regulated protein kinase (ERK) but not JNK or p38 mitogen-activated protein kinase (MAPK). Ras/Raf-1/MEK1/2 and KSR proteins, which are upstream mediators of ERK, were down-regulated, whereas SREBP-2/caveolin and cholesterol were up-regulated. In SM-unmodulated adipocytes treated with DL-1-Phenyl-2-Palmitoylamino-3-morpholino-1-propanol (PPMP), where the ceramide level increased, the expression levels of SREBPs and ERK were modulated in an opposite direction relative to the SM-enriched cells. SM inhibited the insulin-induced expression of SREBP-1. Rosiglitazone, which is an anti-diabetic agent and potent activator of PPARγ, reversed the effects of SM on SREBP-1, PPARγ and CREB. Taken together, these findings provide novel insights indicating that excess membrane SM might be critical for regulating SREBPs in adipocytes via a MAPK-dependent pathway.

Functional differences in Leuconostoc sensitive and resistant strains to mesenterocin 52A, a class IIa bacteriocin.

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR52. The interaction of Mes 52A with bacterial membranes of sensitive, resistant and insensitive Leuconostoc strains has been investigated. The degree of insertion of Mes 52A on the phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane, and the consequence for K(+) efflux and proton motive force was analyzed. Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH in the membrane of the sensitive strain L. mesenteroides ssp. mesenteroides LMA 7, indicating that Mes 52A inserts into the cytoplasmic membrane of this sensitive strain. This insertion leads to K(+) efflux, without perturbation of DeltapH and a weak modification of DeltaPsi, and is consistent with pore formation. With the high-level resistant strain L. mesenteroides ssp. mesenteroides LMA 7AR, or with the insensitive strain Leuconostoc citreum CIP 103405, no modification of TMA-DPH or DPH anisotropy occurred, even in the presence of high Mes 52A levels. The membrane potential was not modified and no K(+) efflux was detected. There is a clear correlation between the physico-chemical characteristics of the membrane, the degree of Mes 52A penetration, the mechanism of action and the resistance or insensitivity characteristic of the target strains.

Fluorescence anisotropy analysis of the mechanism of action of mesenterocin 52A: speculations on antimicrobial mechanism.

Mesenterocin 52A (Mes 52A) is a class IIa bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides FR52, active against Listeria sp. The interaction of Mes 52A with bacterial membranes of two sensitive Listeria strains has been investigated. The Microbial Adhesion to Solvents test used to study the physico-chemical properties of the surface of the two strains indicated that both surfaces were rather hydrophilic and bipolar. The degree of insertion of Mes 52A in phospholipid bilayer was studied by fluorescence anisotropy measurements using two probes, 1-(4-trimethylammonium)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and DPH, located at different positions in the membrane. TMA-DPH reflects the fluidity at the membrane surface and DPH of the heart. With Listeria ivanovii CIP 12510, Mes 52A induced an increase only in the TMA-DPH fluorescence anisotropy, indicating that this bacteriocin affects the membrane surface without penetration into the hydrophobic core of the membrane. No significant K(+) efflux was measured, whereas the Delta Psi component of the membrane potential was greatly affected. With Listeria innocua CIP 12511, Mes 52A caused an increase in the fluorescence of TMA-DPH and DPH, indicating that this peptide inserts deeply in the cytoplasmic membrane of this sensitive strain. This insertion led to K(+) efflux, without perturbation of Delta pH and a weak modification of Delta Psi, and is consistent with pore formation. These data indicate that Mes 52A interacts at different positions of the membrane, with or without pore formation, suggesting two different mechanisms of action for Mes 52A depending on the target strain.

Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes.

Sphingomyelin pathway has been linked with insulin signaling through insulin-dependent GLUT-4 glucose transporter, but a relationship between sphingomyelin and the GLUT-1 transporter responsible for the basal (insulin-independent) glucose transport has not been clearly established. As GLUT-1 is mainly distributed to the cell surface, we explored the effects of changes in membrane sphingomyelin content on glucose transport through GLUT-1. The addition of exogenous sphingomyelin or glutathione (an inhibitor of endogenous sphingomyelinase) to the culture medium increased membrane sphingomyelin and cholesterol contents. Basal glucose uptake was enhanced and positively correlated to sphingomyelin (SM), cholesterol (CL) and SM/CL ratio. The exposure of 3T3-L1 preadipocytes to sphingomyelinase (SMase) significantly increased basal glucose uptake, membrane fluidity and decreased membrane sphingomyelin and cholesterol contents 60 min after SMase addition. There was no significant change in the abundance of GLUT-1 at the cell surface. The membrane sphingomyelin and cholesterol contents, fluidity and basal glucose transport returned to baseline levels within 2 h. The basal glucose uptake was negatively correlated with cholesterol contents and positively with SM/CL ratio. The SM/CL ratio might represent an important parameter controlling basal glucose uptake and a mechanism by which insulin resistance might be induced.

Erythrocyte membrane phospholipid composition is related to hyperinsulinemia in obese nondiabetic women: effects of weight loss.

The complex mechanisms by which obesity predisposes to insulin resistance are not clearly understood. According to a cell membrane hypothesis of insulin resistance, the defects in insulin action could be related to changes in membrane properties. The purpose of this work was to examine the relationship between 2 markers of insulin resistance (fasting plasma insulin [FPI] and homeostasis model assessment [HOMA IR]) and erythrocyte membrane lipid composition. In the first cross-sectional study, 24 premenopausal nondiabetic overweight women (body mass index [BMI], 32.5 +/- 0.9 kg/m(2); age, 35.7 +/- 2.2 years) were compared to 21 lean healthy women (BMI, 21 +/- 0.4 kg/m(2); age, 35.4 +/- 2.2 years). The second study examined whether a 3-month diet-induced weight loss, which usually improves insulin resistance, could also affect the membrane phospholipid (PL) composition and fluidity in the overweight group. Overweight women had significantly higher FPI levels (P <.0001), HOMA IR (P <.0001), membrane sphingomyelin (SM) (P <.05), and cholesterol (P <.05) contents than lean women. Baseline FPI and HOMA IR were positively correlated with membrane SM (P <.005), phosphatidylethanolamine (PE) (P <.005), and phosphatidylcholine (PC) (P <.05) contents, and negatively with phosphatidylinositol (PI) (P <.05) contents in the whole population. Multivariate regression analyses showed that 2 membrane parameters, PE and SM, were among the independent predictors of FPI or HOMA IR in the whole population, but also in the lean and the obese groups separately. Intervention induced a significant reduction in body weight (-5.7% +/- 0.7%), fat mass (-11.3% +/- 1.4%), and FPI (-10.2% +/- 5.4%). An improvement in membrane lipid composition was only observed in the insulin resistant subgroup (FPI > 9.55 mU/L). The reduction in FPI or HOMA IR was directly associated with reduction in SM and PE contents, a finding independent of the reduction in fat mass. A stepwise multiple regression analysis indicated that the changes in SM accounted for 26.6% of the variance in the changes in FPI as an independent predictor, with the changes in fat mass and PE as other determinants (27.8% and 20%, respectively, adjusted r(2) =.704, P <.0001). These results suggest that the abnormalities in the membrane PL composition could be included in the unfavorable lipid constellation of obesity which correlated with impaired insulin sensitivity.