Aims. The purpose of this study was to determine the effect of insulin on ceramide metabolism in skeletal muscle. Methods. Skeletal muscle cells were treated with insulin with or without palmitate for various time periods. Lipids (ceramides and TAG) were isolated and gene expression of multiple biosynthetic enzymes were quantified. Additionally, adult male mice received daily insulin injections for 14 days, followed by muscle ceramide analysis. Results. In muscle cells, insulin elicited an increase in ceramides comparable to palmitate alone. This is likely partly due to an insulin-induced increase in expression of multiple enzymes, particularly SPT2, which, when knocked down, prevented the increase in ceramides. In mice, 14 days of insulin injection resulted in increased soleus ceramides, but not TAG. However, insulin injections did significantly increase hepatic TAG compared with vehicle-injected animals. Conclusions. This study suggests that insulin elicits an anabolic effect on sphingolipid metabolism in skeletal muscle, resulting in increased ceramide accumulation. These findings reveal a potential mechanism of the deleterious consequences of the hyperinsulinemia that accompanies insulin resistance and suggest a possible novel therapeutic target to mitigate its effects.

At high temperature, the presence of cholesterol in phospholipid membranes alters the influence of membrane dipoles, including water molecules, on naphthalene-based fluorescent probes such as Laurdan and Patman (solvatochromism). Although both of these probes report identical changes to their emission spectra as a function of temperature in pure phosphatidylcholine bilayers, they differ in their response to cholesterol. Computer simulations of the spectra based on a simple model of solvatochromism indicated that the presence of cholesterol reduces the probability of bilayer dipole relaxation and also blunts the tendency of heat to enhance that probability. While the overall effect of cholesterol on membrane dipoles was detected identically by the two probes, Laurdan was influenced much more by the additional effect on temperature sensitivity than was Patman. A comparison of the fluorescence data with simulations using a coarse-grained bilayer model (de Meyer et al., 2010) suggested that these probes may be differentially sensitive to two closely related properties distinguishable in the presence of cholesterol. Specifically, Patman fluorescence correlated best with the average phospholipid acyl chain order. On the other hand, Laurdan fluorescence tracked more closely with the area per lipid molecule which, although affected generally by chain order, is also impacted by additional membrane-condensing effects of cholesterol. We postulate that this difference between Laurdan and Patman may be attributed to the bulkier charged headgroup of Patman which may cause the probe to preferentially locate in juxtaposition to the diminutive headgroup of cholesterol as the membrane condenses.

Using coarse-grained molecular dynamics simulations, we demonstrate the nature of the membranophobic effect -a proposed phenomenon that governs self-assembly of inclusions within a lipid bilayer, inspired by the statistical mechanics of the hydrophobic effect. We study the nature of this effect on membrane inclusions of various chemistries and sizes. We identify the range of hydrophobic thicknesses over which this phenomenon occurs and characterize the effects of the proposed phenomenon on small inclusions such as cholesterol versus larger, multidomain transmembrane proteins. Our results show that this effect can provide a force for assembly and reorganization in a lipid bilayer based on the in-plane size and hydrophobic thickness of the inclusion, and the melting temperature of the surrounding lipids. We propose that this effect provides a physical framework that can explain lipid raft formation. Wide interest in the phase behaviour of amphiphilic bilayers has arisen since the realisation that it can be exploited by nature, and engineers, to design-in function via membrane domains. A key confounding feature is the presence of two separate, yet coupled, leaflets within the bilayer. The question of inter-leaflet domain alignment (registration) or otherwise is central to proposed cellular roles such as protein localisation and, more fundamentally, creates a zoo of phase behaviour that can only be captured by properly considering the coupled leaflets. Experiment and simulation yield intriguingly disparate observations, but a full theoretical picture is lacking; existing phenomenological theories provide insight but do not link large-scale behaviour to small-scale features. We introduce a theory for phase separation in coupled leaflets by explicitly coarse-graining a lattice model that includes molecule-level structuring and interactions. We show that accounting for hydrophobic mismatch between the mixed species leads to a complex competition of inter-leaflet coupling energies. The free energy obtained helps unify some prima facie contradictory observations by showing that domain antiregistration typically occurs as a metastable state, but can be kinetically preferred during the initial demixing of a bilayer. The role of kinetics in governing registration/antiregistration is explored, and we find that a bilayer in the usual ''spinodal region'' may in fact require a nucleation process to equilibrate. Our results provide a tractable coarse-grained model that explicitly depends on simplified molecular interactions, providing novel insight and encouraging important future work in which the intra-and inter-leaflet behaviour of mixed bilayers is carefully investigated. Even though glycolipids are present at increased concentrations in the outer leaflet of eukaryotic biomembranes, their influence on the mechanical properties of the membrane has not been studied in much detail. In this work, we investigate the effect of GM1, a prominent example among glycolipids, on the physical characteristics such as phase state and bendin...

The naphthalene-based fluorescent probes Patman and Laurdan detect bilayer polarity at the level of the phospholipid glycerol backbone. This polarity increases with temperature in the liquid-crystalline phase of phosphatidylcholines and was observed even 90°C above the melting temperature. This study explores mechanisms associated with this phenomenon. Measurements of probe anisotropy and experiments conducted at 1M NaCl or KCl (to reduce water permittivity) revealed that this effect represents interactions of water molecules with the probes without proportional increases in probe mobility. Furthermore, comparison of emission spectra to Monte Carlo simulations indicated that the increased polarity represents elevation in probe access to water molecules rather than increased mobility of relevant bilayer waters. Equilibration of these probes with the membrane involves at least two steps which were distinguished by the membrane microenvironment reported by the probe. The difference in those microenvironments also changed with temperature in the liquid-crystalline phase in that the equilibrium state was less polar than the initial environment detected by Patman at temperatures near the melting point, more polar at higher temperatures, and again less polar as temperature was raised further. Laurdan also displayed this level of complexity during equilibration, although the relationship to temperature differed quantitatively from that experienced by Patman. This kinetic approach provides a novel way to study in molecular detail basic principles of what happens to the membrane environment around an individual amphipathic molecule as it penetrates the bilayer. Moreover, it provides evidence of unexpected and interesting membrane behaviors far from the phase transition.

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