Mechanisms of the Anti-Obesity Effects of Oxytocin in Diet-Induced Obese Rats

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid β-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid β-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes

Membrane permeation of arginine-rich cell-penetrating peptides independent of transmembrane potential as a function of lipid composition and membrane fluidity

Cell-penetrating peptides (CPPs) are prominent delivery vehicles to confer cellular entry of (bio-) macromolecules. Internalization efficiency and uptake mechanism depend, next to the type of CPP and cargo, also on cell type. Direct penetration of the plasma membrane is the preferred route of entry as this circumvents endolysosomal sequestration. However, the molecular parameters underlying this import mechanism are still poorly defined. Here, we make use of the frequently used HeLa and HEK cell lines to address the role of lipid composition and membrane potential. In HeLa cells, at low concentrations, the CPP nona-arginine (R9) enters cells by endocytosis. Direct membrane penetration occurs only at high peptide concentrations through a mechanism involving activation of sphingomyelinase which converts sphingomyelin into ceramide. In HEK cells, by comparison, R9 enters the cytoplasm through direct membrane permeation already at low concentrations. This direct permeation is strongly reduced at room temperature and upon cholesterol depletion, indicating a complex dependence on membrane fluidity and microdomain organisation. Lipidomic analyses show that in comparison to HeLa cells HEK cells have an endogenously low sphingomyelin content. Interestingly, direct permeation in HEK cells and also in HeLa cells treated with exogenous sphingomyelinase is independent of membrane potential. Membrane potential is only required for induction of sphingomyelinase-dependent uptake which is then associated with a strong hyperpolarization of membrane potential as shown by whole-cell patch clamp recordings. Next to providing new insights into the interplay of membrane composition and direct permeation, these results also refute the long-standing paradigm that transmembrane potential is a driving force for CPP uptake

Etude biophysique de peptides amyloïdes en présence de membranes (caractérisation de leurs interactions et détermination de leurs structures)

Le peptide amyloïde IAPP, impliqué dans le diabète de type 2, possède la propriété de s agréger, passant d un état monomérique initial à des fibres amyloïdes matures, via des espèces oligomériques. Ce processus d agrégation, qui se produit au contact de la membrane, a été étudié par fluorescence, microscopie électronique, dichroïsme circulaire et RMN. Tout d abord, l influence du modèle membranaire a été mise en évidence, en termes de forme, taille (micelles, bicelles, SUV, LUV) et composition lipidique (chaînes et têtes différentes) sur les cinétiques d agrégation et de changement conformationnel et sur la morphologie des fibres. Nous avons cherché à comprendre le rôle du cholestérol dans les interactions peptide/membranes, du point de vue du peptide et de la membrane, en utilisant des vésicules contenant entre 0 et 30% de cholestérol. Il a ainsi été observé qu un pourcentage élevé de cholestérol semble accélérer la cinétique d agrégation. De plus, des expériences de RMN liquide ont été réalisées dans le but de déterminer la structure du peptide IAPP en présence de bicelles. Les premiers résultats montrent que l extrémité C terminale ne s insère pas dans la membrane et possède une flexibilité importante. Enfin, le peptide IAPP a également été comparé à un peptide antimicrobien aux propriétés amyloïdes, la dermaseptine S9. Ces travaux indiquent que les mécanismes de fibrillation et de perméabilisation membranaire ne sont pas reliés et que le mode d action de la dermaseptine S9 repose sur la formation de pores transitoires impliquant des espèces oligomériques.The amyloid peptide IAPP, which is implicated in type 2 diabetes mellitus, aggregates from an initial monomeric state to amyloid fibrils, via oligomeric species. Peptide aggregation, which takes place through membrane contact, was studied using fluorescence, electron microscopy, circular dichroism and NMR. The effect of membrane model was highlighted, in terms of shape, size (micelles, bicelles, SUV, LUV) and composition (lipid headgroups and acyl chains), on aggregation kinetics, conformational change kinetics and fibril morphology. Next, we wanted to elucidate the role of cholesterol in peptide/membranes interactions using vesicles composed of 0 to 30% cholesterol. High cholesterol content was shown to increase aggregation kinetics. Furthermore, IAPP in the presence of bicelles was studied by liquid state NMR in order to solve its structure under these conditions. First results indicate that the C terminus does not insert into the membrane and has an important flexibility. Finally, IAPP was compared with an antimicrobial and amyloid-like peptide, dermaseptin S9. This study shows that fibril formation and membrane permeabilisation mechanisms are not linked and that dermaseptin S9 binds to membrane in an aggregated state, maybe leading to the formation of a transient pore.PARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Biophysical investigation of the membrane-disrupting mechanism of the antimicrobial and amyloid-like peptide dermaseptin S9.

Dermaseptin S9 (Drs S9) is an atypical cationic antimicrobial peptide with a long hydrophobic core and with a propensity to form amyloid-like fibrils. Here we investigated its membrane interaction using a variety of biophysical techniques. Rather surprisingly, we found that Drs S9 induces efficient permeabilisation in zwitterionic phosphatidylcholine (PC) vesicles, but not in anionic phosphatidylglycerol (PG) vesicles. We also found that the peptide inserts more efficiently in PC than in PG monolayers. Therefore, electrostatic interactions between the cationic Drs S9 and anionic membranes cannot explain the selectivity of the peptide towards bacterial membranes. CD spectroscopy, electron microscopy and ThT fluorescence experiments showed that the peptide adopts slightly more β-sheet and has a higher tendency to form amyloid-like fibrils in the presence of PC membranes as compared to PG membranes. Thus, induction of leakage may be related to peptide aggregation. The use of a pre-incorporation protocol to reduce peptide/peptide interactions characteristic of aggregates in solution resulted in more α-helix formation and a more pronounced effect on the cooperativity of the gel-fluid lipid phase transition in all lipid systems tested. Calorimetric data together with (2)H- and (31)P-NMR experiments indicated that the peptide has a significant impact on the dynamic organization of lipid bilayers, albeit slightly less for zwitterionic than for anionic membranes. Taken together, our data suggest that in particular in membranes of zwitterionic lipids the peptide binds in an aggregated state resulting in membrane leakage. We propose that also the antimicrobial activity of Drs S9 may be a result of binding of the peptide in an aggregated state, but that specific binding and aggregation to bacterial membranes is regulated not by anionic lipids but by as yet unknown factors

Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus

Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet β-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of β-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined

Surface pressure profile (A) after injecting a sample of Drs–S9 into a monolayer of DOPC (solid line), DOPC/DOPG (dashed line) or DOPG (dashed dotted line).

<p>The peptide was injected into the stirred sub–phase at t = 0 min. Surface pressure increase (B) induced by the interaction of Drs–S9 with DOPC (squares, solid line), DOPC/DOPG (triangles, dashed line) or DOPG (diamonds, dashed dotted line) monolayers as a function of the initial surface pressure. The straight lines were obtained by linear regression. Experimental error is estimated at ± 0.5 mN/m.</p

DSC thermograms illustrating the effect of the addition (dashed line) or the incorporation (dashed dotted line) of Drs S9 to (A) DMPC MLVs, (B) DMPC/DMPG 7∶3 MLVs, and (C) DMPG MLVs.

<p>Solid lines correspond to pure lipids.</p

CD spectra (190–270 nm) of Drs S9 (25 µM) in (A) phosphate buffer at pH 7.4, (B) DOPC LUVs, (C) DOPC/DOPG 7∶3 LUVs, and (D) DOPG LUVS.

<p>Peptide was either added (solid line) or incorporated (dashed line) to vesicles. Peptide/lipid ratio was 1∶20.</p

Order parameter profile calculated for de-Paked spectra of (A) POPC-d₃₁, (B) POPC-d₃₁/POPG 7∶3, and (C) POPG-d₃₁, in the absence (diamond) and in the presence (cross) of Drs S9.

<p>Order parameter profile calculated for de-Paked spectra of (A) POPC-d₃₁, (B) POPC-d₃₁/POPG 7∶3, and (C) POPG-d₃₁, in the absence (diamond) and in the presence (cross) of Drs S9.</p

Membrane permeabilization induced by 1 µM Drs S9 of calcein-containing DOPC (solid line), DOPC/DOPG 7∶3 (dashed line) or DOPG (dashed dotted line) LUVs.

<p>Data are the average (± standard deviation) from 4 independent experiments performed in triplicate.</p