Membrane Cholesterol in Skeletal Muscle: A Novel Player in Excitation-Contraction Coupling and Insulin Resistance.

Membrane cholesterol is critical for signaling processes in a variety of tissues. We will address here current evidence supporting an emerging role of cholesterol on excitation-contraction coupling and glucose transport in skeletal muscle. We have centered our review on the transverse tubule system, a complex network of narrow plasma membrane invaginations that propagate membrane depolarization into the fiber interior and allow nutrient delivery into the fibers. We will discuss current evidence showing that transverse tubule membranes have remarkably high cholesterol levels and we will address how modifications of cholesterol content influence excitation-contraction coupling. In addition, we will discuss how membrane cholesterol levels affect glucose transport by modulating the insertion into the membrane of the main insulin-sensitive glucose transporter GLUT4. Finally, we will address how the increased membrane cholesterol levels displayed by obese animals, which also present insulin resistance, affect these two particular skeletal muscle functions.

Potential role of skeletal muscle glucose metabolism on the regulation of insulin secretion.

Pancreatic beta cells sense glucose flux and release as much insulin as required in order to maintain glycaemia within a narrow range. Insulin secretion is regulated by many factors including glucose, incretins, and sympathetic and parasympathetic tones among other physiological factors. To identify the mechanisms linking obesity-related insulin resistance with impaired insulin secretion represents a central challenge. Recently, it has been argued that a crosstalk between skeletal muscle and the pancreas may regulate insulin secretion. Considering that skeletal muscle is the largest organ in non-obese subjects and a major site of insulin- and exercise-stimulated glucose disposal, it appears plausible that muscle might interact with the pancreas and modulate insulin secretion for appropriate peripheral intracellular glucose utilization. There is growing evidence that muscle can secrete so-called myokines that can have auto/para/endocrine actions. Although it is unclear in which direction they act, interleukin-6 seems to be a possible muscle-derived candidate protein mediating such inter-organ communication. We herein review some of the putative skeletal muscle-derived factors mediating this interaction. In addition, the evidence coming from in vitro, animal and human studies that support such inter-organ crosstalk is thoroughly discussed.

Incidencia de seroma postoperatorio tras reparación de la fascia de Scarpa en abdominoplastia-liposucción / Postoperatory seroma incidence after repair of Scarpa fascia in abdominoplasty-lipoaspiration

La fascia de Scarpa es un hito anatómico de la pared abdominal y divide el tejido graso de ésta en una capa superficial y en otra profunda. Existen publicaciones que aseguran que en una abdominoplastía, la preservación in situ de esta estructura mediante disección superficial a la misma puede prevenir la formación de seromas, pero en la ejecución de ésta técnica se han observado peores resultados estéticos. Consideramos que al reparar el defecto creado, mediante la sutura de la fascia de Scarpa, podemos emular el efecto de disminución de seromas pero sin comprometer el resultado estético. Mediante el uso de la base de datos del primer autor, seleccionamos pacientes que cumplan como criterios de inclusión: ser menores de 65 años, en rangos normales de peso, sometidas a abdominoplastia con lipoaspiración en el período comprendido entre abril del 2004 y diciembre del 2009, divididas en 2 grupos cuya diferencia es el cierre de la fascia de Scarpa (que hacemos de rutina desde julio del 2007). En total, 2 grupos de 97 pacientes cada uno en los que se midió el débito del drenaje que se retira al tercer día, y en controles médicos posteriores si se sospechaba la presencia de seroma, realizando punciones hasta obtener menos de 50 ml. Al comparar el débito de drenajes, número de punciones y cantidad de fluido obtenido en las punciones, encontramos diferencias estadísticamente significativas entre ambos grupos. No hubo diferencia en el índice de masa corporal (IMC), comorbilidad y complicaciones entre los grupos. En conclusión, planteamos que con la reparación de la fascia de Scarpa mediante su sutura en los procedimientos de abdominoplastia con lipoaspiración se remeda el efecto de prevención de formación de seroma de una disección más superficial, pero sin compromiso estético secundario; además, creemos que la retirada de los drenajes al tercer día postoperatorio es una práctica segura. Sin embargo, se requieren más estudios para profundizar y confirmar los hallazgos

The Scarpa fascia is an anatomic hint in the abdominal wall, and divides the fat tissue in a superficial and a deep layer. Other publications assert that in abdominoplasty, preserve this structure and making a more superficial dissection can prevent the seroma formation, but with worse aesthetic results. We consider that repairing the defect created by suturing the Scarpa fascia we can emulate the effects in diminishing the seroma, but without the aesthetic compromise. Using the first author´s data base, we select patients that complies with the following inclusion criteria of abdominoplasty plus liposuction: women, younger than 65, with no overweight, operated between April 2004 and December 2009, divided in 2 groups, with and without Scarpa´s fascia closure (that became a routine in July 2007). Two groups, 97 patients each. We measured drain content (all the drains were took away by day 3), and in subsecuent medic control if a seroma was clinically suspected, it was punctioned untill the amount of serum obtained was under 50 cc. Comparing both groups, there is statiscally significant difference between them in drainage and amount of fluid obtained by punctions; also in the number of punctions and number of patients that needed the procedure. There is no difference between groups in age, body mass index (BMI), co-morbidity or complications. We think that repairing the defect by stitching the Scarpa fascia redeems the effect of preserving it but without the aesthetic defect, and all drainages can be safely took away by day 3. Anyway, further studies are needed in order to confirm this aspect

Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. a comparative study.

Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13-16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to beta-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.

Large chloride channel from pre-eclamptic human placenta.

Chloride transport involving conductive pathways participates in numerous epithelial functions, such as membrane voltage maintenance, solute transport and cell volume regulation. Evidence points to involvement of transepithelial chloride transport in such functions in placental syncytiotrophoblast. A molecular candidate for physiologic conductive chloride transport in apical syncytiotrophoblast membrane is a Maxi-chloride channel with distinct biophysical properties: conductance over 200 pS, multiple substates, voltage dependent open probability, and permeation to anionic amino acids. Pre-eclampsia, a high incidence pathology of pregnancy, exerts great impact on fetal morbi-mortality. This relies, among others, on intrauterine growth restriction (IUGR), thought to be mediated by diminished blood flow to the placenta, with growing knowledge regarding contribution of other factors. The Maxi-chloride channel's properties suggest it could be altered in this pathology. We have characterized the apical chloride channels from pre-eclamptic placentae, reconstituted in giant liposomes suitable for patch-clamp electrophysiological studies. In n=33 experiments from n=6 pre-eclamptic placentae we observed a chloride-permeable channel with similar biophysical properties to the channel from normal tissue (n=29 experiments from n=15 placentae). However, the main conductance state showed diminished magnitude (<150 pS), and the open probability versus voltage relationship exhibited a flattened curve instead of the bell-shaped curve of normal placentae. These results are the first evidence of a functionally altered ionic channel from placental syncytiotrophoblast in pre-eclampsia. Considering the abundance of chloride-conducting channel activity in human apical membrane and their relevance in epithelial function in general, these alterations could greatly disturb numerous placental functions that rely on syncytiotrophoblast integrity.

A low conductance, non-selective cation channel from human placenta.

Non-selective cation channels have been identified in the plasma membranes of many different cells. Previous research using fluorescent techniques has demonstrated the presence of cation conductances in membranes from human trophoblast. The purpose of this work was to explore, by electrophysiological methods, a non-selective cation channel in apical membranes from human placenta. Human placental apical membranes were purified by differential centrifugation and reconstituted in giant liposomes. These giant liposomes were then used for electrophysiological studies and were probed for the presence of cation channels by the patch-clamp method. The channel identified had a linear current-potential relationship with a conductance of around 16 pS in symmetrical Na(+) solution. Under asymmetrical conditions the reversal potential was close to the reversal potential for Na(+). The channel was equally permeable to sodium and potassium and the permeability sequence was NH+4>Cs(+) approximately Rb(+)>Na(+) approximately K(+)>Li(+). The channel also showed permeability to calcium and barium. The channel was insensitive to calcium but was blocked by millimolar concentration of Mg(2+). We have demonstrated the presence of a low conductance, non-selective cation channel in placental apical membranes. These channels share some properties with non-selective cation channels previously described in other different cells. The precise role of these channels in placental physiology has yet to be determined.