Identification of an inward Na+ current activated by acetylcholine in mouse pancreatic -cells

Our aim was to identify and characterize the current by which acetylcholine (ACh) depolarizes the plasma membrane of pancreatic beta-cells. The observations that this depolarization is abrogated by omission of Na+, and that ACh increases cytosolic [Na+] suggested that the current is carried by Na+. [...

New targets to control skeletal muscle inflammation: MicroRNAs regulated by adiponectin

Background and aims: Low-grade pro-inflammatory state contributes to the metabolic syndrome (MS). Adiponectin (ApN), which is reduced in the MS, has emerged as a master regulator of inflammation/immunity. We wished to identify whether microRNAs (miRNAs) may mediate the antiinflammatory action of ApN on skeletal muscle. Materials and methods: miRNA expression profiling was performed in tibialis anterior muscles of ApN-knockout (ApN-KO) mice: one leg was electrotransferred with a plasmid coding for the ApN gene, while the contralateral leg received an empty plasmid and served as control. Mice were next challenged by lipopolysaccharide (LPS) to induce inflammation. Role of specific miRNAs was analyzed by gain-of or loss-of function approaches in C2C12 myotubes and in vivo by muscle electrotransfer. miRNA expression was also studied in human myotubes. Results: Expression of miR-711 was up-regulated by muscle electrotransfer of ApN, which concomitantly reduced inflammation (TNFα, IL-1β) and oxidative stress (peroxiredoxin-3) markers. Likewise, in C2C12 cells, ApN treatment upregulated miR-711 expression. Transfection of miR-711 mimic reproduced the anti-inflammatory effects of ApN, while miR-711 blockade attenuated its protective effects. We found that miR-711 repressed the expression of 4 genes belonging to the Toll-like receptor-4 (TLR4) pathway. This pathway is activated by LPS and ultimately leads to stimulation of NF-kB, a pro-inflammatory transcription factor. As expected, NF-kB activity, measured via a luciferase reporter plasmid, was reduced by the miR mimic and enhanced by miR silencing. This protection against inflammation was recapitulated in ApN-KO mice by in vivo muscle electrotransfer of a plasmid coding for miR-711. Eventually, miR-711 expression was also upregulated in human myotubes after ApN treatment. Conclusion: miR-711, which is up-regulated by ApN, represses TLR4 signaling and NF-kB, acting therefore as a major mediator of the antiinflammatory action of ApN on muscle. This novel miRNA may open new therapeutic perspectives for the MS or other inflamed muscle condition

Potent anti-inflammatory properties of ApN on the skeletal muscle

Background and aims: Adiponectin (ApN) is an adipokine, which is decreased in the metabolic syndrome, thereby playing a key role in the pathogenesis of these disorders. We have previously shown that ApN exerts important anti-inflammatory effects on skeletal muscle in mice exposed to acute inflammation (LPS injection) or metabolic stress (obesogenic diet). The aim of this work is to test the potency of the anti-inflammatory properties of ApN by using a model with severe and sustained inflammation. Materials and methods: Mdx mice (a mouse model of Duchenne muscular dystrophy, where persistent inflammation worsens the consequences of the genetic deficiency) were crossed with transgenic mice that over express adiponectin (Tg-ApN mice) in order to generate mdx-Tg-ApN mice. Different markers of inflammation/oxidative stress (TNF-α, IL-1β, NF-κB, CD68, CD3, peroxiredoxin 3/5 (PRDX3/5), 4 hydroxynonenal (HNE)) were studied and quantified by immunohistochemistry, ELISA and western blot. In vivo functional tests were also carried out to determine the global force of mice. Finally, the extent of muscle damage was quantified by Evans Blue Dye (EBD) injection following an eccentric exercise. Results: Compared to WT mice, muscles of mdx mice presented significant increases (+ 10 to 15 fold; p<0.001) in the expression of pro-inflammatory factors (TNF-α, IL-1β, NF-κB) and oxidative stress markers (PRDX3/5, HNE) as well as a massive infiltration of macrophages and T lymphocytes as shown by CD68 and CD3 immunolabeling respectively. All of these abnormalities were drastically reduced in mdx-Tg-ApN mice (– 60 to 75 % vs mdx; p<0.001). In addition, mdx-Tg-ApN mice exhibited higher global muscular force (+ 50 % vs mdx; p<0.01) along with a significant decrease in EBD content in their muscle fibers (– 50 to 60 % vs mdx; p<0.001). Conclusion: Adiponectin proves to be an extremely potent anti-inflammatory agent that protects muscle against injury. These anti-inflammatory properties of ApN are of interest in the metabolic syndrome as well as in other diseases where inflammation plays a triggering or worsening pathogenic rol

Glucose controls glucagon secretion with minor changes in alpha cell [Ca2+]c whereas sulfonylureas control glucagon secretion by increasing alpha cell [Ca2+]c and via somatostatin

BACKGROUND AND AIMS: The mechanisms by which glucose (G) and KATP channels control glucagon release are poorly understood. A rise in [Ca2+]c is considered as a signal triggering glucagon secretion. However, the effects of glucose and KATP channels on α-cell [Ca2+]c are hotly debated. In the present study, we tested the effects of glucose and KATP channel blockers (sulfonylureas) on α-cell [Ca2+]c using mice expressing the Ca2+indicator, GCAMP6f, specifically in α-cells, and we studied the correlation between α-cell [Ca2+]c and glucagon secretion. We also investigated the paracrine influence of somatostatin (SST) using Sst-/- mice. [...

The influence of cell number on Ca2+ oscillations in clusters of B-cells

During continuous stimulation with glucose, intact islets display regular oscillations of cytosolic [Ca2+], whereas the oscillations are Inconsistent and variable in single B-cells. Here, we tested the hypothesis that a minimum number of B-cells must be coupled for appearance of regular oscillations [...

Effects of increasing glucose concentrations on gene mRNA expression in cultured rat pancreatic islets

Background and Aims, Materials and Methods: Survival and function of rat β-cells are best preserved by culture at 10 mM glucose (G10) and rapidly deteriorate at lower or higher glucose concentrations. To evaluate how glucose exerts these effects on the β-cell phenotype, we measured gene mRNA levels in rat islets precultured for 1wk in G10 and further cultured 18h in G2, G5, G10 or G30 (Affymetrix Rat Genome 230 2.0 oligonucleotide arrays, n=4). Match/mismatch probe signal intensities and differences in mRNA abundance, expressed as absolute signal log2 ratios (SLR), were calculated with GCOS software. Results: From the 31099 probe sets on the arrays, 18081 were reliably detected in at least one glucose concentration. The SLR for at least one of the comparisons between the different glucose concentrations was ≥ 1 and ≥ 0.5 in 1039 and 3530 probe sets respectively. Using Self-Organizing Map cluster analysis, 3120 of the 3530 affected probe sets were classified in 6 clusters with monophasic concentration-dependencies that differed in overall direction (increase/decrease) and glucose threshold. The remaining 410 probe sets were grouped into 5 small clusters with complex mRNA expression profiles (V-shaped or inverted V-shaped with minimum or maximum in G5 or G10). Analysis of gene function in each cluster suggested the presence of glucose co-regulated metabolic and signalling pathways (glycolysis, cholesterol synthesis, unfolded protein response…). Conclusion: These results will help identifying key molecules that maintain the differentiated β-cell phenotype as well as those responsible for β-cell deterioration after exposure to chronic hypo- or hyperglycemi