The Constitutive Lack of α7 Nicotinic Receptor Leads to Metabolic Disorders in Mouse.

Type 2 diabetes (T2D) occurs by deterioration in pancreatic β-cell function and/or progressive loss of pancreatic β-cell mass under the context of insulin resistance. α7 nicotinic acetylcholine receptor (nAChR) may contribute to insulin sensitivity but its role in the pathogenesis of T2D remains undefined. We investigated whether the systemic lack of α7 nAChR was sufficient to impair glucose homeostasis. We used an α7 nAChR knock-out (α7 <sup>-/-</sup> ) mouse model fed a standard chow diet. The effects of the lack of α7 nAChR on islet mass, insulin secretion, glucose and insulin tolerance, body composition, and food behaviour were assessed in vivo and ex vivo experiments. Young α7 <sup>-/-</sup> mice display a chronic mild high glycemia combined with an impaired glucose tolerance and a marked deficit in β-cell mass. In addition to these metabolic disorders, old mice developed adipose tissue inflammation, elevated plasma free fatty acid concentrations and presented glycolytic muscle insulin resistance in old mice. Finally, α7 <sup>-/-</sup> mice, fed a chow diet, exhibited a late-onset excessive gain in body weight through increased fat mass associated with higher food intake. Our work highlights the important role of α7 nAChR in glucose homeostasis. The constitutive lack of α7 nAChR suggests a novel pathway influencing the pathogenesis of T2D

Measurement of protein digestibility in humans by a dual-tracer method

Background: Recent evaluations of the risk of dietary protein deficiency have indicated that protein digestibility may be a key limiting factor in the provision of indispensable amino acids (IAAs), particularly for vulnerable populations living in challenging environments where intestinal dysfunction may exist. Since the digestion of protein occurs only in the small intestine, and the metabolic activity of colonic bacteria confounds measurements at the fecal level, there is a need to develop noninvasive protein digestibility measurements at the ileal level. Objective: We used a dual-tracer method with stable isotopes to characterize the digestibility of uniformly labeled [13C]-spirulina protein as a standard protein, in comparison to a mixture of 2H-labeled crystalline amino acids, and then demonstrated the use of this standard protein to measure the digestibility of selected legumes (chick pea and mung bean) through the use of proteins that were intrinsically labeled with 2H. Design: The digestibility of uniformly labeled [13C]-spirulina was first measured in 6 healthy volunteers (3 males and 3 females) by feeding it along with a standard mixture of 2H-labeled amino acids, in a dual-tracer, plateau-fed test meal approach. Next, intrinsically labeled legume protein digestibility was studied with a similar dual-tracer approach, with uniformly labeled [13C]-spirulina as the standard, when processed differently before consumption. Results: The average digestibility of IAA in spirulina protein was 85.2%. The average IAA digestibility of intrinsically 2H-labeled chick pea and mung bean protein was 56.6% and 57.7%, respectively. Dehulling of mung bean before ingestion increased the average IAA digestibility by 9.9% in comparison to whole mung bean digestibility. Conclusions: An innovative, minimally invasive “dual-stable-isotope” method was developed to measure protein digestibility, in which the ingestion of an intrinsically 2H-labeled test protein along with a 13C-labeled standard protein of known digestibility allows for an accurate measure of digestion and absorption of the intrinsically labeled protein. This minimally invasive method is critical to redefining protein quality and will aid in revisiting human protein requirements in different settings and in vulnerable populations. This trial was registered at Clinical Trials Registry—India as CTRI/2017/11/010468

Th17-related mammary immunity, but not a high systemic Th1 immune response is associated with protection against E. coli mastitis

International audienceVaccination against bovine mastitis lags behind despite high demand from the dairy industry and margin for efficacy improvement. We previously compared two immunization protocols against E. coli using either only the intramuscular route or a combination of intramuscular and mammary ductal routes, also known as 'prime and pull' strategy. A homologous mammary challenge during the memory phase showed that immunization favorably modified the mastitis course, notably in locally immunized cows in comparison to intramuscular and control adjuvant-only groups. Here, we performed whole-blood profiling through RNA-seq transcriptome and plasma cytokine 15-plex analyses at time points of the E. coli mastitis that showed significant clinical and laboratory differences among the groups. Diminished production of inflammatory cytokines and increased IFN gamma were detected in the blood of immunized cows, where a T lymphocyte activation profile was evidenced at 12-h post infection. Acute phase neutropenia was less severe in these cows, and pathways related to neutrophil diapedesis and monocyte activation were also present. Furthermore, three intramammary-immunized cows showing faster healing and shorter mastitis duration had gene profiles that differed from their counterparts, but without any clue for the mastitis susceptibility difference. Inasmuch, when gene expression of CD4 T cells was assessed in mammary tissue, enrichment of IL-17-associated pathways was identified in the quarters of intramammary-immunized cows not only after challenge but also in the control quarters that were not infected. These findings indicate that local immunization mobilizes protective mechanisms that rely on the settlement of type 3 immunity-related CD4 T cells prior to infection

Increasing habitual protein intake results in reduced postprandial efficiency of peripheral, anabolic wheat protein nitrogen use in humans

International audienceThe postprandial retention of dietary protein decreases when the prevailing protein intake increases. We investigated the influence of the prevailing protein intake on the regional utilization and anabolic use of wheat protein during the postprandial non-steady state in humans. Healthy adults (n ҃ 8) were adapted for 7 d, first to a normal-protein diet (NP: 1 g ⅐ kg Ҁ1 ⅐ d Ҁ1) and then to a high-protein diet (HP: 2 g ⅐ kg Ҁ1 ⅐ d Ҁ1). After each adaptation period, the subjects received the same single, solid mixed meal containing [ 15 N]-labeled wheat protein. The postprandial kinetics of dietary nitrogen were then measured for 8 h in blood and urine. These data were further analyzed by using a multicompartmental model to predict the postprandial kinetics of dietary nitrogen in unsampled pools. The postprandial whole-body retention of wheat protein nitrogen, measured 8 h after meal ingestion, decreased by 10% when the subjects switched from the NP diet to the HP diet. According to modeling results, this resulted from an increased splanchnic utilization of dietary nitrogen for urea production, whereas its incorporation into splanchnic proteins was unchanged, leading to a 20-30% decrease in peripheral availability and anabolic use in HP-adapted compared with NP-adapted subjects having ingested the same protein load. By combining clinical experimentation with compartmental modeling, we provide a global overview of postprandial dietary protein metabolism. Increasing prior protein intake was shown to reduce the postprandial retention of wheat protein nitrogen, mainly by diminishing the efficiency of its peripheral availability and anabolic use

Stimulation of autophagy with PP242 reduces desmin aggregation in myoblasts.

<p>(A) C2C12 cells were transiently transfected for 4 h with GFP-Desmin D399Y mutant, washed, and treated for 16 h with PP242 (10 μM). They were fixed and several fields photographed. A typical field is displayed. Green dots are cell aggregates and blue dots are cell nuclei visualized with DAPI. Cells from the control panel (CNTL) were treated with DMSO. Scale bar, 30 μm. (B) Quantification of 3 independent experiments (n = 1200 total cells for each condition in each experiment). The left panel displays results obtained with the GFP-Desmin Q389P construct, and in the right panel, from GFP-Desmin D399Y transfections. (C) Same as for (B), except that myc-Desmin Q389P (left panel) and myc-Desmin D399Y (right panel) constructs were used. Cells were transiently transfected for 4 h with these constructs, washed, and treated 16 h with PP242 (10 μM) or DMSO as solvent (CNTL). They were fixed and then processed for immunocytochemistry with an anti-myc antibody. Numbers of cells with aggregates per field and number of myc-positive cells without aggregates but displaying a normal desmin network were counted. The percentage of cells with aggregates among all myc-positive cells was calculated, taking into account 5 fields per experimental condition (n = 1400) in 3 independent experiments. Transfection efficiency was 25%. Significant differences from the control are indicated with asterisk (p<0.05 calculated with a non-parametric test).</p

Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy

<div><p>Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the <i>desmin</i> gene (<i>DES</i>), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant <i>DES</i>. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.</p></div

Modulation of cell signaling pathways related to the cytoskeleton reduces desmin aggregation.

<p>(A) C2C12 cells were co-transfected with a GFP-tagged desmin mutant D399Y and constructs coding for either wild type (WT) or dominant-negative mutant (DN) kinases or kinase-modulating proteins [i.e., Rac1, p21-activated protein kinase (PAK1), Rho kinase (ROCK), mammalian Diaphanous (mDia), protein kinase C (PKC), p38-regulated/activated protein kinase (PRAK) and transforming growth factor β activated kinase 1 (TAK1)]. At 20 h after transfection, cells were fixed and the total number of cells (n = 1500) and the number of cells with aggregates were counted. Experiments were performed 4 times. The percentage of cells with aggregates is displayed on a box plot graph (Tukey's diagram). Asterisk indicates a result statistically different from the control co-transfected with the desmin mutant and the empty vector pcDNA3 (p < 0.05 calculated with a non-parametric test). The black horizontal bar represents the median value; limits of the rectangle represent the first (25% lower values) and the third (75% lower values) quartiles, respectively. Error bars, Tukey's adjacent values. (B) Same treatment as for (A) except that cells were fixed 48 h after transfection.</p

Triple treatment with therapeutic drugs cooperates to reduce desmin aggregation.

<p>C2C12 cells were transiently transfected with the GFP-Desmin WT- (left panel) or D399Y- (right panel) expressing vectors for 4 h. Cells were washed and subsequently incubated with PP242 (5 μM), NSC23766 (50 µM), Trolox (200 μM), all three compounds, or DMSO (CNTL) for 16 h. Cells were then fixed and counted under a microscope for the percentage having aggregates. A box plot representing 3 independent experiments is shown (n = 100). Statistical analysis with a non-parametric test revealed significant differences indicated by a simple asterisk (p < 0.05), and an asterisk above an horizontal bar indicates a significant difference between the double and simple treatments (p < 0.05).</p