Evaluation of glucose tolerance test and surrogate indexes of insulin sensitivity to detect insulin resistance syndrome in transition dairy cows

Healthy multiparous Holstein-Fresian cows (n=101, parity ≥ 2) from 3 large-scale dairy herds in Italy were subjected to an intravenous glucose tolerance test (GTT) 14-1 d before (Week -1) and 3-9 d after calving (Week +1). A single blood collection was repeated 10-16 d after calving. Several plasma metabolites and insulin were determined at basal samples (T0); Revised Quantitative Insulin Sensitivity Check Index (RQUICKI) and Homeostasis Model Assessment (HOMA) for the estimation of peripheral insulin sensitivity were also calculated at T0. Insulin and glucose were also measured 10 (T10) and 80 (T80) minutes after glucose infusion. T80/T0 glucose ratio in Week -1 was used for cows’ classification in two categories: GTT class 1 = cows with T80/T0 > 1.2; GTT class 0 = cows with T80/T0 ≤ 1.2. First milk test productions, diseases and mastitis cases were recorded. Cows in GTT class 0 and 1 in Week -1 differed both for glucose concentration at T0 and glucose at T80 (P < 0.01). Cows in GTT class 1 had a relative risk of 2.18 of having NEFA higher than 0.5 mEq/l pre-partum (P < 0.05). There was a significant herd effect over NEFA, Glucose, Insulin, Albumins, Urea, GOT-AST and GPT-ALT at T0 pre-partum (P < 0.05); post-partum herd effect was confirmed for Glucose and RQUICKI (P < 0.05). In the pre-partum weeks interestingly different levels of plasma insulin at T10 were evidenced within GTT class with lower hormone concentrations in class 1 (P < 0.01). Those cows also reported post-partum higher HOMA values (P < 0.01) and lower RQUICKI (P < 0.05), which reflect decreased insulin sensitivity. In conclusion, our study demonstrates a promising opportunity for application of surrogate indices of insulin sensitivity and GTT in field trials to detect insulin resistance syndrome in dairy cows in the future. The T80/T0 ratio derived by GTT seemed to be useful in detecting a low insulin secretion as a likely complication of altered glucose uptake following glucose load in transition cows

The amphoterin (HMGB1)/receptor for advanced glycation end products (RAGE) pair modulates myoblast proliferation, apoptosis, adhesiveness, migration, and invasiveness. Functional inactivation of RAGE in L6 myoblasts results in tumor formation in vivo.

We reported that RAGE (receptor for advanced glycation end products), a multiligand receptor of the immunoglobulin superfamily expressed in myoblasts, when activated by its ligand amphoterin (HMGB1), stimulates rat L6 myoblast differentiation via a Cdc42-Rac-MKK6-p38 mitogen-activated protein kinase pathway, and that RAGE expression in skeletal muscle tissue is developmentally regulated. We show here that inhibition of RAGE function via overexpression of a signaling deficient RAGE mutant (RAGEΔcyto) results in increased myoblast proliferation, migration, and invasiveness, and decreased apoptosis and adhesiveness, whereas myoblasts overexpressing RAGE behave the opposite, compared with mocktransfected myoblasts. These effects are accompanied by a decreased induction of the proliferation inhibitor, p21Waf1, and increased induction of cyclin D1 and extent of Rb, ERK1/2, and JNK phosphorylation in L6/RAGEΔcyto myoblasts, the opposite occurring in L6/RAGE myoblasts. Neutralization of culture medium amphoterin negates effects of RAGE activation, suggesting that amphoterin is the RAGE ligand involved in RAGE-dependent effects in myoblasts. Finally, mice injected with L6/RAGEΔcyto myoblasts develop tumors as opposed to mice injected with L6/RAGE or L6/mock myoblasts that do not. Thus, the amphoterin/RAGE pair stimulates myoblast differentiation by the combined effect of stimulation of differentiation and inhibition of proliferation, and deregulation of RAGE expression in myoblasts might contribute to their neoplastic transformation

Report and Abstracts of the 20th Meeting of IIM, the Interuniversity Institute of Myology: Assisi, October 12-15, 2023

The 2023 represented a milestone for the Interuniversity Institute of Myology (IIM) since it marked twenty years of IIM activity joined with the 20th annual meeting organized by the association. The 20th IIM meeting took place in the fascinating town of Assisi, in the heart of central Italy, from 12 to 15 October. The commemorative 20th edition of the meeting represented a success in terms of participation and contributions as it brought together 160 myologists, clinicians, pharmaceutical companies, and patient organization representatives from Italy, several European countries (especially France), the United Kingdom, Brazil, and the USA. Four main scientific sessions hosted 36 oral communications and 54 always-on-display posters reporting original and unpublished results. Four main lectures from internationally renowned invited speakers and talks from delegates of the Societé Française de Myologie gave particular interest and emphasis to the scientific discussion. In line with the traditional policy of the IIM to encourage the participation of young researchers, about 50% of the attendees were under 35 years old. Moreover, the 20th IIM meeting was part of the high-training course in “Advanced Myology Update 2023”, reserved to young trainees and managed by the University of Perugia (Italy) in collaboration with the IIM. In addition to the meeting scientific sessions, the 29 attendees to the course had a dedicated round table and dedicated lessons with the IIM invited speakers as teachers. Awards for the best talk, best poster blitz, and best poster have been conferred to young attendees, who became part of the IIM Young Committee, involved in the scientific organization of the IIM meetings. To celebrate the 20th IIM anniversary, a special free-access educational convention on “Causes and mechanisms of muscle atrophy. From terrestrial disuse to Space flights” has been organized, in which IIM experts in the field have illustrated the current knowledge about the muscle atrophy process in several atrophying conditions, and the former Italian astronaut, Paolo Nespoli shared his incredible experience in Space fascinating the large audience attending both in presence and online live stream. The meeting was characterized by a vibrant, friendly, and inclusive atmosphere, and stimulated discussion on emerging areas of muscle research, fostering international collaborations, and confirming the IIM meeting as an ideal venue to discuss around muscle development, function, and diseases pointing to the development of efficacious therapeutic strategies. Here, the abstracts of the meeting illustrate the most recent results on basic, translational, and clinical research in the myology field. Some abstracts are missing as per authors’ decision due to the patentability of the results

Targeting RAGE prevents muscle wasting and prolongs survival in cancer cachexia

Background: Cachexia, a multifactorial syndrome affecting more than 50% of patients with advanced cancer and responsible for ~20% of cancer-associated deaths, is still a poorly understood process without a standard cure available. Skeletal muscle atrophy caused by systemic inflammation is a major clinical feature of cachexia, leading to weight loss, dampening patients' quality of life, and reducing patients' response to anticancer therapy. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily and a mediator of muscle regeneration, inflammation, and cancer. Methods: By using murine models consisting in the injection of colon 26 murine adenocarcinoma&nbsp;(C26-ADK) or Lewis lung carcinoma (LLC) cells in BALB/c and C57BL/6 or Ager−/− (RAGE-null) mice, respectively, we investigated the involvement of RAGE signalling in the main features of cancer cachexia, including the inflammatory state. In vitro experiments were performed using myotubes derived from C2C12 myoblasts or primary myoblasts isolated from C57BL/6 wild type and Ager−/− mice treated with the RAGE ligand, S100B (S100 calcium-binding protein B), TNF (tumor necrosis factor)α±IFN (interferon) γ, and tumour cell- or masses-conditioned media to analyse hallmarks of muscle atrophy. Finally, muscles of wild type and Ager−/− mice were injected with TNFα/IFNγ or S100B in a tumour-free environment. Results: We demonstrate that RAGE is determinant to activate signalling pathways leading to muscle protein degradation in the presence of proinflammatory cytokines and/or tumour-derived cachexia-inducing factors. We identify the RAGE ligand, S100B, as a novel factor able to induce muscle atrophy per se via a p38 MAPK (p38 mitogen-activated protein kinase)/myogenin axis and STAT3 (signal transducer and activator of transcription 3)-dependent MyoD (myoblast determination protein 1) degradation. Lastly, we found that in cancer conditions, an increase in serum levels of tumour-derived S100B and HMGB1 (high mobility group box 1) occurs leading to chronic activation/overexpression of RAGE, which induces hallmarks of cancer cachexia (i.e. muscle wasting, systemic inflammation, and release of tumour-derived pro-cachectic factors). Absence of RAGE in mice translates into reduced serum levels of cachexia-inducing factors, delayed loss of muscle mass and strength, reduced tumour progression, and increased survival. Conclusions: RAGE is a molecular determinant in inducing the hallmarks of cancer cachexia, and molecular targeting of RAGE might represent a therapeutic strategy to prevent or counteract the cachectic syndrome

S100B Protein, A Damage-Associated Molecular Pattern Protein in the Brain and Heart, and Beyond

S100B belongs to a multigenic family of Ca2+-binding proteins of the EF-hand type and is expressed in high abundance in the brain. S100B interacts with target proteins within cells thereby altering their functions once secreted/released with the multiligand receptor RAGE. As an intracellular regulator, S100B affects protein phosphorylation, energy metabolism, the dynamics of cytoskeleton constituents (and hence, of cell shape and migration), Ca2+ homeostasis, and cell proliferation and differentiation. As an extracellular signal, at low, physiological concentrations, S100B protects neurons against apoptosis, stimulates neurite outgrowth and astrocyte proliferation, and negatively regulates astrocytic and microglial responses to neurotoxic agents, while at high doses S100B causes neuronal death and exhibits properties of a damage-associated molecular pattern protein. S100B also exerts effects outside the brain; as an intracellular regulator, S100B inhibits the postinfarction hypertrophic response in cardiomyocytes, while as an extracellular signal, (high) S100B causes cardiomyocyte death, activates endothelial cells, and stimulates vascular smooth muscle cell proliferation

S100B's double life: Intracellular regulator and extracellular signal

AbstractThe Ca2+-binding protein of the EF-hand type, S100B, exerts both intracellular and extracellular functions. Recent studies have provided more detailed information concerning the mechanism(s) of action of S100B as an intracellular regulator and an extracellular signal. Indeed, intracellular S100B acts as a stimulator of cell proliferation and migration and an inhibitor of apoptosis and differentiation, which might have important implications during brain, cartilage and skeletal muscle development and repair, activation of astrocytes in the course of brain damage and neurodegenerative processes, and of cardiomyocyte remodeling after infarction, as well as in melanomagenesis and gliomagenesis. As an extracellular factor, S100B engages RAGE (receptor for advanced glycation end products) in a variety of cell types with different outcomes (i.e. beneficial or detrimental, pro-proliferative or pro-differentiative) depending on the concentration attained by the protein, the cell type and the microenvironment. Yet, RAGE might not be the sole S100B receptor, and S100B's ability to engage RAGE might be regulated by its interaction with other extracellular factors. Future studies using S100B transgenic and S100B null mice might shed more light on the functional role(s) of the protein

Influence of Ageing Time and Method on Beef Quality and Safety

The effectiveness of dry ageing with regard to retaining meat quality is still subject to debate. At 4 d post mortem, samples of boneless strip loins were excised from young Charolais carcasses and then stored for a further 26 d in a cooler, either vacuum-packaged (VP) or dried-aged (DA). Loin samples were also dissected 7 d post mortem as a control treatment (CT). Chemical, instrumental and microbiological data (n = 18) were determined in longissimus dorsi and underwent ANOVA to estimate the differences in the ageing fixed factor split into two orthogonal contrasts: control vs. aged and VP vs. DA. Ageing loss (both surface dehydration and water purge) was greater in DA compared to VP samples, resulting in the lowest moisture content and highest crude protein and fat percentage in DA loins. The ageing method did not affect meat surface colour, except for redness, which had the lowest value in DA samples. Meat tenderness improved a similar amount following both VP and DA ageing treatments. Compared to the control, prolonged ageing raised both the peroxide value and the total microbial count, especially in DA samples, though both remained within the recommended limits. In summation, both ageing methods improved beef meat tenderisation, preserving its shelf life

Absence of RAGE in an animal experimental model of Duchenne muscular dystrophy results in reduced muscle necrosis and inflammation

Duchenne muscular dystrophy (DMD) is a lethal X-linked neuromuscular disorder characterized by progressive muscle degeneration due to lack of dystrophin, a protein essential for the integrity of sarcolemma during contraction. Chronic inflammation is a hallmark of muscles in DMD subjects, and contributes to progressive muscle wasting. RAGE (receptor for advanced glycation end-products) is a multiligand receptor of the immunoglobulin superfamily involved in physiological and pathological processes including inflammation and myogenesis [1]. While absent in healthy adult muscle tissue, RAGE is expressed in regenerating myofibers during muscle regeneration [2,3], in dystrophic muscles and activated immune cells. To have information about the role of RAGE in the pathophysiology of DMD we generated a double mutant mouse lacking dystrophin and RAGE (mdx/Ager–/– mouse) by cross-breeding dystrophic (mdx) mice with RAGE-null (Ager-/-) mice. Comparison of Quadriceps femoris of mdx and mdx/Ager–/– mice at different ages (i.e., 2, 3, 4 and 5 weeks, and 6 and 12 months of age) showed that the absence of RAGE in dystrophic mice did not affect the onset of the pathology. However, compared with age-matched mdx mice, muscles of 5 week- and 6 and 12 month-old mdx/Ager–/– mice showed i) significantly reduced numbers of necrotic myofibers, ii) a shift towards higher values of the cross-sectional areas (CSA) of myofibers, which was also evident in regenerating (centrally-nucleated) myofibers, and iii) reduced areas of immune cell infiltrate. The expression of MAC3, a marker of activated macrophages, was strongly reduced in muscles of mdx/Ager–/– mice compared with mdx mice. Moreover, muscles of mdx/Ager–/– mice exhibited significantly reduced PAX7+ve and myogenin+ve cell numbers, suggesting a reduced recruitment of muscle precursor cells and more efficient regeneration in dystrophic mice lacking RAGE. Our results suggest that RAGE may sustain inflammatory and degenerative processes in dystrophic muscles, and the inhibition of its expression/activity might represent a potential therapeutic approach in DMD patients.This work was supported by grants from MIUR 2012N8YJC3, AFM-Téléthon 16812 and Fondazione CRP 2015.0325.021

Identification of withania somnifera-silybum marianum-trigonella foenum-graecum formulation as a nutritional supplement to contrast muscle atrophy and sarcopenia

Background: Muscle atrophy, i.e., the loss of skeletal muscle mass and function, is an unresolved problem associated with aging (sarcopenia) and several pathological conditions. The im-balance between myofibrillary protein breakdown (especially the adult isoforms of myosin heavy chain, MyHC) and synthesis, and the reduction of muscle regenerative potential are main causes of muscle atrophy. Methods: Starting from one-hundred dried hydroalcoholic extracts of medical plants, we identified those able to contrast the reduction of C2C12 myotube diameter in well-characterized in vitro models mimicking muscle atrophy associated to inflammatory states, glucocorticoid treatment or nutrient deprivation. Based on their ability to rescue type II MyHC (MyHC-II) expression in atrophying conditions, six extracts with different phytochemical profiles were selected, mixed in groups of three, and tested on atrophic myotubes. The molecular mechanism underpinning the effects of the most efficacious formulation, and its efficacy on myotubes obtained from muscle biopsies of young and sarcopenic subjects were also investigated. Results: We identified WST (Withania som-nifera, Silybum marianum, Trigonella foenum-graecum) formulation as extremely efficacious in protecting C2C12 myotubes against MyHC-II degradation by stimulating Akt (protein kinase B)-dependent protein synthesis and p38 MAPK (p38 mitogen-activated protein kinase)/myogenin-dependent myoblast differentiation. WST sustains trophism in C2C12 and young myotubes, and rescues the size, developmental MyHC expression and myoblast fusion in sarcopenic myotubes. Conclusion: WST strongly counteracts muscle atrophy associated to different conditions in vitro. The future validation in vivo of our results might lead to the use of WST as a food supplement to sustain muscle mass in diffuse atrophying conditions, and to reverse the age-related functional decline of human muscles, thus improving people quality of life and reducing social and health-care costs

S100B protein regulates myoblast and macrophage functions in skeletal muscle regeneration

Regeneration of acutely injured skeletal muscles relies on a tightly controlled chain of cellular and molecular events, but a complete picture of factors concurring to the regeneration process is still missing. Extracellular S100B protein inhibits myoblast differentiation and stimulates myoblast proliferation by activating its canonical receptor, RAGE (receptor for advanced glycation endproducts), or bFGF/FGFR1 depending on myoblast density (1-4). S100B is released by damaged muscle tissue early after injury in advance of bFGF release, with declining release thereafter (4). We show that S100B is required for correct timing of skeletal muscle regeneration after acute injury. S100B expands the myoblast population, attracts macrophages to damage sites, promotes macrophage polarization into M2 (pro-regenerative) phenotype and reduces fibroblast proliferation. Also, S100B is transiently induced in and released by infiltrating macrophages under the action of proinflammatory and antiinflammatory cytokines, and effects of macrophage-derived S100B sum up with those of myofiber-released S100B. S100B’s effects are mediated by RAGE during the first 3 days after injury, however during the myoblast proliferation phase/macrophage M2 phase (i.e. at days 4-6 post-injury) S100B also activates bFGF-FGFR1 to stimulate myoblast proliferation and macrophage M1/M2 transition. Thus, S100B is a major molecular determinant of timed muscle regeneration after acute injury by virtue of its regulatory effects on myoblasts and macrophages.This work was supported by grants from MIUR PRIN-2010R8JK2X_004, AFM-Téléthon 16260 and Fondazione CRP 2012.0241.021