Cholecalciferol (vitamin D3) has a direct protective activity against interleukin 6-induced atrophy in C2C12 myotubes.

We previously determined that different vitamin D metabolites can have opposite effects on C2C12 myotubes, depending on the sites of hydroxylation or doses. Specifically, 25(OH)D3 (25VD) has an anti-atrophic activity, 1,25(OH)2D3 induces atrophy, and 24,25(OH)2D3 is anti-atrophic at low concentrations and atrophic at high concentrations. This study aimed to clarify whether cholecalciferol (VD3) too, the non-hydroxylated upstream metabolite, has a direct effect on muscle cells. Assessing the effects of VD3 treatment on mouse C2C12 skeletal muscle myotubes undergoing atrophy induced by interleukin 6 (IL6), we demonstrated that VD3 has a protective action, preserving C2C12 myotubes size, likely through promoting the differentiation and fusion of residual myoblasts and by modulating the IL6-induced autophagic flux. The lack, in C2C12 myotubes, of the hydroxylase transforming VD3 in the anti-atrophic 25VD metabolite suggests that VD3 may have a direct biological activity on the skeletal muscle. Furthermore, we found that the protective action of VD3 depended on VDR, implying that VD3 too might bind to and activate VDR. However, despite the formation of VDR-RXR heterodimers, VD3 effects do not depend on RXR activity. In conclusion, VD3, in addition to its best-known metabolites, may directly impact on skeletal muscle homeostasis.

Haptoglobin Phenotypes Are Associated with the Postload Glucose and Insulin Levels in Pediatric Obesity.

PURPOSE: Haptoglobin (Hp) is a protein involved in the acute-phase reaction of inflammation. Humans have three major phenotypes (Hp1-1, Hp1-2, and Hp2-2). Several studies have shown altered Hp regulation in adults with obesity and metabolic alterations. The Hp2-2 phenotype is associated with a high cardiovascular risk. Our aim was to investigate if Hp levels and the Hp2-2 phenotype are associated with glucose metabolism in pediatric obesity. METHODS: We retrospectively studied 192 participants (92 males and 100 females), aged 4-18 years. Clinical and biochemical data were collected. The Hp phenotype (Hp1-1, Hp1-2, and Hp2-2) was identified through Western immunoblot. RESULTS: Subjects carrying Hp1-1, Hp1-2, and Hp2-2 phenotypes were 13.6, 50.8, and 35.6%, respectively. Hp serum, fasting glucose, and insulin levels, as well as HOMA-IR, were similar among groups. Postload glucose and insulin levels (as insulin AUC) were progressively higher from the Hp1-1 to Hp2-2 phenotype. CONCLUSION: To our knowledge, this is the first study on Hp phenotypes conducted in a pediatric population with obesity. We showed that the presence of Hp2 allele is associated with a worse response of glucose load in terms of both glucose and insulin levels. Thus, the Hp2-2 phenotype could predispose in pediatrics, at the same degree of obesity, to a worse glycemic and insulinemic compensation.

Both ghrelin deletion and unacylated ghrelin overexpression preserve muscles in aging mice.

Sarcopenia, the decline in muscle mass and functionality during aging, might arise from age-associated endocrine dysfunction. Ghrelin is a hormone circulating in both acylated (AG) and unacylated (UnAG) forms with anti-atrophic activity on skeletal muscle. Here, we show that not only lifelong overexpression of UnAG (Tg) in mice, but also the deletion of ghrelin gene (Ghrl KO) attenuated the age-associated muscle atrophy and functionality decline, as well as systemic inflammation. Yet, the aging of Tg and Ghrl KO mice occurs with different dynamics: while old Tg mice seem to preserve the characteristics of young animals, Ghrl KO mice features deteriorate with aging. However, young Ghrl KO mice show more favorable traits compared to WT animals that result, on the whole, in better performances in aged Ghrl KO animals. Treatment with pharmacological doses of UnAG improved muscle performance in old mice without modifying the feeding behavior, body weight, and adipose tissue mass. The antiatrophic effect on muscle mass did not correlate with modifications of protein catabolism. However, UnAG treatment induced a strong shift towards oxidative metabolism in muscle. Altogether, these data confirmed and expanded some of the previously reported findings and advocate for the design of UnAG analogs to treat sarcopenia.

Identification of Haptoglobin as a Readout of rhGH Therapy in GH Deficiency.

BACKGROUND: GH deficiency (GHD) is characterized by a cluster of cardiovascular risk factors and subtle inflammation. We aimed to demonstrate, through a proteomic approach, molecules directly modulated by GHD and involved in the inflammatory state. METHODS: Ten children with isolated GHD were studied before and after 1 year of treatment with rhGH and compared with 14 matched controls. A two-dimensional electrophoresis plasma proteomics analysis was performed at baseline and after GH treatment to identify the top molecules modulated by GH. In vitro studies on human hepatoma (HepG2) cells were performed to validate the data. RESULTS: Twelve of 20 proteomic spots were predicted to be isoforms α and ß of haptoglobin (Hp) and confirmed by liquid chromatography tandem mass spectrometry and Western immunoblot analyses. Hp levels were higher in patients with GHD than controls at baseline (P < 0.001) and were reduced following GH treatment (P < 0.01). In HepG2 cells, both GH and IGF-1 were able to downregulate IL-6-induced Hp secretion. Moreover, Hp secretion was restored in pegvisomant-treated HepG2 cells. CONCLUSIONS: Hp is a molecule acting in the inflammatory state of GHD and a possible biomarker for GH treatment. Nevertheless, the contribution of other factors and the molecular pathways involved in the GH downregulation of Hp remain to be clearly defined.

Opposing effects of 25-hydroxy- and 1α,25-dihydroxy-vitamin D3 on pro-cachectic cytokine-and cancer conditioned medium-induced atrophy in C2C12 myotubes.

AIM: Loss of skeletal muscle is one of the main features of cancer cachexia. Vitamin D (VD) deficiency is associated with impairment of muscle mass and performance and is highly prevalent in cachectic patients; therefore, VD supplementation has been proposed to counteract cancer cachexia-associated muscle loss. However, in both cachectic cancer patients and tumour-bearing animals, VD supplementation led to disappointing results, urging the need for a better understanding of VD activity on skeletal muscle. METHODS: Cancer-associated muscle wasting was reproduced in vitro by treating C2C12 myotubes with cancer cell conditioned medium, a combination of TNF-α and IFNγ or IL-6 pro-cachectic cytokines. The biological effects and mechanisms of action of 1,25-dihydroxy VD (1,25 VD) and its precursor 25-hydroxy VD (25 VD) on myotubes were explored. RESULTS: We demonstrated that only 25 VD was able to protect from atrophy by activating Akt signalling, inducing protein synthesis, and stimulating the autophagic flux, while 1,25 VD had an atrophic activity per se, increasing FoxO3 levels, inducing the expression of atrogenes, and blocking the autophagic flux. Furthermore, we showed that the contrasting activities of these VD metabolites on C2C12 myotubes depend on a differential induction of VD-24-hydroxylase and transformation of VD metabolites in pro-atrophic 24-hydroxylated products, as silencing of VD-24-hydroxylase reduced the atrophic activity of 1,25 VD. CONCLUSIONS: Altogether these data might explain the lack of efficacy of VD treatment in vivo for the protection of muscle mass in cancer.

Ghrelin knockout mice display defective skeletal muscle regeneration and impaired satellite cell self-renewal.

PURPOSE: Muscle regeneration depends on satellite cells (SCs), quiescent precursors that, in consequence of injury or pathological states such as muscular dystrophies, activate, proliferate, and differentiate to repair the damaged tissue. A subset of SCs undergoes self-renewal, thus preserving the SC pool and its regenerative potential. The peptides produced by the ghrelin gene, i.e., acylated ghrelin (AG), unacylated ghrelin (UnAG), and obestatin (Ob), affect skeletal muscle biology in several ways, not always with overlapping effects. In particular, UnAG and Ob promote SC self-renewal and myoblast differentiation, thus fostering muscle regeneration. METHODS: To delineate the endogenous contribution of preproghrelin in muscle regeneration, we evaluated the repair process in Ghrl-/- mice upon CTX-induced injury. RESULTS: Although muscles from Ghrl-/- mice do not visibly differ from WT muscles in term of weight, structure, and SCs content, muscle regeneration after CTX-induced injury is impaired in Ghrl-/- mice, indicating that ghrelin-derived peptides actively participate in muscle repair. Remarkably, the lack of ghrelin gene impacts SC self-renewal during regeneration. CONCLUSIONS: Although we cannot discern the specific Ghrl-derived peptide responsible for such activities, these data indicate that Ghrl contributes to a proper muscle regeneration.