RESUMO
BACKGROUND: The maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. The mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate the effects of Nr2f6 modulation on muscle biology in vivo and in vitro. METHODS: Global RNA-seq was performed in Nr2f6 knockdown C2C12 myocytes (N = 4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and Nr2f6 overexpression C2C12 cell lines (N = 3-6). Nr2f6 content was evaluated in lipid overload models in vitro and in vivo (N = 3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N = 4), ex vivo contractility experiments (N = 5), and histological analysis (N = 7). The conservation of Nr2f6 depletion effects was confirmed in primary skeletal muscle cells of humans and mice. RESULTS: Nr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 knockdown significantly increased the expression of myosin heavy chain genes (two-fold to three-fold) and siRNA-mediated depletion of Nr2f6 increased maximal C2C12 myocyte's lipid oxidative capacity by 75% and protected against lipid-induced cell death. Nr2f6 content decreased by 40% in lipid-overloaded myotubes and by 50% in the skeletal muscle of mice fed a high-fat diet. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofibre content (18%), followed by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an inflammation-like molecular signature and a decrease in the expression of genes involved in muscle contractility and oxidative metabolism, which was associated with the repression of the uncoupling protein 3 (20%) and PGC-1α (30%) promoters activity following Nr2f6 overexpression in vitro. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation. CONCLUSIONS: Our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These results have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.
Assuntos
Contração Muscular , Músculo Esquelético , Animais , Camundongos , Músculo Esquelético/metabolismo , Contração Muscular/fisiologia , Humanos , Linhagem Celular , MasculinoRESUMO
Human islet amyloid polypeptide (hIAPP or amylin) is a hormone co-secreted with insulin by pancreatic ß-cells, and is the main component of islet amyloid. Islet amyloid is found in the pancreas of patients with type 2 diabetes and may be involved in ß-cell dysfunction and death, observed in this disease. Thus, counteracting islet amyloid toxicity represents a therapeutic approach to preserve ß-cell mass and function. In this sense, thiazolidinediones (TZDs), as rosiglitazone, have shown protective effects against other harmful insults to ß-cells. For this reason, we investigated whether rosiglitazone could protect ß-cells from hIAPP-induced cell death and the underlying mechanisms mediating such effect. Here, we show that rosiglitazone improved the viability of hIAPP-exposed INS-1E cells. This benefit is not dependent on the insulin-degrading enzyme (IDE) since rosiglitazone did not modulate IDE protein content and activity. However, rosiglitazone inhibited hIAPP fibrillation and decreased hIAPP-induced expression of C/EBP homologous protein (CHOP) (CTL 100.0 ± 8.4; hIAPP 182.7 ± 19.1; hIAPP + RGZ 102.8 ± 9.5), activating transcription factor-4 (ATF4) (CTL 100.0 ± 3.1; hIAPP 234.9 ± 19.3; hIAPP + RGZ 129.6 ± 3.0) and phospho-eukaryotic initiation factor 2-alpha (p-eIF2α) (CTL 100.0 ± 31.1; hIAPP 234.1 ± 36.2; hIAPP + RGZ 150.4 ± 18.0). These findings suggest that TZDs treatment may be a promising approach to preserve ß-cell mass and function by inhibiting islet amyloid formation and decreasing endoplasmic reticulum stress hIAPP-induced.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Rosiglitazona , Amiloide/metabolismo , Animais , Apoptose , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Ratos , Rosiglitazona/farmacologiaRESUMO
The medicinal uses of Calotropis procera are diverse, yet some of them are based on effects that still lack scientific support. Control of diabetes is one of them. Recently, latex proteins from C. procera latex (LP) have been shown to promote in vivo glycemic control by the inhibition of hepatic glucose production via AMP-activated protein kinase (AMPK). Glycemic control has been attributed to an isolated fraction of LP (CpPII), which is composed of cysteine peptidases (95%) and osmotin (5%) isoforms. Those proteins are extensively characterized in terms of chemistry, biochemistry and structural aspects. Furthermore, we evaluated some aspects of the mitochondrial function and cellular mechanisms involved in CpPII activity. The effect of CpPII on glycemic control was evaluated in fasting mice by glycemic curve and glucose and pyruvate tolerance tests. HepG2 cells was treated with CpPII, and cell viability, oxygen consumption, PPAR activity, production of lactate and reactive oxygen species, mitochondrial density and protein and gene expression were analyzed. CpPII reduced fasting glycemia, improved glucose tolerance and inhibited hepatic glucose production in control animals. Additionally, CpPII increased the consumption of ATP-linked oxygen and mitochondrial uncoupling, reduced lactate concentration, increased protein expression of mitochondrial complexes I, III and V, and activity of peroxisome-proliferator-responsive elements (PPRE), reduced the presence of reactive oxygen species (ROS) and increased mitochondrial density in HepG2 cells by activation of AMPK/PPAR. Our findings strongly support the medicinal use of the plant and suggest that CpPII is a potential therapy for prevention and/or treatment of type-2 diabetes. A common epitope sequence shared among the proteases and osmotin is possibly the responsible for the beneficial effects of CpPII.