Investigation of pharmacological compounds to increase skeletal muscle glucose uptake in vitro and in vivo

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Background and aims: A healthy metabolism and proper regulation of blood sugar levels are key factors to maintain the quality of our daily lives. In these, a significant role can be played by skeletal muscle, which constitutes a substantial portion of the body. Beside adipose tissue, skeletal muscle also expresses the unique type 4 glucose transporter (GLUT4) responsible for glucose uptake. The GLUT4 translocation to the plasma membrane, and consequently glucose uptake involves complex regulatory mechanisms. Initiators of this process include insulin binding to the insulin receptor, which can regulate Akt substrate of 160 kDa (AS160; also known as TBC1D4) through the phosphoinositide 3-kinase (PI3K)/ 3-phosphoinositide-dependent protein kinase 1 (PDK1)/ Akt axis. AMP-activated protein kinase (AMPK), which senses cellular energy status, is also capable of regulating GLUT4 translocation via AS160. However, in case of insulin resistance and consequently in diabetes the GLUT4 translocation becomes impaired. As a result, blood glucose levels remain consistently high, giving rise to numerous long-term complications such as cardiovascular diseases, peripheral neuropathy, retinopathy, and diabetic nephropathy. Although the dramatically increasing number of patients makes it increasingly urgent to find a solution, so far, only a few compounds have been discovered capable of improving GLUT4 translocation and normalizing blood glucose levels. The possibility has emerged that some less explored pathways in GLUT4 translocation,such as the bone morphogenetic protein (BMP) signaling pathway, might influence blood glucose regulation. Recently, it has been discovered that BMPs can sensitize tissues to insulin, increase the amount of GLUT4, reduce blood glucose levels through the DK1/PI3K/Akt pathway, thus enhancing GLUT4 translocation, and changes in their serum levels correlate with diabetes. Since oxidative stress is known to play a role in the development of insulin resistance and diabetes, maintaining redox balance is of paramount importance. Astaxanthin is an exceptionally potent antioxidant capable of reducing high glucose- or fatty acid-induced reactive oxygen species (ROS) formation, improving insulin sensitivity, and enhancing glucose uptake. Moreover, astaxanthin is also easily available, as it is present in numerous marine organisms and consequently in our seafoods. Therefore, our aim was to investigate the impact of a BMP-inducing agent, tilorone, on skeletal muscle glucose uptake in vitro and in vivo. Additionally, we aimed to explore how 8 astaxanthin, as a potent antioxidant, can influence the regulation of proteins crucial for GLUT4 translocation. Materials and methods: The tilorone treatment was conducted at two concentrations (20 and 35 nM) on C2C12 myoblasts for a duration of 40 hours. Additionally, after 5 days of differentiation, tilorone was applied only at the lower concentration (20 nM) on myotubes to understand its acute effects at 2 and 5 hours on myotubes. In both of our in vivo experiments, we used 3-month-old C57BL/6 mice. Two intraperitoneal tilorone injections (25 mg/kg body weight) were administered with a 3-day interval. For the astaxanthin feeding model, the astaxanthin-containing preparation dissolved in ethanol was added to the mice's normal food at a dosage of 4 g/kg for a duration of 4 weeks. We used qRT-PCR to determine the transcription of BMPs, as well as GLUT1 and GLUT4. Western blot technique was used to examine Smad1/5/8 activation, the levels of GLUT1 and GLUT4, and the phosphorylation changes of molecules involved in GLUT4 translocation. Immunofluorescence labeling and microscopy were also utilized to assess GLUT4 quantities. Using glucose analogue, 18F-fluoro-2-deoxyglucose (18FDG), we investigated cellular glucose uptake. The specific inhibitor of GLUT1 was used to specify GLUT1-associated 18FDG uptake. Myoblast itochondrial function was assessed through high-resolution respirometry. Additionally, tissue glucose uptake was measured in vivo using 18FDG-PET/CT imaging. Key results: Tilorone is capable of inducing multiple BMPs (BMP2, BMP4, BMP7, and BMP14) in myoblasts and activating the BMP-mediated Smad1/5/8 and Smad4. As a result of tilorone treatment, GLUT4 levels increased, and Akt2/AS160 signaling was activated. The uptake of the labeled glucose also increased, which was not accompanied by an increase in mitochondrial O2 consumption; in fact, the basal and ATP-linked respiration decreased. AS160 phosphorylation and glucose uptake were elevated in myotubes as well. Additionally, tilorone was able to further enhance insulin-stimulated Akt2 phosphorylation and 18FDG uptake. In vivo, tilorone increased 18FDG uptake in skeletal muscle, adipose tissue, and liver. Furthermore, astaxanthin feeding elevated the activation of proteins important in glucose uptake and metabolism in an in vivo mouse model.

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