Effects of Vitamin D and Resistance Exercise Training on Neuromuscular Health and Function in an Obese and Pre-Diabetic Mouse Model

Obesity is a known pathological condition associated with impaired glucose homeostasis leading to type 2 diabetes and increased susceptibility to loss of skeletal muscle mass and function. Recent investigations have demonstrated potential beneficial effects of vitamin D on insulin sensitivity and skeletal muscle mass and function similarly to the adaptations induced by resistance exercise training (RT). The main objectives of the present study were 1) to investigate physical and metabolic characteristics of a newly introduced mouse model of obesity and insulin resistance, p62 knock-out (KO) mice, in comparison to wildtype control mice (WC) and 2) to evaluate the efficacy of 10 weeks of vitamin D administration with or without RT to reduce adiposity and to alleviate the adverse effects of obesity on neuromuscular function, systemic insulin sensitivity, muscle/myofiber hypertrophy, and myofiber insulin signaling/inflammatory/myogenic regulators in p62 KO mice. In the present study, forty 24-week old p62 KO mice and ten sex-/age-match non-obese WC were utilized. In addition to non-obese, age-/gender-matched WC (n=10), p62 KO mice (n=40) were randomly assigned into one of four experimental groups (10/group) for 10 weeks: p62 KO control group (p62C, no treatment), vitamin D administration group (VD, 75 IU of vitamin D3 (cholecalciferol) 1/3d), resistance exercise training group (RX, ladder climbing 3/wk), or combined treatment group (VRX, VD + RX). Body weight and food intake were monitored in all groups biweekly over the 10-week intervention. In vivo measurements including body composition (dual-energy X-ray absorptiometry, DXA), skeletal muscle function (i.e., grip strength and inclined plane test), and oral glucose tolerance test (OGTT), were performed in all groups at pre- and post-intervention. Upon completion of the intervention, blood samples, the hindlimb muscles, and the spleen were collected from mice in all groups for in vitro analysis including serum vitamin D concentration, tissue wet weights, myofiber cross-sectional area (CSA), and myofiber protein expression levels of regulatory molecules for muscle growth [i.e., mammalian target of rapamycin (mTOR), protein kinase B (AKT), atrogin-1, muscle RING-finger protein-1 (MuRF-1)] and insulin signaling [i.e., insulin receptor ß (IR-ß) and glucose transporter type 4 (GLUT4)], pro-inflammation [i.e., tumor necrosis factor-α (TNF-α)] and bioactivity of vitamin D [i.e., vitamin D receptor (VDR)]. In the present study, p62C exhibited greater daily food intake (+17.1%) as well as increased adiposity compared to WC which involved greater total body mass (TBM) (24 weeks; +18.0%, 34 weeks: 26.2%), fat mass (FM) (24 weeks: +46.7%, 34 weeks: +69.3%), body fat percentage (%FM) (24 weeks: +28.8%, 34 weeks: +31.6%) as well as bone mineral density (BMD) (24 weeks: +8.5%, 34 weeks: +16.5%). In addition, p62C showed significantly increased fasting blood glucose levels (fBG) (+30.0%) and diminished blood glucose clearance leading to greater blood glucose levels during the OGTT [post-15 min blood glucose level (p15BG): +40.5%, post-30 min blood glucose level (p30BG): +28.3%, post-60 min blood glucose level (p60BG): +27.5%] compared to WC. p62C showed significantly lower relative grip strength (-21.7%) and sensorimotor function (-11.4%) despite greater myofiber CSA (+33.3%) compared to WC at the age of 34 weeks. The spleen mass was greater in p62C (+63.7%) than WC. Protein expression levels of total mTOR (+27.1%) and phosphorylated mTOR (+96.6%) in the soleus, total AKT in the soleus (+12.8%) and the gastrocnemius (+53.8%) were greater in p62C compared to WC. Muscle protein expression levels of MuRF-1 (+35.4%) and TNF-α (+200.4%) were higher in p62C compared to WC. Daily food intake, body weight (BW), and BMD were not different between p62C, VD, RX, and VRX. TBM elevation in p62C was attenuated in the intervention groups (VD: +13.5%, RX: +8.3%, VRX: +8.7% vs. p62C: +16.8%), which was associated with preserved FM and %FM in VD, RX, and VRX. VD (+61.1%) and VRX (+37.6%) exhibited greater circulating vitamin D levels compared to p62C. p62C exhibited significant loss of grip strength (absolute: -22.5%, relative: -38.9) and sensorimotor function (-12/8%) over time. In contrast, RX maintained both grip strength and sensorimotor function, and VD and VRX maintained grip strength after the 10-week intervention. Only RX and VRX displayed improved glucose tolerance which suppressed increases in p15BG (p62C: +16.0% vs. RX & VRX: no change), and lowered p30BG (RX: -40.0%, VRX: -36.9%) compared to p62C. No difference was observed between VD and p62C for tissue wet weights of the hindlimb muscles and the spleen, and myofiber CSA. However, RX exhibited lower spleen mass compared to p62C (-26.0%). Muscle protein expression profiles related to myofiber growth, protein degradation, glucose uptake, pro-inflammation, and the bioactivity of vitamin D were not changed by the interventions. In the present study adult p62 KO mice developed obesity, shown as increased adiposity, impaired glucose homeostasis, attenuated physical function, and an enlarged spleen. In addition, the present findings demonstrated that 10 weeks of vitamin D administration with or without RT or RT alone attenuated the progression of obesity and improved neuromuscular function in p62 KO mice. In addition, RT with or without vitamin D was effective in improving insulin sensitivity and systemic inflammation in p62 KO mice. In contrary, vitamin D administration alone was unable to induce favorable changes in insulin sensitivity, wet weights of skeletal muscle and spleen, myofiber CSA, and protein expression profiles of regulatory factors. Moreover, neither synergistic nor additive effects were noted when vitamin D administration was combined with RT.