title: Lifestyle influences on cardiometabolic health: systemic and cellular mechanisms
Creators: name: McCullough, D
Publisher: name: Liverpool John Moores University
Year: 2022

description: Introduction: Cardiometabolic disease (CMD) encompasses all metabolic and cardiovascular disorders including metabolic abnormalities such as dyslipidaemia, hypertension, insulin resistance, abdominal obesity and inflammation. Skeletal muscle accounts for the largest increase in insulin stimulated glucose uptake. In accordance with this, skeletal muscle mass and function has an inverse association with CMD, type 2 diabetes (T2D) and cardiovascular disease (CVD). Poor lifestyle choices can disrupt the metabolic environment causing skeletal muscle insulin resistance further exacerbating CMD and risk of T2D and CVD. Low carbohydrate (LC) diets have shown to improve markers of CMD. However, the impact of the metabolic environment on skeletal muscle insulin dynamics are yet to be investigated. Anabolic-androgenic steroid (AAS) are derived from the hormone testosterone which plays a key role in regulating skeletal muscle mass and cardiometabolic health. Paradoxically, although AAS increase muscle mass, supraphysiological levels of testosterone increase CMD and risk of CVD. Further research is warranted on markers of CMD among AAS users in addition to its impact on the metabolic environment and subsequent skeletal muscle insulin mechanisms. Therefore, this thesis aims to investigate how markers of CMD are impacted by diets differing in carbohydrate intake and anabolic-androgenic steroid use and how their subsequent metabolic environment impacts skeletal muscle cellular mechanisms. Methods: In a randomised parallel design, willing participants followed either a VLC or high carbohydrate (HC) and provided anthropometrics, blood samples, physical activity and food diaries at 0, 4 and 8 weeks. In a cross-sectional design, resistance-trained males who use AAS and did not use (NAT) provided similar measures. Blood samples were processed for plasma and serum and analysed for markers of CMD. Mechanisms of insulin signalling were investigated using the murine C2C12 skeletal muscle cell line. Under standard cell culture procedures (Differentiating media: DM), C2C12 myoblasts and myotubes were stimulated with insulin to investigate cellular signalling and glucose uptake. To investigate the impact of diet or AAS use on insulin signalling, C2C12 myoblasts and myotubes were incubated in serum derived from participants and stimulated with insulin. The impact of human serum on C2C12 metabolism was also determined. Results: Both diets significantly (P &lt; 0.05) improved triglyceride, insulin, HOMA IR and leptin:adiponectin levels. Compared with HC, the VLC diet showed significantly (P &lt; 0.05) greater improvements in blood pressure, fat mass, leptin and small dense low-density lipoprotein cholesterol (sdLDL-C) low-density lipoprotein cholesterol ratio. The VLC diet also reported trends in improving sdLDL-C, FGF21 and waist circumference (P = 0.06). In myoblasts, serum from both groups showed a tendency (P = 0.07) of increasing Akt phosphorylation (p-AKT) but had no effect on the insulin stimulated fold-change. In myotubes, both diets showed a significant (P &lt; 0.01) decrease in p-Akt but had no effect on the insulin stimulated fold-change. Insulin stimulated glucose uptake also did not significantly change with either diet. In myotubes, both diets resulted in a significant (P &lt; 0.05) decrease in p-AMPKthr172 and which may have caused the reduction in p-Akt in myotubes. The AAS group showed significantly (P &lt; 0.05) lower levels of high-density lipoprotein cholesterol (HDL-C), apolipoprotein A1 (ApoA1) and plasminogen activator inhibitor 1. Myoblasts and myotubes significantly (P &lt; 0.01) increased p-Aktser473 in response to insulin stimulation in NAT and AAS conditions. No difference was observed between groups. CK activity significantly (P &lt; 0.01) increased comparably between conditions yet protein abundance was significantly (P = 0.04) higher in AAS serum vs. DM after 96 hr. In myoblasts, serum significantly (P &lt; 0.05) increased Aktser473, mTORser2448, ERK1/2T202/Y204 and P38t180/Y182 phosphorylation. ERK and P38 phosphorylation was significantly (P &lt; 0.05) higher compared to DM. In myotubes, all conditions similarly significantly (P &lt; 0.05) increased p-Aktser473 and p-ERKT202/Y204 at 30 mins. MPS and the rate of MPB was similar between groups at all timepoints. Conclusion: In summary, both a VLC and HC diet showed similar improvements in some markers of CMD. However, a VLC diet induced greater improvements in blood pressure, fat mass and sdLDL/LDL-C. Serum from both groups reduced cellular energy stress as shown by the reduction in p-AMPK which is likely to have increased insulin sensitivity at baseline. AAS users display reduced HDL-C and ApoA1 leading to increased CVD risk. Serum derived from AAS users does not impair insulin signalling or glucose uptake compared to non-users. Serum derived from resistance trained males may enhance skeletal muscle differentiation similarly to AAS users via increased P38 and ERK activity compared with DM.