The influence of sex and the human serum environment on fuel storage, skeletal muscle metabolism, and insulin sensitivity

Abstract

Type 2 diabetes (T2D) is a growing global health concern with notable sex-based differences in its pathology. Despite having lower muscle mass and greater fat mass, pre-menopausal females are more insulin sensitive and have a lower prevalence of T2D than males. These discrepancies may be the result of incomplete understanding of the molecular mechanisms underlying glycemic control, insulin resistance and the relationship between sex and insulin sensitivity. As skeletal muscle is the predominate tissue responsible for peripheral insulin sensitivity, the overall purpose of this thesis was to delineate the contribution of sex on factors that affect skeletal muscle insulin sensitivity. The primary aims of this thesis were to determine if 1) sex differences existed in mitochondrial or lipid storage characteristics in aerobically matched participants, 2) sex hormones correlate with metabolic changes in skeletal muscle, and 3) the human serum environment influences proteins associated with metabolism and insulin signaling in mouse skeletal muscle cells (C2C12). In study 1 and 2, muscle biopsies were taken for the determination of mitochondrial and intramyocellular (IMCL) content using electron microscopy and content and activity of proteins related to mitochondrial function and lipid metabolism using western blot and activity assays. Both mitochondrial and IMCL storage characteristics were similar between females and males. However, females have a greater number of mitochondria interacting with IMCL in the subsarcolemmal region (SS) of muscle (p=0.05). For studies 3, 4, and 5, blood was collected from females in the follicular phase and luteal phase of the menstrual cycle and males. Samples were analyzed for estradiol, progesterone, testosterone, insulin, and glucose using commercially available assays. Differentiated C2C12 cells were incubated in 2% human serum for 24 hours and mRNA expression (RT-qPCR), glucose uptake (2-NBDG assay), and protein expression and activation (WB) were measured. We determined that human serum altered mRNA expression, such that estrogen receptor alpha (ERα) expression correlated with estradiol in male serum (Pearson’s r=0.64, p=0.05), but not in female follicular or female luteal serum in C2C12 cells relative to cells incubated with horse serum. Myosin heavy chain I (MHCI) gene expression correlated with estradiol in female follicular (Pearson’s r=0.71, p=0.05) and male serums (Pearson’s r=0.67, p=0.02), but not in female luteal serum in C2C12 cells. Interestingly, the ratio between testosterone and estradiol was positively correlated with ERα gene expression in female follicular serum (Pearson’s r=0.82, p=0.03), but not in female luteal or male serums in C2C12 cells. Further, in C2C12 cells, the activation of protein kinase B (AKT) sans insulin was greater following treatment with female luteal serum than males (p=0.02), but not than female follicular serum, with no difference between female follicular compared to male serums despite similarities in fasting insulin concentrations. Similarly, in C2C12 cells, the activation of phosphatase and tensin homolog (PTEN) without insulin stimulation was lower with female luteal serum when compared to female follicular serum (p=0.05), but not when compared with male serum, with no difference between female follicular and male serum. Finally, glucose uptake with insulin stimulation was increased in female follicular serum (p=0.006), but not in C2C12 cells treated with female luteal or male serums. These findings suggest that insulin sensitivity of skeletal muscle is likely, at least in part, controlled by the hormonal environment in which it is surrounded. Overall, the findings of this thesis found that: 1) aerobically matched females and males have similar mitochondrial characteristics, 2) aerobically matched females and males have similar lipid storage characteristics, 3) sex hormone environment correlates with changes in mRNA, 4) human serum did influence the activation status of proteins associated with insulin-stimulated glucose uptake, and 5) human serum alters insulin-dependent glucose uptake in a similar pattern as seen in whole body trials. These findings provide new insights into the distinct roles of skeletal muscle morphology and serum composition in regulating insulin sensitivity, helping to clarify the interplay between sex, metabolism, and insulin resistance.