To controls [20]. Thus, in the current study, it appears that compensatory dilatory mechanisms served to maintain normal blood flow under baseline conditions in PD. The presence of high blood lactate (a potent vasodilator [35]) in PD likely contributed to buffering the effects of augmented sympathetic vascular modulation. In support of our data, others have shown that insulin resistance [36] and type 2 diabetes [37] are associated with heightened lactate levels. Our observations of augmented baseline Y1R and a1R activation in PD are complemented by our findings that PD had greater NPY concentration and Y1R and a1R expression in hindlimb skeletal muscle. Neuropeptide Y is produced in sympathetic neuronal cell soma and packaged into secretory large dense-cored vesicles and undergoes axonal transport (the rate of which is SNA level dependent) to the axon terminal where it is released and eventually degraded by enzymes in the synaptic cleft [38]. This is in contrast to NE, which is produced in sympathetic nerve terminal, released, and eventually taken back up into the nerve terminal [39]. Based on the unique origin and fate of NPY, it can be reasonably inferred that increased skeletal muscle NPY concentration measured in PD was a result of one or a combination of the following: i) augmented sympathetic neuronal density; ii) increased production and axonal transport of NPY; and/or iii) increased NPY release into skeletal muscle interstitium. This line of reasoning falls in line with work by others who reported sympathetic nerve hyperactivity in insulin resistant and type 2 diabetic subjects, as well as heightened plasma NPY levels in type 2 diabetic patients [40,41]. Beyond this, in vivo studies investigating NPY levels and Y1R/a1R expression in pre-diabetes are limited, however 
increased Y1R mRNA expression has been reported in cardiac I-BRD9 cost tissue of diabetic rats [42] and it was shown that rat vascular smooth muscle cells treated with high levels of insulin resulted in upregulation of a1R [43].LimitationsWe used hindlimb muscle homogenate in order to quantify the receptors located along downstream resistance arterioles, as these vessels are responsible for modulating flow at the level of the femoral artery. Previous work indicates that peripheral Y1Rs are predominantly associated with vasculature [44]. In contrast, a1Rs have been identified on skeletal muscle fibers in rats, however the density of those located in muscle fibers is negligible compared to a1R expression on resistance arterioles [45]. Based on past reports and the internal consistency between our functional and cellular data, we are confident that our reported differences in ligand concentration and receptor expression reasonably reflect what is occurring at the level of the vasculature.Figure 7. a1R expression is augmented in PD. Western blot analysis of a1R expression (,42 kDa) in hindlimb muscle homogenate of CTRL (n = 6 per muscle group) and PD (n = 6 per muscle group). PD had greater a1R expression in red vastus muscle, compared to CTRL. * Indicates BTZ-043 biological activity different from CTRL (p,0.05). doi:10.1371/journal.pone.0046659.gPre-Diabetes and Sympathetic Vascular ControlWe measured skeletal muscle tissue NPY concentration instead of plasma NPY levels for several reasons. Indeed, repeated blood sampling poses the risk of evoking hypotension and increases in sympathetic nerve activity. As well, plasma NPY levels represent a mixed sample originating from several sources throughout the body. I.To controls [20]. Thus, in the current study, it appears that compensatory dilatory mechanisms served to maintain normal blood flow 
under baseline conditions in PD. The presence of high blood lactate (a potent vasodilator [35]) in PD likely contributed to buffering the effects of augmented sympathetic vascular modulation. In support of our data, others have shown that insulin resistance [36] and type 2 diabetes [37] are associated with heightened lactate levels. Our observations of augmented baseline Y1R and a1R activation in PD are complemented by our findings that PD had greater NPY concentration and Y1R and a1R expression in hindlimb skeletal muscle. Neuropeptide Y is produced in sympathetic neuronal cell soma and packaged into secretory large dense-cored vesicles and undergoes axonal transport (the rate of which is SNA level dependent) to the axon terminal where it is released and eventually degraded by enzymes in the synaptic cleft [38]. This is in contrast to NE, which is produced in sympathetic nerve terminal, released, and eventually taken back up into the nerve terminal [39]. Based on the unique origin and fate of NPY, it can be reasonably inferred that increased skeletal muscle NPY concentration measured in PD was a result of one or a combination of the following: i) augmented sympathetic neuronal density; ii) increased production and axonal transport of NPY; and/or iii) increased NPY release into skeletal muscle interstitium. This line of reasoning falls in line with work by others who reported sympathetic nerve hyperactivity in insulin resistant and type 2 diabetic subjects, as well as heightened plasma NPY levels in type 2 diabetic patients [40,41]. Beyond this, in vivo studies investigating NPY levels and Y1R/a1R expression in pre-diabetes are limited, however increased Y1R mRNA expression has been reported in cardiac tissue of diabetic rats [42] and it was shown that rat vascular smooth muscle cells treated with high levels of insulin resulted in upregulation of a1R [43].LimitationsWe used hindlimb muscle homogenate in order to quantify the receptors located along downstream resistance arterioles, as these vessels are responsible for modulating flow at the level of the femoral artery. Previous work indicates that peripheral Y1Rs are predominantly associated with vasculature [44]. In contrast, a1Rs have been identified on skeletal muscle fibers in rats, however the density of those located in muscle fibers is negligible compared to a1R expression on resistance arterioles [45]. Based on past reports and the internal consistency between our functional and cellular data, we are confident that our reported differences in ligand concentration and receptor expression reasonably reflect what is occurring at the level of the vasculature.Figure 7. a1R expression is augmented in PD. Western blot analysis of a1R expression (,42 kDa) in hindlimb muscle homogenate of CTRL (n = 6 per muscle group) and PD (n = 6 per muscle group). PD had greater a1R expression in red vastus muscle, compared to CTRL. * Indicates different from CTRL (p,0.05). doi:10.1371/journal.pone.0046659.gPre-Diabetes and Sympathetic Vascular ControlWe measured skeletal muscle tissue NPY concentration instead of plasma NPY levels for several reasons. Indeed, repeated blood sampling poses the risk of evoking hypotension and increases in sympathetic nerve activity. As well, plasma NPY levels represent a mixed sample originating from several sources throughout the body. I.
