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Pre-diabetes affects approximately 25% of North Americans, and its prevalence continues to rise with increased cases of obesity and sedentary lifestyle (Canadian Diabetes Association, 2011, Disease Control and Prevention, 2011). Inherently, pre- diabetes is associated with impairments in cardiovascular health that manifest prior to the onset of overt type 2 diabetes (Faeh, William, Yerly, Paccaud, & Bovet, 2007; Haffner, Stern, Hazuda, Mitchell, & Patterson, 1990; Shin, Lee, & Lee, 2011). Characterized by hyperinsulinemia, insulin resistance, elevated blood glucose and frequently accompanied by obesity, the pathological metabolic characteristics of pre-diabetes play a role in the initiation of cardiovascular complications centrally and peripherally (DeFronzo & Abdul- Ghani, 2011; Gupta et al., 2012; Schaefer et al., 2010); however, our knowledge regarding the effects of pre-diabetes on skeletal muscle arteriolar function is limited.

Skeletal muscle makes up approximately 40% of body mass and contains the greatest proportion of arterioles than any other organ (Janssen et al., 2000). Comprising approximately 20% of the body’s total baseline systemic vascular resistance, skeletal muscle arterioles play a key role in blood pressure regulation at rest and blood flow redistribution during exercise. A hallmark of musculoskeletal health is ability to rapidly match skeletal muscle blood supply to metabolic demand at rest and during physical activity and research in the past decade provides evidence of arteriolar dysregulation in pre-diabetes (Gorczynski et al., 1978, Laughlin and Armstrong, 1982, Fuglevand and Segal, 1997, Lesniewski et al., 2008, Ellis et al., 2010). In 2008, Lesniewski et al. reported increases in vasoconstrictor responsiveness to norepinephrine and endothelin-1 in 1st order arterioles isolated from the gastrocnemius of normotensive pre-diabetic

Zucker Diabetic Fatty (ZDF) rats (Lesniewski et al., 2008). In congruence, we have shown that, despite having normal resting blood flow, sympathetic influences on baseline vascular control are augmented in normotensive pre-diabetic ZDF rats in vivo (Novielli, Al-Khazraji, Medeiros, Goldman & Jackson, 2012). Taken together, it is reasonable to conclude that skeletal muscle arteriolar dysregulation in early pre-diabetes has little to no effect on systemic blood pressure or bulk blood flow to skeletal muscle under resting conditions. However, the conditions described above may render skeletal muscle microcirculation opposable to arteriolar dilation under exercise conditions, leading to microvascular perfusion deficits.

Studies directly investigating the impact of pre-diabetes on skeletal muscle microvascular control during exercise are limited. Certainly, human and animal studies have illustrated impaired skeletal muscle perfusion and O2 delivery/uptake, and compromised blood flow regulation at rest and during exercise/muscle contraction in overt type 2 diabetes, the metabolic syndrome, and obesity (Padilla et al., 2006a, Padilla et al., 2006b, Musa, Torrens & Clough, 2014, Blain, Limberg, Mortensen & Schrage, 2012, Vinet et al., 2011, Karpoff et al., 2009, Frisbee, 2003, Frisbee, 2004, MacAnaney, Reilly, O’Shea, Egana & Green, 2011, Kingwell, Formosa, Muhlmann, Bradley & McConell, 2003). However, differences in the experimental models and methodological limitations generally constrain the current understanding of vascular control in metabolic diseases to bulk blood flow measures. Although such studies have merit, measures of bulk blood flow provide no information on the site or nature of arteriolar dysregulation. Furthermore, models of overt type 2 diabetes, metabolic syndrome, and obesity are accompanied by chronic states of cardiovascular compromise and overt vascular disease;

where early pre-diabetes represents the primary stage of diabetic disease progression, where vascular complications may not be as clear-cut.

Direct observations of arteriolar networks using intravital video microscopy (IVVM) illustrate that healthy microvascular responses to contraction are non-uniform, with greater (relative) arteriolar dilation occurring in distal versus proximal regions (Dodd & Johnson, 1991; Marshall & Tandon, 1984; VanTeeffelen & Segal, 2006). As well, it has been shown that arterioles respond differently to vasoactive substances associated with muscle contraction [(e.g. potassium (M. L. Armstrong, Dua, & Murrant, 2007), adenosine (Murrant & Sarelius, 2002), acetylcholine (VanTeeffelen & Segal, 2006), lactate (Chen, Wolin, & Messina, 1996), nitric oxide (Silveira, Pereira-Da-Silva, Juel, & Hellsten, 2003)] depending on where they reside in the network. Distal arterioles closest to the capillaries are the first to dilate, as the sensitivity of these vessels to metabolic vasoactive substances has been shown to be greater than proximal arterioles (Davis, Hill, & Kuo, 2008). Furthermore, distal arterioles are able to dilate and overcome sympathetic activation more readily than proximal arterioles within the muscle (Anderson & Faber, 1991). Finally, studies demonstrate that sympathetic receptors located on arterioles (responsible for vasoconstriction and maintaining arteriolar tone) are differentially distributed at different branch orders of arteriolar networks, indicating distinct spatial sympathetic arteriolar control (Anderson & Faber, 1991; Moore, Jackson, & Segal, 2010). Since these studies illustrate heterogeneous arteriolar regulation in skeletal muscle under healthy conditions, then it would be ideal to investigate arteriolar function at different levels of continuously branching arteriolar networks in pre-diabetes. Interestingly, deficits in post-exercise capillary perfusion have been demonstrated in

overt type 2 diabetic subjects, where blood flow in the supplying conduit artery was not compromised (Womack et al., 2009). These findings suggest that deficiencies in arteriolar regulation may be apparent in the distal microcirculation before impairments of exercise-evoked blood flow can be detected in large vessels (Kingwell et al., 2003; MacAnaney et al., 2011). However, in an effort to determine the effects of early pre- diabetes on the onset of skeletal muscle microvascular dysregulation, an appropriate model enabling concurrent observation of multiple arteriolar orders is needed.

The gluteus maximus (GM) preparation, developed by Bearden et al. (Bearden, Payne, Chisty, & Segal, 2004), provides a unique model for investigating skeletal muscle arteriolar control using IVVM. Unlike many other experimental skeletal muscle models, the GM is common to both sexes, is found in all mammalian species, and is recruited during locomotion (Bearden et al., 2004). Furthermore, due to its planar arrangement of microvessels and uniform tissue thinness it is optically ideal for IVVM. These properties enable comprehensive evaluation of complete arteriolar networks within a single focal plane.

In an effort to understand the impact of early pre-diabetes on arteriolar network regulation in skeletal muscle, our lab has refined and adapted the GM preparation in a novel murine model of pre-diabetes, The Pound Mouse. Using this model we investigated the effects of pre-diabetes on GM muscle branching arteriolar network function in response to muscle contraction. Herein, we tested the hypothesis that arteriolar dilation and blood flow in response to single tetanic and rhythmic (steady state) muscle contractions would be blunted in pre-diabetes. Furthermore, we predicted that the greatest

decrements in contraction-evoked vasodilation would occur in distal pre-capillary arterioles versus proximal arterioles.