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. 2015 Jan 12;594(8):2223–2231. doi: 10.1113/jphysiol.2014.283549

Muscle microvascular blood flow responses in insulin resistance and ageing

Michelle A Keske 1,✉, Dino Premilovac 1,2, Eloise A Bradley 1, Renee M Dwyer 2, Stephen M Richards 2, Stephen Rattigan 1
PMCID: PMC4933123  PMID: 25581640

Abstract

Insulin resistance plays a key role in the development of type 2 diabetes. Skeletal muscle is the major storage site for glucose following a meal and as such has a key role in maintenance of blood glucose concentrations. Insulin resistance is characterised by impaired insulin‐mediated glucose disposal in skeletal muscle. Multiple mechanisms can contribute to development of muscle insulin resistance and our research has demonstrated an important role for loss of microvascular function within skeletal muscle. We have shown that insulin can enhance blood flow to the microvasculature in muscle thus improving the access of glucose and insulin to the myocytes to augment glucose disposal. Obesity, insulin resistance and ageing are all associated with impaired microvascular responses to insulin in skeletal muscle. Impairments in insulin‐mediated microvascular perfusion in muscle can directly cause insulin resistance, and this event can occur early in the aetiology of this condition. Understanding the mechanisms involved in the loss of microvascular function in muscle has the potential to identify novel treatment strategies to prevent or delay progression of insulin resistance and type 2 diabetes.

Abbreviations

Akt

protein kinase B

eNOS

endothelial nitric oxide synthase

GLUT4

glucose transporter 4

IRS1

insulin receptor substrate 1

IRS2

insulin receptor substrate 2

MAPK

mitogen‐activated protein kinase

NO

nitric oxide

PI3K

phosphoinositide 3‐kinase

TNFα

tumour necrosis factor α

VEGF

vascular endothelial growth factor

Introduction

Skeletal muscle is responsible for up to 80% of insulin‐mediated glucose uptake in the post‐prandial state (Thiebaud et al. 1982). Exchange of nutrients between blood (or plasma) and tissue depends on (i) the permeability of the microvasculature, (ii) the surface area of the microvasculature, and (iii) the rate of blood flow through these vessels (Renkin, 1968). Here we review the literature demonstrating that control of the microvasculature in skeletal muscle is an important response for insulin and its action to enhance muscle glucose uptake.

Insulin enhances glucose uptake by muscle in three ways (Fig. 1). Firstly, insulin binds to receptors on the vascular endothelium activating processes leading to increased microvascular blood flow thus enhancing delivery of both insulin and glucose to the myocyte. Secondly, insulin transport from the vasculature to the interstitial space occurs by an insulin receptor‐dependent process. Thirdly, insulin binds to receptors on myocytes and increases glucose transporter 4 (GLUT4) translocation to the cell surface membrane, enhancing glucose uptake. All of these responses are impaired in insulin resistance and type 2 diabetes resulting in diminished glucose disposal by muscle. Here we review the literature (summarised in Table 1) demonstrating that loss of microvascular insulin action not only accompanies muscle insulin resistance and ageing, but can occur prior to the development of myocyte insulin resistance.

Figure 1. Effects of ageing and insulin resistance on glucose and insulin exchange between muscle compartments .

Figure 1

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In this model there are a number of rate‐limiting transport steps for glucose and/or insulin between compartments: (1) the extent of microvascular recruitment; (2) trans‐endothelial transport (TET); and (3) sarcolemmal transport. The appearance of insulin and glucose in the microvascular compartment is limited by poor microvascular blood flow in both ageing and insulin‐resistant states. This may reflect the widespread impairment of NO‐dependent signalling in larger vessels in obesity and insulin resistance. Recent evidence suggests that insulin action at the myocyte is limited by reduced insulin‐stimulated TET in insulin resistance. Once at the myocyte glucose transport into the cytoplasm is also limited by reduced GLUT4 translocation across the sarcolemma in insulin‐resistant animals and humans. The effect of ageing on step 1 has only been partially investigated. Effects of ageing on steps 2 and 3 are either not known or have conflicting evidence. Clearly these are areas that require further investigation.

Table 1.

Key findings on effects of insulin resistance and ageing on skeletal muscle microvascular responses

Species Experimental model Muscle microvascular perfusion References
Healthy Rat Standard chow ↑ Insulin (Dawson et al. 2002; Vincent et al. 2002, 2003 a, 2004; St‐Pierre et al. 2010; Premilovac et al. 2013)
Mouse Standard chow ↑ Insulin (Kubota et al. 2011)
Human BMI < 30 kg m–2, normal fasting plasma glucose and insulin levels ↑ Insulin ↑ Mixed meal (Coggins et al. 2001; Clerk et al. 2006; Vincent et al. 2006; Keske et al. 2009)
Insulin resistant Rat High fat diet ↔ Insulin (St‐Pierre et al. 2010; Premilovac et al. 2013)
Rat High salt diet ↔ Insulin (Premilovac et al. 2014)
Rat Zucker obese ↔ Insulin (Wallis et al. 2002)
Rat Acute infusion of agents to cause insulin resistance (vasoconstrictors, elevated FFA, TNFα) ↔ Insulin (Rattigan et al. 1999; Youd et al. 2000; Clerk et al. 2002; Ross et al. 2007)
Mouse High fat diet ↔ Insulin (Kubota et al. 2011)
Mouse IRS2 KO ↔ Insulin (Kubota et al. 2011)
Mouse VEGF KO ↔ Glucose uptake during insulin infusion (Bonner et al. 2013)
Human BMI > 30 kg m–2 ↔ Insulin (Clerk et al. 2006; Keske et al. 2009)
Insulin resistant defined by insulin clamp or elevated insulin levels during mixed meal challenge ↔ Mixed meal
Ageing Human 29 ± 2 years ↑ Mixed meal delayed ↑ Mixed meal (Vincent et al. 2006; Keske et al. 2009)
41 ± 4 years
Human 32 ± 2 years ↑ Insulin (Timmerman et al. 2010 a,b)
71 ± 2 years ↔ Insulin
Human 21 ± 1 years ↑ Essential amino acid meal (Mitchell et al. 2013)
70 ± 2 years
↔ Essential amino acid meal
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Ages are means ± SEM. BMI, body mass index; KO, knockout. ↑ stimulation; ↔ no change.

Vascular actions of insulin in muscle

In skeletal muscle vasculature (and prior to insulin reaching the myocyte) insulin binds to receptors on the vascular endothelium causing vasodilatation through the production of nitric oxide (NO) (Vincent et al. 2003 b). Quon and colleagues determined that upon binding to insulin receptors on endothelial cells, insulin activates the insulin receptor substrate 1 (IRS1)/phosphoinositide 3‐kinase (PI3K)/phosphoinosi‐tide‐dependent kinase‐1/protein kinase B (Akt)/endoth‐elial nitric oxide synthase (eNOS) signalling molecules leading to NO production (Montagnani et al. 2001, 2002 a,b). This endothelial insulin signalling pathway shares common steps with the myocyte insulin signalling pathway that leads to GLUT4 translocation and glucose uptake (Vincent et al. 2003 b). The similarity between these two pathways suggests a common function and highlights the important link between the vascular and metabolic actions of insulin.

Insulin‐stimulated NO production induces vasodilatation thus increasing total blood flow to muscle in vivo. Baron and colleagues were among the first to champion the notion that insulin acts to increase total muscle blood flow to facilitate access of glucose and insulin to myocytes (Baron, 1994; Steinberg et al. 1994). However, the importance of this to the metabolic actions of insulin was initially controversial as the change in total blood flow to muscle was not always observed when physiological doses (typically seen after a meal) of insulin were used (Raitakari et al. 1996). Given this inconsistency we hypothesised that physiological doses of insulin may selectively increase nutritive microvascular blood flow in muscle. This may occur regardless of changes in total blood flow by redistributing flow to enhance perfusion of capillaries intimately associated with myocytes. This increase in capillary blood flow to myocytes increases the surface area available for nutrient exchange (hence ‘nutritive’) thus enabling enhanced glucose disposal by the myocytes.

Our research group, together with our collaborators at the University of Virginia, USA, have developed two techniques for assessing microvascular blood flow in vivo. The first technique relies on stoichiometric metabolism of exogenously infused 1‐methylxanthine to 1‐methylurate by microvascular xanthine oxidase (Rattigan et al. 1997, 1999; Youd et al. 2000; Vincent et al. 2002; Wallis et al. 2002; Zhang et al. 2004; St‐Pierre et al. 2010). The second technique is an adaptation of an ultrasound imaging technique (contrast‐enhanced ultrasound, CEU) to skeletal muscle (Coggins et al. 2001; Dawson et al. 2002; Vincent et al. 2002, 2003 a, 2004, 2006; Clerk et al. 2006, 2007; Keske et al. 2009). Using both techniques we have demonstrated that physiological doses of insulin increase microvascular blood flow and that this increase is associated with enhanced glucose uptake by muscle (Rattigan et al. 1997; Dawson et al. 2002; Vincent et al. 2002, 2003 a, 2004, 2006; Clerk et al. 2006; Keske et al. 2009). This microvascular insulin response has been observed in both humans and experimental animals (Rattigan et al. 1997; Coggins et al. 2001; Dawson et al. 2002; Vincent et al. 2002, 2003 a, 2004, 2006; Clerk et al. 2006; Keske et al. 2009) and we have shown that it is independent of insulin's macrovascular actions in muscle (Dawson et al. 2002; Vincent et al. 2002, 2004; Zhang et al. 2004). Additionally, we have shown that insulin increases microvascular blood flow earlier (within 10–15 min of infusion) than its effect to increase total blood flow to muscle (60–90 min of infusion) (Vincent et al. 2004). We have also observed that low doses of insulin, which do not elicit an increase in total blood flow to muscle, can still increase microvascular blood flow (Vincent et al. 2004; Zhang et al. 2004).

Systemic (Vincent et al. 2003 a, 2004) or local hindleg (Bradley et al. 2013) infusion of a NO synthase inhibitor blocks most, if not all, of the insulin‐mediated microvascular blood flow in muscle and inhibits ∼40% of muscle glucose uptake. Therefore, insulin‐mediated increases in microvascular blood flow in muscle are, at least in part, NO dependent.

Although it has been argued by others (Poole et al. 2013) that these measures may not actually represent capillary blood flow it is without doubt that the changes in microvascular perfusion that these techniques detect contribute to the insulin‐mediated glucose uptake in muscle.

Impaired microvascular blood flow and insulin resistance

The above findings suggest that increased microvascular perfusion by insulin is an important physiological response, and if so, this may be impaired in insulin‐resistant states. By manipulating insulin's vascular response using vasoconstrictors such as α‐methylserot‐onin (Rattigan et al. 1999) and endothelin‐1 (Ross et al. 2007) we have shown that insulin‐stimulated microvascular blood flow can be inhibited, resulting in impaired insulin‐mediated glucose uptake in vivo. This loss of vascular insulin function is also apparent during acute infusions of factors known to be elevated in various insulin‐resistant states, such as tumour necrosis factor α (TNFα) (Youd et al. 2000) and free fatty acids (elevated by infusion of Intralipid and heparin) (Clerk et al. 2002).

The importance of insulin's microvascular actions is further evidenced in chronic animal models of insulin resistance, including the high fat‐fed (St‐Pierre et al. 2010), Zucker obese (Wallis et al. 2002) and Zucker diabetic fatty (Clerk et al. 2007) rats. These animal models all display reduced microvascular responsiveness during insulin infusion and exhibit impairment of insulin‐mediated muscle glucose uptake. In humans, we have also shown that insulin infusion (Clerk et al. 2006) or the ingestion of a mixed meal (Keske et al. 2009) act to similarly increase microvascular blood flow and that this response is blunted in obese insulin‐resistant subjects (Clerk et al. 2006; Keske et al. 2009). Together, these studies highlight the important link between microvascular and metabolic actions of insulin in muscle and indicate that loss of microvascular insulin sensitivity may contribute to development or worsening of insulin resistance in skeletal muscle.

Microvascular‐derived insulin resistance

The mechanisms of skeletal muscle insulin resistance are multifactorial and there has been much focus on the myocyte per se as the main defect. Most animal models of insulin resistance (such as those described above) develop myocyte insulin resistance in addition to microvascular insulin resistance. Thus it has been difficult to distinguish the importance of microvascular versus myocyte insulin resistance. However, recent evidence has emerged that defects in the microvasculature can be an independent event leading to development of muscle insulin resistance (Kubota et al. 2011; Bonner et al. 2013; Premilovac et al. 2013, 2014).

Diet‐induced insulin resistance models

In our laboratory we have recently characterised two dietary animal models of insulin resistance (Premilovac et al. 2013, 2014). Both of these models support the concept that microvascular insulin resistance develops before, and contributes to, reduced muscle glucose uptake in vivo. The first of these models is the moderately raised dietary fat model in which dietary fat in rats is increased by 2‐fold (from 5% to 9% w/w), rather than the more common 5‐ to 7‐fold increase employed in many studies (Kraegen et al. 1986; St‐Pierre et al. 2010; Turner et al. 2013). After 4 weeks of moderate fat feeding, these animals develop whole body, muscle and microvascular insulin resistance in vivo, but retain normal increases in femoral artery blood flow in response to insulin (Premilovac et al. 2013). When we assessed myocyte insulin sensitivity ex vivo using the constant‐flow pump‐perfused hindleg technique (in which insulin and glucose are delivered to the myocyte in the absence of vascular actions of insulin) we found that insulin‐mediated myocyte glucose uptake was similar between 5 and 9% fat‐fed animals (Premilovac et al. 2013). Therefore, the moderate fat‐fed model provides evidence that the reduction in insulin‐stimulated muscle glucose disposal in vivo can be driven by impairment of microvascular actions of insulin without loss of macrovascular or myocyte insulin responsiveness. This is the first piece of evidence that suggests defects in microvascular insulin action are an early, independent event that directly contributes to fat‐induced muscle insulin resistance.

The second animal model we have characterised pertinent to this discussion is the high salt‐fed rat model (Premilovac et al. 2014). After 4 weeks of high salt feeding (8.0% NaCl w/w) these animals develop whole body, skeletal muscle and microvascular insulin resistance in vivo when compared to normal salt‐fed rats (0.3% NaCl w/w). In contrast to the moderate fat model above, high salt‐fed rats exhibit reduced basal femoral artery blood flow compared to control diet animals. Despite this reduction, insulin stimulated a comparable percentage increase in femoral artery blood flow in both normal and high salt‐fed rats. Assessing myocyte insulin sensitivity in this model using the constant‐flow perfused hindleg preparation, we found no difference in muscle glucose uptake between high and normal salt‐fed rats. Together, these data indicate that myocyte insulin sensitivity is normal in high salt‐fed animals and that impairment of microvascular insulin responsiveness is sufficient to induce muscle insulin resistance in vivo.

Therefore, we have identified two distinct dietary models that both develop skeletal muscle insulin resistance in vivo as a consequence of impaired microvascular insulin action. These data position the loss of normal microvascular function as an early driver in the development of muscle insulin resistance and provide a possible early therapeutic target for prevention of insulin resistance within skeletal muscle.

Knockout mouse models

Other researchers have develo‐ped knockout mouse models that provide important links between vascular and metabolic actions of insulin. Kubota and colleagues demonstrated that endothelial insulin receptor substrate 2 (IRS2) knockout mice (which have normal levels of IRS2 in skeletal muscle, liver and white adipose tissue) had normal liver insulin sensitivity, but whole body and muscle insulin resistance in vivo (Kubota et al. 2011). This mouse displayed microvascular insulin resistance, and impaired insulin‐mediated muscle glucose uptake in vivo, and this was associated with reduced acti‐vation of both Akt and eNOS in endothelial cells. When muscles from these animals were isolated and incubated in vitro with insulin (where delivery occurs by diffusion rather than via the microvasculature), insulin‐mediated myocyte glucose uptake was not different to control animals. These data suggest that impaired insulin signalling in endothelial cells reduces insulin‐mediated muscle glucose uptake by decreasing insulin‐mediated microvascular blood flow in skeletal muscle. These data implicate reduced insulin‐mediated endothelial NO production (via reduced eNOS activation) as a contributor to reduced insulin‐mediated microvascular blood flow and muscle glucose uptake.

The degree of capillarisation of skeletal muscle is another important factor that can influence insulin‐mediated glucose uptake by myocytes. Muscle‐specific vascular endothelial growth factor (VEGF) knockout animals have reduced capillary density in skeletal muscle and display whole body and muscle insulin resistance in vivo (Bonner et al. 2013). However, in vitro (muscle incubation) assessment of insulin‐mediated glucose uptake revealed no difference compared with muscle from wild‐type animals. Additionally, studies involving human subjects with reduced capillary density have also yielded results indicating reduced insulin‐mediated muscle glucose uptake in these individuals (Gavin et al. 2005). Thus, even in the presence of normal myocyte insulin sensitivity, reduced delivery of glucose and insulin to the myocyte through decreased capillary number is sufficient to significantly reduce insulin‐stimulated muscle glucose uptake in vivo.

Trans‐endothelial transport of insulin to the interstitial space

The movement of insulin from the vasculature to the interstitial space is another potential rate‐limiting step for insulin's metabolic actions in skeletal muscle (Fig. 1). The concentration of insulin in the interstitium (measured by lymphatic sampling or microdialysis) is substantially lower (∼50%) than the concentration in plasma (Yang et al. 1989, 1992; Sjöstrand et al. 1999). The time‐course for insulin action to augment muscle glucose disposal is delayed in insulin‐resistant and type 2 diabetics during a euglycaemic hyperinsulinaemic clamp (Nolan et al. 1997). Thus, these data are consistent with a delay in the transit of insulin from the vasculature into the interstitium in insulin‐resistant and type 2 diabetic individuals.

Barrett et al. have demonstrated that trans‐endothelial transport of insulin into the endothelium (cell culture) is insulin receptor mediated (Barrett et al. 2009; Barrett & Liu, 2013). The uptake of insulin into endothelial cells is dependent on various insulin signalling cascades (PI3K and MAPK) and the activation of eNOS (Wang et al. 2008). Cytokines such as TNFα and interleukin 6, which are elevated during states of insulin resistance, impair insulin uptake into the endothelium (Wang et al. 2011). Thus, these observations suggest a mechanism linking insulin resistance and impaired or delayed insulin delivery to the interstitial space in contact with myocytes.

Ageing and microvascular actions in muscle

Ageing is associated with an altered response to both vasodilators and vasoconstrictors (Celermajer et al. 1994; Seals et al. 2006; Barrett‐O'Keefe et al. 2013) suggesting a loss of normal function of the vascular system as age increases. In a young healthy cohort of subjects (29 ± 2 years (mean ± SEM)) we have demonstrated that a mixed meal challenge, which increases plasma insulin concentrations approximately 10‐fold, significantly increases microvascular blood flow in muscle by 60 min (Vincent et al. 2006). In contrast, when the same mixed meal was given to an older cohort (41 ± 4 years), microvascular blood flow remained unchanged at 60 min, but was significantly stimulated by 120 min post‐meal (Keske et al. 2009). However, these studies were performed independently of each other and therefore only provide indirect evidence of an association between ageing and reduced (or temporally delayed) insulin sensitivity in the microvasculature. Timmerman and colleagues have reported that insulin‐mediated microvascular responses to local infusion of insulin to one leg is markedly impaired in older (71 ± 2 years) versus younger (32 ± 2 years) people (Timmerman et al. 2010 a,b). However, similarly, these studies were performed independently of each other, and therefore only provide indirect evidence. Taken together these studies indicate that as age increases, the microvascular responsiveness to insulin decreases. Other investigators have looked more closely at this association.

Age‐related loss of microvascular responsiveness was recently confirmed by Mitchell and colleagues (Mitchell et al. 2013). Participants were given an essential amino acid meal, which raised plasma insulin concentrations by approximately 3‐fold. This amino acid meal stimulated an increase in microvascular blood flow in muscle by 45 min in the younger (21 ± 1 years) cohort and this effect was completely absent in the older (70 ± 1 years) cohort. Others have reported reductions in resting microvascular blood volume in older (67 ± 2 years) versus younger (30 ± 2 years) people (Durham et al. 2010). This study also showed that 60 min after a bout of exercise both younger and older participants had elevated microvascular blood flow; however, the increase was significantly smaller in the older participants (Durham et al. 2010). Whilst the mechanisms that lead to increased blood flow probably differ between insulin and post‐exercise, the attenuated microvascular blood flow response seen in the older participants indicates a loss of microvascular responsiveness in these individuals.

Evidence for the effect of ageing on insulin resistance in terms of glucose uptake is conflicting, with some investigators reporting no changes in whole body or muscle insulin‐mediated glucose uptake in both humans (Lind et al. 2001; Rasmussen et al. 2006; Chevalier et al. 2011) and experimental animals (Schulman et al. 2007), while others have shown impairment with age (Escriva et al. 1997; Luzi et al. 2001; Bhashyam et al. 2007). Likewise, myocyte insulin‐mediated glucose uptake in vitro during ageing is also equivocal (Frøsig et al. 2013; Ropelle et al. 2013). Therefore it is unclear whether ageing per se causes insulin resistance (reduced glucose uptake) directly, or whether it is due to other risk factors such as a sedentary lifestyle, increased adiposity or hypertension that often coexist with ageing (Hildrum et al. 2007). Clearly this is an area that requires further investigation.

Conclusions

Obesity and declining vascular function in ageing frequently accompanies insulin resistance. Prior to the discovery of insulin's important microvascular responses in muscle it was unclear whether vascular dysfunction had any bearing on the development of insulin resistance. It is now clear that vascular dysfunction is likely to contribute substantially to muscle insulin resistance, particularly in the early stages of insulin resistance and type 2 diabetes. Although vascular dysfunction has been reported with ageing there is a need for further studies in aged populations to determine whether the commonly observed loss of insulin sensitivity in this population is also due to loss of the insulin‐mediated microvascular actions in muscle. A further key question that remains is to establish whether correcting this microvascular defect (i.e. restoring insulin‐mediated microvascular responses) is an effective therapeutic option for the treatment of more advanced type 2 diabetic states, either alone or in combination with existing therapies that address skeletal myocyte and/or liver insulin resistance.

Additional information

Competing interests

There are no competing interests.

Funding

This work was funded by various grants awarded from the National Health and Medical Research Council of Australia, Australian Research Council, Heart Foundation of Australia, Diabetes Australia, and the US National Institutes of Health.

Biography

Michelle A. Keske is a senior research fellow at the Menzies Institute for Medical Research, University of Tasmania. Her research has shown that (i) microvascular function in muscle plays an important role in blood glucose regulation, and (ii) microvascular dysfunction in muscle can cause insulin resistance. Her current research focuses on interventions to prevent or reverse insulin resistance by regulating microvascular blood flow within muscle. Stephen Rattigan is a professor and Deputy Director at the Menzies Institute for Medical Research. His research interests have focused on the control of skeletal muscle metabolism. Collaborative studies with investigators in the USA, Denmark and The Netherlands have elucidated the important role that the vascular system plays in regulating muscle glucose uptake. He has pioneered novel methods to investigate microvascular blood flow in vivo in experimental animals and humans.

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This review was presented at the symposium Impact of physical activity, ageing, obesity and metabolic syndrome on muscle microvascular perfusion and endothelial metabolism, which took place at Physiology 2014, the annual meeting of The Physiological Society, London, UK on 1 July 2014.

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