The vascular actions of insulin control its delivery to muscle and regulate the rate-limiting step in skeletal muscle insulin action
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Evidence suggests that insulin delivery to skeletal muscle interstitium is the rate-limiting step in insulin-stimulated muscle glucose uptake and that this process is impaired by insulin resistance. In this review we examine the basis for the hypothesis that insulin acts on the vasculature at three discrete steps to enhance its own delivery to muscle: (1) relaxation of resistance vessels to increase total blood flow; (2) relaxation of pre-capillary arterioles to increase the microvascular exchange surface perfused within skeletal muscle (microvascular recruitment); and (3) the trans-endothelial transport (TET) of insulin. Insulin can relax resistance vessels and increase blood flow to skeletal muscle. However, there is controversy as to whether this occurs at physiological concentrations of, and exposure times to, insulin. The microvasculature is recruited more quickly and at lower insulin concentrations than are needed to increase total blood flow, a finding consistent with a physiological role for insulin in muscle insulin delivery. Microvascular recruitment is impaired by obesity, diabetes and nitric oxide synthase inhibitors. Insulin TET is a third potential site for regulating insulin delivery. This is underscored by the consistent finding that steady-state insulin concentrations in plasma are approximately twice those in muscle interstitium. Recent in vivo and in vitro findings suggest that insulin traverses the vascular endothelium via a trans-cellular, receptor-mediated pathway, and emerging data indicate that insulin acts on the endothelium to facilitate its own TET. Thus, muscle insulin delivery, which is rate-limiting for its metabolic action, is itself regulated by insulin at multiple steps. These findings highlight the need to further understand the role of the vascular actions of insulin in metabolic regulation.
KeywordsBlood flow Capillary Caveolae Endothelium Insulin resistance Insulin transport Microvascular recruitment Nitric oxide Nitric oxide synthase Skeletal muscle
Endothelial nitric oxide synthase
Extracellular receptor kinase
Insulin-like growth factor-I
Insulin-mediated glucose disposal
In this review we first examine the evidence that insulin delivery to muscle is rate-limiting for insulin-mediated glucose disposal (IMGD). Classical studies of the kinetics of whole body handling of infused insulin and the kinetic response of glucose disposal to insulin in vivo are considered. We then examine data regarding insulin access to muscle interstitium as estimated by lymphatic sampling, by microdialysis methods, and by estimates of insulin uptake by muscle. Combined, these data make the case that the delivery of insulin to muscle interstitium is rate-limiting for muscle IMGD and that insulin resistance may slow the rate of insulin delivery. We then provide an overview of several steps at which insulin, by acting on vascular tissue, may potentially regulate its own delivery to muscle interstitium. Finally, we consider evidence that one or more of these processes is disturbed in insulin-resistant states.
Insulin delivery to muscle—estimates from whole body insulin kinetics
Kinetics of insulin-stimulated glucose disposal and suppression of glucose production
Steady-state glucose disposal rate t1/2 as a function of insulin infusion rate in lean and obese humans
Insulin infusion rate (mU min−1 m−2)
Lean Rdt1/2 (min)
Obese Rdt1/2 (min)
Temporal dissociation between in vivo and in vitro insulin action on muscle
It is conceivable that the slow onset of insulin action on glucose disposal in vivo could be secondary to a slow response of the cellular machinery within the myocyte, the cell responsible for glucose uptake (e.g. activation of the insulin receptor or recruitment of GLUT4 to the membrane surface). However, when studied in vitro using isolated cells (which eliminates physical barriers to the binding of insulin to its receptor), these processes appear to be fully active in 2–5 min [10, 11]—a much more rapid time-course than that observed in vivo. Thus, the kinetics of insulin delivery determines the in vivo time-course for IMGD and this time dependence is quite slow relative to the time required for IMGD in isolated cell systems.
Measurements of muscle insulin concentrations: interstitial vs plasma insulin
The effect the time-dependent access of insulin to muscle interstitial space has on IMGD was clarified by the demonstration that insulin concentrations in thoracic duct lymph were persistently lower than those in plasma throughout a 3 h euglycaemic–hyperinsulinaemic clamp . Furthermore, it was observed that, throughout the study, whole body IMGD correlated strongly with lymphatic, but not with plasma, insulin concentrations . This study also demonstrated that the concentration dependence of insulin-stimulated glucose disposal (presumably occurring in muscle) is similar to that for the suppression of hepatic glucose production by insulin when the muscle lymph insulin concentration is considered . Subsequently, these same investigators observed that the temporal relationship of IMGD to hind leg lymphatic insulin concentration was even stronger than that seen for thoracic duct insulin . Additional canine studies have confirmed that insulin activation of hepatic insulin signalling pathways occurs more promptly than insulin activation of muscle insulin signalling pathways . Together, these findings strongly suggest an important role for insulin delivery to interstitium in the regulation of muscle glucose uptake.
Support for the relevance of these findings to humans has been provided by a clinical study  that involved cannulation of lymphatic ductules in the foot to sample subcutaneous lymph during a euglycaemic–hyperinsulinaemic clamp. In both lean and obese humans, interstitial insulin temporally lagged behind arterial insulin concentrations, and a large arterial–interstitial insulin gradient was present throughout a 150 min insulin clamp. They also observed that the delay in transfer of insulin could not alone account for the decreased IMGD seen in the obese, insulin-resistant participants, indicating that compromised insulin action on the myocyte was also an important contributor to insulin resistance.
Studies comparing the time-course for insulin receptor tyrosine kinase activation with that for changes in interstitial insulin concentration during a euglycaemic–hyperinsulinaemic clamp confirmed that in vivo (in both dogs and humans) kinase activation occurs very promptly following increases in interstitial insulin, but is delayed relative to increases in plasma concentrations [15, 17]. This is in agreement with the extensive in vitro evidence demonstrating that activation of insulin signalling processes in isolated target cells and tissues occurs within a few minutes of the addition of insulin [10, 11].
Measurement of muscle interstitial insulin by microdialysis
Steady-state relationship between plasma and interstitial muscle insulin
Plasma insulin (pmol/l)
Interstitial insulin (% of plasma value)
Sjostrand et al. 
Gudbjornsdottir et al. 
Herkner et al. 
Castillo et al. 
Yang et al. 
Holmang et al. 
Use of the limb balance technique to measure muscle insulin uptake
There are scant data from several early studies in which investigators measured forearm muscle insulin uptake using the limb balance method . These studies reported that there was a positive net balance (uptake) of insulin by muscle, and that this was increased by exercise. As exercise increases muscle blood flow, this finding suggests that ‘perfusion’ may affect insulin delivery. However, these studies were not followed-up in a systematic fashion. As a result, careful measurements of the kinetics of insulin uptake by skeletal muscle tissue are lacking.
Muscle total blood flow and the ‘delivery’ of insulin to muscle
Effects of insulin on total blood flow to skeletal muscle
As the above studies were building evidence for an important role for muscle interstitial insulin in regulating the onset of insulin action, Baron and colleagues introduced the novel concept that insulin could regulate its own delivery, and that of glucose, by increasing blood flow to muscle . Indeed, they showed a remarkable correlation between the effect of insulin on whole body glucose uptake and the effect of insulin on leg blood flow over a broad range of insulin sensitivities in normal and insulin-resistant states [30, 31, 32]. These correlations were typically observed under steady-state conditions, several hours after the initiation of an insulin clamp, and did not specifically address the kinetic properties of insulin delivery described above. The relationship between muscle blood flow and glucose uptake was investigated by a number of other laboratories. Some [33, 34, 35, 36], but by no means all [37, 38, 39], of these studies supported a regulatory role for insulin as a mediator of increases in total limb blood flow and, consequently, of glucose and insulin delivery to muscle.
Additional, and perhaps more compelling, evidence for a role for blood flow in regulating muscle glucose uptake are the observations that increasing muscle blood flow with methacholine infusion during either a high- or low-dose insulin clamp enhances both leg blood flow and leg glucose uptake . The vasodilatory action of insulin is dependent on nitric oxide generation. Inhibiting insulin-induced increases in blood flow with the nitric oxide synthase inhibitor l-NG-monomethyl arginine diminished both blood flow and glucose uptake [41, 42]. These studies have provided the most direct evidence for a role for total limb blood flow in regulating insulin-stimulated muscle glucose uptake.
Physiological relevance of the effect of insulin on total limb blood flow
As noted above, under physiological conditions, insulin has not uniformly been found to increase limb blood flow in humans [33, 34, 35, 36, 39, 43, 44, 45]. In addition, while there is a report of physiological levels of insulin increasing muscle blood flow within 30 min , more typically, 2 h or more are required to see the effects of insulin on limb blood flow. This is not easily reconciled with a significant metabolic role for limb blood flow in regulating muscle insulin delivery . There is consensus that pharmacological insulin concentrations can more quickly increase limb blood flow and glucose uptake (Table 1). However, the significance of this is uncertain. Interestingly, increasing limb blood flow in insulin-resistant individuals by co-infusion of nitroprusside or bradykinin during a euglycaemic–hyperinsulinaemic clamp did not increase limb glucose uptake [46, 47, 48], arguing against a significant contribution of total blood flow to insulin resistance.
It is unfortunate that none of the limb balance studies examining the effects of insulin on limb blood flow and muscle glucose metabolism included measurements of muscle insulin uptake or lymphatic or muscle interstitial insulin concentrations. Such data could have provided information on whether insulin resistance, as seen in diabetes or obesity, affects peripheral insulin handling. Nonetheless, on balance, there appears to be evidence that insulin can promote increases in limb blood flow in humans. Whether this occurs sufficiently rapidly at physiologically encountered insulin concentrations to contribute to IMGD in healthy individuals is uncertain. Consequently, whether impairments in the effect of insulin on total muscle blood flow play an important role in the derangements in IMGD seen in obesity and type 2 diabetes remains to be determined. However, these findings, and the controversy that surrounded them, prompted the re-examination of the discrete processes involved in the delivery of insulin to muscle.
Extraction ratios of insulin and glucose across skeletal muscle
Conditions are only slightly more favourable for insulin uptake than for glucose uptake by muscle in the post-absorptive state. In healthy humans the insulin extraction ratio across the human forearm is 10–15% after an overnight fast [24, 25]. This ratio falls to ~7% during a 1 mU min−1 kg−1 insulin clamp . As a result, increasing total muscle blood flow could increase the intravascular–interstitial insulin gradient and thereby increase tissue insulin uptake only modestly. Such observations serve to emphasise the importance of blood flow in circumstances in which the extraction ratio is high (e.g. glucose extraction by muscle can approach 50% under maximally insulinised circumstances) and the relatively minor role of total blood flow changes under circumstances in which the extraction ratio for the observed metabolite or hormone is small.
Insulin action on muscle microvasculature: microvascular recruitment to increase muscle insulin delivery
This process of microvascular recruitment involves the dilation of terminal arterioles, each of which feeds 12–20 capillaries. Under basal conditions, vasomotion appears to determine the extent of vasodilation of terminal arterioles and, by consequence, the extent to which a capillary network is perfused at any time. Insulin may act to simply modulate the dilation or constriction rate constant to extend the period of perfusion. The cellular mechanisms responsible for insulin- or exercise-induced microvascular recruitment cannot be fully addressed using the methods available at present. Efforts to examine this issue using video-microscopic methods have shown that insulin can relax very small muscle arterioles . However, actually visualising the recruitment process has been problematic. Efforts to use video-microscopy of thin muscle preparations (e.g. cremaster, spinotrapezius) have been hampered because the sensitivity of these preparations to the process of surgical exposure and superfusate O2 and CO2  is such that in the ‘basal state’ the microvasculature is extensively recruited, which means that further changes with insulin are not reproducibly observed .
At present, successful efforts to visually examine the microvascular action of insulin have come from work examining skin microvasculature. Using capillary video-microscopy and laser Doppler flowmetry, Stehouwer and colleagues have reported that, in healthy individuals, systemic hyperinsulinaemia increases skin microvascular perfusion and increases the number of nailbed capillaries carrying erythrocytes . This provides direct evidence for the regulation of capillary perfusion by insulin in humans. The same investigators have demonstrated that capillary recruitment in both the nailbed and skin is blunted by the insulin resistance seen in hypertension , obesity [65, 66], elevated plasma NEFA concentrations  and the metabolic syndrome . These last two observations are particularly interesting as they suggest that altered microvascular responses may be an early dysfunction in individuals at risk of diabetes.
There is concern as to whether the vascular responses observed in skin reflect those in muscle, as the vasculature of skin is highly specialised for processes such as heat exchange. Nevertheless, these studies provide direct visual evidence that insulin can regulate the microvasculature and, specifically, can expand the surface area available for nutrient exchange.
Relevance of microvascular recruitment to muscle insulin resistance
Impaired microvascular recruitment leading to a decreased surface area available for nutrient exchange may be important to skeletal muscle insulin resistance. Muscle insulin resistance is considered to involve an impairment in the recruitment of GLUT4 glucose transporters to the myocyte sarcolemma [69, 70]. This appears to be secondary to alterations early in the insulin signalling cascade, involving the phosphorylation of serine residues of insulin receptor substrates . Available data in humans and animals suggest that these myocyte processes occur in parallel with impairments in insulin action within skeletal muscle microvasculature. While studies have shown that diminished IMGD is accompanied by functional changes in the microvasculature, data demonstrating biochemical correlates of this within microvascular cells in vivo are lacking. For example, the infusion of TNFα  or NEFA  into rodents inhibits insulin-stimulated microvascular recruitment and interferes with insulin signalling to nitric oxide synthase in cultured ECs [74, 75]. Their effects on NO generation in situ have not been defined, but in isolated vessels, TNFα  and NEFA  are known to block NO-dependent vasodilation. Clearly, there may be interrelated aspects to these observations, in that impaired insulin transport into muscle interstitium, through diminished total blood flow, decreased capillary recruitment or impaired movement of insulin across the endothelial barrier (see below), would affect muscle interstitial insulin concentrations and therefore insulin action at the myocyte.
Relevance to hypertension
The EC responds to insulin by increasing the phosphorylation of endothelial nitric oxide synthase (eNOS) on serine 1177, and this increases Ca2+-independent nitric oxide synthase activity [78, 79]. Interestingly, insulin also activates the mitogen-activated protein kinase pathway in ECs, which enhances the generation of the vasoconstrictor endothelin-1 . This can lead to insulin-stimulated vasoconstriction if signalling from the insulin receptor to eNOS is inhibited pharmacologically or downregulated by insulin resistance [62, 68, 76]. In this manner, endothelial insulin resistance may contribute to the development of hypertension and account in part for the epidemiological relationship between hypertension and insulin resistance .
Movement of insulin across the endothelial lining of the microvasculature within muscle
Beyond the relaxing effect of insulin on resistance vessels (increasing flow) and terminal arterioles (recruiting the microvasculature), insulin may exert a third highly significant endothelial action to promote its own movement across the EC barrier. As with studies on the effects of insulin on total limb blood flow, studies on the trans-endothelial transport (TET) of insulin have at times yielded seemingly conflicting findings. For example, early work by King and Johnson using cultured ECs reported that the trans-endothelial movement of insulin was saturable and blocked by antibodies to the exofacial domain of the insulin receptor , and several additional studies supported this finding [27, 82, 83, 84]. However, two other studies, both of which reported observations in vivo, suggested that the TET of insulin was not saturable and involved a passive diffusional process, perhaps similar to the TET of inulin [85, 86]. Indeed, whether insulin crosses the endothelium by a cellular or a paracellular pathway has been a source of some controversy. The continuous endothelium of muscle forms a relatively tight barrier that could prevent insulin from freely diffusing and thus sustain the significant plasma–interstitial insulin gradient in muscle, discussed above.
ECs express substantially more insulin-like growth factor-I (IGF-I) receptors than insulin receptors . Experiments using fluorochrome-labelled insulin and cultured bovine aortic ECs grown on transwell plates suggested that insulin TET is inhibited by insulin, IGF-I and antibody to the IGF-I receptor . Early studies demonstrated that ECs accumulate and only slowly metabolise 125I-labelled insulin [27, 28]. Such observations certainly suggest that a receptor-mediated trans-cellular insulin transport process may mediate insulin TET.
Intravenously infused fluorescein isothiocyanate (FITC)-labelled insulin was shown by confocal microscopy to be rapidly localised within the vascular endothelium of skeletal muscle in vivo . There was no evidence for movement of the labelled-insulin between ECs, and the intensity of the EC labelling was substantial, suggesting that the uptake process may even concentrate insulin within the EC.
Role of caveolin-1
Further microscopic studies suggested the co-localisation of insulin with the insulin receptor and the caveolar structural protein caveolin-1 in ECs. Furthermore, the insulin receptor co-immunoprecipitated with caveolin-1, and disruption of the caveolae using filipin, cyclodextrin  or, more recently, small interfering RNA directed against caveolin-1  inhibited insulin uptake by cultured ECs. In an early electron microscopic investigation, exposure of the eel rete mirabili to 0.4 mmol/l immunogold-labelled insulin showed that insulin associates with caveolae-like structures within the EC . Whether this reflects pathways used at physiological insulin concentrations is unclear.
Evidence for a saturable insulin TET pathway
From the dimensions of caveolae (~800 Å diameter), one can estimate that passive EC insulin uptake by caveolar potocytosis would provide only one insulin molecule per 5,000 caveolae at physiological insulin concentrations. The localisation of high-affinity insulin receptors within caveolae would allow efficient insulin trapping and could greatly increase the likelihood of insulin moving across the endothelium. Beyond simply trapping insulin, which can then shuttle between the luminal and ablumenal face of the EC, an intriguing question is whether insulin acts on the cell to stimulate caveolar-mediated insulin transport. By parallel, albumin stimulates a Src kinase-mediated pathway to enhance caveolar-mediated albumin uptake. Multiple studies show that insulin can stimulate classical insulin signalling pathways in ECs [74, 75, 93, 94]. As mentioned above, eNOS is a substrate for Akt in the EC . Nearly all studies that have examined EC insulin signalling have used insulin concentrations (10–100 nmol/l) that activate both insulin and IGF-1 receptors. In a dose–response study, 100 pmol/l insulin enhanced the phosphorylation of Akt, eNOS and ERK , and this was not blocked by pre-incubation of the cells with an antibody to the exofacial domain of the IGF-I receptor, arguing for a role for physiological insulin concentrations. Recent data indicate that inhibition of either phosphatidylinositol 3-kinase (wortmannin), ERK (PD 98059) or tyrosine kinase activity (genistein) diminishes endothelial uptake of FITC-labelled insulin , suggesting that insulin, like albumin, can promote its own movement into ECs .
Based on the data presented, it is reasonable to hypothesise that insulin exerts yet a third action on endothelium—facilitation of TET by binding to its receptor. Indeed, beyond binding, it may also act on the EC to stimulate its own uptake and transport across the vascular endothelium. In this line, inflammatory factors (e.g. TNFα) and oxidative stress, which provoke insulin resistance both in vivo and in vitro, have been observed to diminish both insulin-induced activation of Akt and eNOS and insulin uptake by cultured bovine aortic ECs .
A seeming difficulty with hypothesising a major role for EC insulin action in the overall effects of insulin in muscle is the that EC-specific insulin receptor deletion in mice did not alter insulin sensitivity as assessed by the insulin clamp . It was not addressed whether mechanisms (e.g. greater endothelial permeability, increased IGF-1 receptor concentration) compensated for the loss of insulin receptors. Recent observations that EC-specific knockout of IRS-2  produced insulin resistance (insulin clamp) and decreased both the transport of insulin to muscle interstitium and insulin-induced changes in muscle blood flow suggest an important role for EC insulin action. This important adapter/signalling protein is downstream of both the insulin and IGF-1 receptors.
Over the past two decades, it has been increasingly recognised that vascular tissue, particularly the EC, is a potentially important physiological target for insulin. It appears that insulin can act on ECs at multiple levels of the vasculature. In addition to the vascular actions explicitly addressed in this review, insulin has been shown to acutely relax conduit vessels in healthy, but not in insulin-resistant, individuals . This decreased compliance is unlikely to influence insulin or glucose delivery to muscle, but may, over time, contribute to vascular injury within the walls of conduit vessels. At the level of resistance arterioles, terminal arterioles and capillaries within muscle, resistance to the actions of insulin may impede insulin delivery to skeletal muscle interstitium and thereby contribute significantly to insulin resistance. Postprandially, the resulting compensatory hyperinsulinaemia will promote hepatic triacylglycerol synthesis as an alternative fate for ingested carbohydrate. This could set up a positive feedback loop, whereby elevated triacylglycerol or NEFA levels provoke further increases in vascular insulin resistance . The quantitative contribution of slowed muscle insulin delivery to overall insulin and glucose homeostasis in states of insulin resistance will require considerable further study. Fortunately, the tools necessary to probe this important aspect of insulin action are increasingly becoming available.
This work was supported by National Institutes of Health grants DK-057878, DK-073759, DK063609 (to E. J. Barrett) and RR00847 (to the University of Virginia General Clinical Research Center). We thank M. Thorner (University of Virginia, Charlottesville, VA, USA), J. Yudkin (University College London, London, UK) and E. Eringa (VU University Medical Center, Amsterdam, the Netherlands) for thoughtful suggestions for the manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.