Pflugers Arch - Eur J Physiol (2013) 465:1065–1074 DOI 10.1007/s00424-013-1218-z

INVITED REVIEW

The IGF-1 receptor and regulation of nitric oxide bioavailability and insulin signalling in the endothelium V. Kate Gatenby & Helen Imrie & Mark Kearney

Received: 20 September 2012 / Revised: 19 December 2012 / Accepted: 7 January 2013 / Published online: 22 January 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract The insulin-like growth factor-1 receptor (IGF-1R), like the insulin receptor (IR), plays a significant role in determining bioavailability of the critical signalling molecule nitric oxide (NO) and hence, modulates endothelial cell function, particularly in response to stimulation with insulin. In particular, the ability of the IGF-1R to form hybrid receptors with the IR appears to be highly significant in determining the sensitivity of the endothelial cell to insulin. This review will examine the structure of the IGF-1R and how this, with particular reference to the ability of the IGF-1R and the IR to form hybrid receptors, may have an effect both on endothelial cell function and the development of cardiovascular disease. Keywords Insulin like growth factor-1 receptor . Insulin receptor . Hybrid receptor . Endothelial dysfunction . Nitric oxide

the IGF-1R has been shown to affect endothelial function, in murine studies, and will review the evidence in humans showing a relationship between the IGF-1R and clinical sequelae of endothelial dysfunction, with particular reference to cardiovascular disease.

IGF-1 Receptor The IGF-1R shares significant structural homology with and is part of the same family of trans-membrane tyrosine kinase (TK) receptors as the insulin receptor (IR). It binds IGF-1 with high affinity, and IGF-2 and insulin with lower affinity. From an evolutionary perspective, the emergence of a separate IGF-1R and insulin receptor (IR) appears to have occurred with the transition from protochordates to vertebrates [55], and indeed, the IGF-1R was only recognised as distinct from the IR in 1974 [46].

Introduction Genetics Developments within the last few decades have suggested that the insulin-like growth factor-1 receptor (IGF-1R) modulates endothelial cell function and bioavailability of the signalling radical nitric oxide (NO) [28]. This review will firstly look at the structure and function of the IGF-1R, particularly with reference to its relationship with the insulin receptor, before examining the way in which the IGF-1R may modulate endothelial function. It will be concluded with a review of how manipulation of V. K. Gatenby (*) : H. Imrie : M. Kearney Department of Diabetes and Cardiovascular Research, LIGHT Laboratories, University of Leeds, Clarendon Way, Leeds LS2 9NL, UK e-mail: [email protected]

The IGF-1R gene is located on chromosome15q26 and consists of 4,989 nucleotides, coding a 1,367 amino acid precursor [80]. The IGF-1R and IR share around 70 % DNA sequence identity [2, 16], and there is a notable similarity in both the exon distribution and size of the two genes. After synthesis, the pre-proreceptor undergoes a series of steps involving co-translational cleavage, glycosylation and folding before being transported to the Golgi apparatus. It is here, before transportation to the membrane, that the receptors are cleaved into the characteristic α and β subunits at the tetrabasic protease cleavage site Arg-Lys-Arg-Arg [60, 80]. The subunits subsequently assemble into the mature receptor, a homodimer, comprised of two α subunits and two β subunits

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held together by disulphide bonds [45]. The α subunit is extracellular and contains the ligand binding site, whilst the β subunit comprises extracellular, trans-membrane and intracellular components containing the tyrosine kinase domain. Structure The N terminal of the α subunit comprises two homologous domains (L1 and L2) flanking the cystine-rich (CR) domain [86]. The C terminal of the α subunit contains two fibronectin type III (FnIII) domains, which facilitate protein binding and are common to many membrane-anchored receptors [85]. There is a large insert domain (ID) within the second FnIII which contains the Arg-Lys-Arg-Arg protease cleavage site [43]. The N terminal of the β subunit is both extracellular and trans-membrane and contains two further FnIII domains followed by an intracellular juxta-membrane domain and the tyrosine kinase (TK) domain which spans residues 973– 1,229 [2]. Following phosphorylation, the tyrosine residues function as docking sites for intracellular signalling molecules, particularly insulin receptor substrates 1–4 (IRS 1–4) and the Src homology domain 2 (SH2) of the Shc adaptor protein. The 108 residue C tail domain contains the phosphotyrosine binding sites for signalling molecules. The full function of the C tail domain has yet to be completely elucidated; however, it is understood that it plays a role in modulating tyrosine kinase activity, particularly at the ATP binding site [48]. Fig. 1 Diagrammatic representation of IGF-1R. FnIII fibronectin type III domains, ID insert domain, TK domain tyrosine kinase catalytic domain (residues 973–1,229). Transand juxta-membrane region (residues 930–972). C tail (residues 1,230–1,337). Adapted from Adams et al. [2] and Sehat et al. [76]

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Secondary structure of the mature IGF-1R is comprised of two α subunits and two β subunits, which is controlled by the presence of disulphide bonds. There are inter-α chain disulphide bonds at two locations, a single bond in the FnIII-1 and a triple in the ID [86]. There is only one disulphide bond between α and β subunits located between the first FnIII repeat on the α subunit and the second FnIII repeat on the β subunit [71] (Fig. 1). Ligand binding and activation of TK domain As with the IR, the binding interaction between IGF-1 and the IGF-1R is complex. Scatchard plots for the IGF-1R indicate the presence of both high- and low-affinity binding sites, and the dissociation of the bound ligand is accelerated in the presence of higher concentrations of IGF-1, suggesting that the binding sites display negative co-operativity [14–16, 40]. In contrast with the relationship between insulin and IR, there is no loss of negative co-operativity at high concentrations of IGF-1 [11]. As is the case for the insulin receptor, the IGF-1 receptor has two binding sites. Site 1 is comprised of the N terminal part of the L1 domain, the C terminal part of the FnIII-2 domain and a third area within the CR domain [29, 31, 39, 74, 89]. This third area is distinctive to the IGF-1R; insulin does not have a C domain, and site 1 on the IR contains two areas rather than three. Site 2 is thought to incorporate part of the FnIII-1 domain [16].

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The now largely accepted model of binding to the IR and the IGF-1R suggests that binding to site 1 on one IGF-1R monomer is followed by binding to site 2 on the second monomer, hence causing cross-linking and dimerisation [8, 16]. This has the effect of creating high-affinity binding within the IGF-1R dimer and also moves the two monomers into closer proximity. As is the case with the IR, it is the cooperation between the two receptor halves which creates a site with high affinity. Following ligand binding to the α subunit of the IGF-1R, trans-autophosphorylation occurs in three tyrosine residues in the activation loop (A-loop) of the tyrosine kinase domain [38, 50]. In the unstimulated state, the residues Tyr 1131, Tyr 1135 and Tyr 1136 block the active ATP site. Autophosphorylation of Tyr 1131 and Tyr 1135 destabilises the autoinhibitory A-loop conformation and the catalytically active A-Loop formation becomes stabilised by the autophosphorylation of Tyr 1136 [20, 34]. Autophosphorylation of these three residues has been shown to cause a major conformational change in the activation loop which allows unrestricted access of ATP and protein substrates to the active kinase site [34, 56].

IGF-1R and endothelial cell dysfunction IGF-1 and endothelium The spectrum of diseases associated with endothelial cell dysfunction, particularly those associated with type 2 diabetes, poses a significant global socioeconomic and healthcare problem. It is now well established that insulin resistance itself, which precedes the development of type 2 diabetes by many years [88], is an independent risk factor for the development of premature cardiovascular disease [58, 59]. Insulin-resistant but otherwise healthy South Asian males have been shown to have endothelial cell dysfunction [13, 51], and several studies have demonstrated a significant association between a reduction in the anti-atherosclerotic signalling molecule nitric oxide (NO) bioavailability and insulin resistance in humans [42, 72, 76, 87]. In addition, we have demonstrated a strong inverse correlation between insulin sensitivity and endothelial dysfunction [47, 90, 91], as assessed by NO bioavailability. Mice with expression of a mutated form of insulin receptor expressed only on the vascular endothelium (ESMIRO) exhibit endothelial-specific insulin resistance and associated reduced NO bioavailability [19]. This demonstrates that insulin receptor expression on the surface of the vasculature modulates endothelial function. The role which IGF-1 plays in modulating endothelial function is less clear. The expression and regulation of IGF1 and the IGF-1R within the vasculature are highly complicated (see [17] for review); the role of the IGF-1R and particularly, the hybrid receptor formed by the IR/IGF-1R

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in modulating endothelial function will be the subject for the rest of this article. IGF-1R activation and nitric oxide production Endothelial dysfunction is thought to play a critical role in the development of atherosclerosis [61]. In addition to acting as a semi-permeable barrier between the vessel lumen and tissue, the healthy endothelium functions as a paracrine, autocrine and endocrine organ which, in response to chemical, hormonal and physical stimuli, releases a portfolio of mediators with the function of maintaining vascular homeostasis [57]. The regulation of blood vessel tone by release of compounds with opposing actions, particularly NO and endothelin-1 is of particular relevance when considering the interaction between insulin and IGF-1 and the endothelium. IGF-1 stimulation has been demonstrated to induce NO production in rat renal inter-lobar artery endothelial cells, an effect which was blocked by the nitric oxide synthase inhibitor L-NAME and by an IGF-1 inhibitor [79]. Stimulation of human umbilical vein endothelial cells (HUVECs) with insulin and IGF-1 has been shown to lead to NO production [93]; the IGF-1R blocking antibody αIR3 completely inhibited the NO production in response to IGF-1 but only blocked 50 % of the insulin signal. Furthermore, the same group demonstrated that wortmannin, a PI3-K inhibitor, blocked the NO production seen in response to insulin stimulation. Taken together, these data show that both insulin and IGF-1 lead to NO production via a principle interaction with the canonical receptor. Whilst both IGF-1 and insulin stimulation leads to NO production, insulin is a more potent stimulator of NO production. In human umbilical vein endothelial cells (HUVECs), maximal IGF-1 stimulation produced only 40 % of the level of NO seen with maximal stimulation with insulin [93]. Although there is cross-talk between the IGF-1R and the IR, it is important to note that at physiological concentrations (100-500 pM), insulin appears to only stimulate the IR. Using bovine aortic endothelial cells, Li et al. [44] demonstrated physiological doses of insulin phosphorylated IRβ, Akt1 and eNOS but not the IGF-1Rβ subunit. At supraphysiological concentrations of insulin (1–5 nM), phosphorylation of the IGF-1Rβ subunit occurred; this was blocked by an IGF-1R-neutralizing antibody. Following stimulation with insulin or IGF-1, autophosphorylation of tyrosine residues on the β subunit of the receptor initiates phosphorylation of insulin receptor substrate (IRS) proteins (particularly IRS1) at multiple tyrosine residues [30, 64]. Phosphorylated IRS1 subsequently binds PI3-K, particularly via the SH2 domain of the p85 α subunit. Increased catalytic activity of the p110 subunit of PI3K occurs, initiating the conversion of plasma lipid phosphatidylinositol 3,4,-bisphosphate to phosphatidylinositol

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3,4,5-trisphosphate (PIP3). The activated PIP3 causes proteins containing pleckstrin homology domains to accumulate at the cell membrane. With regard to insulin signalling, the most important of these are seronine−threonine kinases Akt and 3-phosphoinositide dependent protein kinase-1 (PDK-1) [7]. The congregation of these proteins in close proximity promotes the phosphorylation and subsequent activation of Akt by PDK-1 (Fig. 2). Following phosphorylation, Akt induces phosphorylation of the endothelial isoform of nitric oxide synthase (eNOS) at serine 1,177 [18]. Phosphorylated eNOS catalyses the conversion of L-arginine to L-citrulline by the transfer of electrons from nicotinamide adenine dinucleotide phosphate (NADPH); this results in the generation of NO [57]. The reaction requires the essential cofactors tetrahydrobiopterin (BH4) and flavin adenine dinucleotide (FAD). Insufficient quantities of either co-factor or of NADPH itself may lead to the generation of oxygen-free radicals instead of NO [12, 31] (Fig. 3). In addition to activation of the PI3/K pathway, binding of insulin or IGF-1 to the respective or hybrid receptors also leads to Ras-dependent activation of the MAP kinase pathway through interaction with the Src homology domain 2 (SH2) of the Shc adaptor protein [52]. Although activation of the mitogen-activated protein kinase (MAPK) pathway does not appear to modulate endothelial function, IGF-1

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activation of the MAPK pathway has been demonstrated to induce vascular smooth muscle cell proliferation and migration, and hence, may be of importance in maintaining atherosclerotic plaque stability [63, 84]. IGF-1 signalling and angiogenesis Whilst not primarily linked to endothelial function, activation of the IGF-1 signalling cascade is able to modulate angiogenesis, principally by interaction with vascular endothelial growth factor (VEGF). IGF-1 itself promotes endothelial cell migration and tube formation [17], a process which is regulated by IGF-1R mediation of VEGF action; this, in turn, results in an intracellular cascade involving specific mitogen-activated protein kinases (MAPKs) [67]. Knockout of the IGF-1R specific to the vascular endothelium (VENIFARIKO) produces a modest reduction in retinal neovascularisation in response to hypoxia [41], in tandem with a reduction in vascular mediators.

IGF-1R/IR hybrids It is now well recognised that IGF-1R and IR are able to form hybrid receptors, consisting of an IR αβ heterodimer and an IGF-1R αβ heterodimer [49, 69]. The physiological role of

Fig. 2 Initiation of PI3-K/Akt pathway by ligand binding to the IGF-1R. The same applies for binding to the insulin or the IGF-1R/IR hybrid

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Fig. 3 Production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). The reaction requires tetrahydrobiopterin (BH4) which binds to the oxygenase domain of eNOS. L-arginine and O2 also bind to the oxygenase of eNOS; NADPH and flavin mononucleotide and flavin adenine dinucleotide (FAD) bind to the reductase domain of eNOS

hybrids has yet to be established, although it appears increasingly likely that heterodimerisation of the IRαβ and the IGF1Rαβ plays a significant part in determining the cells’ sensitivity to insulin [9, 35]. We have recently published data demonstrating that manipulation of the ratio of IR and IGF1R affects hybrid receptor formation and insulin sensitivity [1, 36]. This will be discussed in greater detail later. Hybrid receptors behave in a similar manner to the IGF1R with respect to the binding capability of IGF-1 and insulin; the hybrid receptor has an affinity for IGF-1 which is 20-fold higher than its affinity for insulin [6, 68, 70]. It is not precisely clear why the hybrid receptor has a significantly higher affinity for IGF-1 than insulin. The determining fact is likely to relate to the ability of the ligand to bind to site 2 on the opposing α subunit. It would appear that IGF-1 is either able to bind to site 2 of the IR as effectively as it can to the IGF-1R or that two site interactions are less important in determining high affinity for IGF1 than for insulin [6, 11]. Interestingly, replacing the L2/Fn domain from the IGF-1R within a hybrid with the equivalent domain of an IR (isoform A) increases the affinity of the hybrid receptor for insulin by 20-fold [6].This suggests that the L2/Fn domain plays a critical role in binding insulin in trans from the primary binding site. Ligand binding to the hybrid receptor via a single α subunit stimulates autophosphorylation of both β subunits, causing activation of the TK domain in the manner described above [27]. Furthermore, it has been demonstrated that formation of a hybrid receptor comprising a wild-type IGF-1R and a kinase inactive IR results in trans-dominant inhibition of kinase activity [78]. This evidence suggests that interaction between the two halves of the hybrid receptor is critical to allow for high-affinity binding and also that loss of kinase activity of either monomer renders the whole hybrid inactive. It remains unclear whether the isoform of the insulin receptor incorporated into the hybrid has an effect on ligand

binding. Preliminary studies appeared to suggest that hybrids composed of IR-A/IGF-1R have a higher affinity for insulin than those comprising the IR-B/IGF-1R variant [54]. In contrast, two studies demonstrated that the IR isoform incorporated into the hybrid receptor has no effect on the affinity for insulin binding [6, 66]. It should be noted that whilst there is good evidence to demonstrate that the hybrid receptor functions as an IGF-1R receptor in respect to its binding capability, there is no direct evidence highlighting the downstream effects following hybrid receptor autophosphorylation. It is perhaps reasonable to suspect that signal transduction occurs through the PI3-K pathway, as is the case for the IR and the IGF-1R, but there is no direct experimental evidence to demonstrate this. In particular, there is no evidence that these hybrid receptors are able to stimulate glucose uptake or NO production. Whether the effect seen in tissues which differentially express hybrid receptors is a function of the hybrid receptor itself or occurs as a result of sequestering the IR or IGF-1R is also not known. It is unclear precisely what regulates the formation of hybrid receptors, although proportions in normal tissue appear to correlate with a random process of assembly [77]. There is, however, a relationship between the molar ratio of receptors and the formation of hybrid receptors [5]; increasing the expression of IGF-1R αβ half receptors in cultured fibroblasts drives IR αβ half receptors to be incorporated into hybrids [27]. We have demonstrated similar findings on endothelial cells [36]; furthermore, we have demonstrated that reducing expression of the IGF-1R on endothelial cells reduces hybrid expression [1]. Hybrid receptors are expressed in many tissues and cell types, with data suggesting that a high number of IGF-1 binding sites are found on hybrid receptors rather than the IGF-1R itself. The distribution of hybrid receptors in mammalian tissue appears to be extensive, with a surprisingly high number of IGF-1 binding sites behaving as hybrid receptor.

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Bailyes et al. [5] found that 74 % of the skeletal muscle IGF1R were incorporated into hybrid receptors, with similar results in the heart (87 %), kidney (70 %), fat (68 %), spleen (53 %) and placental tissue (72 %). It should be noted that this represents results obtained from tissue homogenates rather than in specific cell types. Federici et al. [24] used microwell assays found lower percentages of IGF-1R expressed as hybrid receptors in human tissue—placental (55 %), skeletal muscle (46 %) and adipose tissue (38 %). Using immunoprecipitation and immunoblotting, HUVECs [53], human coronary artery endothelial cells (HCAECs) [10] and human cardiac microvascular endothelial cells (HMVECsC) [4] have all been shown to express hybrid receptors. In each of these cell lines, IGF-1R was noted to be more numerous than IR (mRNA levels of IGF-1R compared with IR were eightfold higher in HCAEC [10], sevenfold higher in freshly isolated HUVEC [53] and 120-fold higher in HMVEC-C) [4]. In common with hybrids found elsewhere, they bind IGF-1 rather than insulin with high affinity. Certainly in these cells, IGF-1Rs are more numerous than IR, and therefore, a significant number of IR are sequestered into hybrid receptors. In human microvascular endothelial cells, phosphorylation of Akt in response to insulin occurred at 10−7 M and at between 10−8 and 10−7 M in response to IGF-1 [37], suggesting that these microvascular endothelial cells are sensitive to IGF-1 but relatively insensitive to insulin, most likely due to an excess of IGF-1R and incorporation of IR into hybrid receptors. Taking these data together, it is clear that the vascular endothelium itself is a relatively insulin-resistant tissue. Our group has recently published work which highlights that the ratio of IR/IGF-1 not only determines hybrid formation but also appears to have an effect on insulin sensitivity at a whole body level and at the level of the endothelium. Transgenic mice with endothelial-specific over-expression of the IGF-1R (hIGFREO) have increased levels of hybrid receptors compared with wild-type counterparts, coupled with endothelial insulin insensitivity (as measured by reduced insulin-stimulated NO production) [36]. Studies performed on a model which crossed the IRKO mouse mice (whole body haplo-insufficiency of in the insulin receptor; which have both whole body and vascular insulin resistance and endothelial dysfunction) with an endothelial-specific knockdown of the IGF-1R (ECIGFRKO) support our hypothesis that manipulation of hybrid numbers may have an effect on insulin sensitivity [1]. The resultant model demonstrated that reducing IGF-1R in IRKO mice restored insulin-mediated vasorelaxation, enhanced insulin-stimulated eNOS activation and enhanced insulin-stimulated NO release in endothelial cells. These data suggest that manipulation of IGF-1 to insulin receptor stoichiometry may be able to restore insulin sensitivity in insulin-resistant states. Although the exact mechanism which regulates the formation of hybrids is not known, there is now a clear association

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with increased expression of hybrid receptors and hyperinsulinaemia [22], hyperglycaemia [21], insulin resistance [81], type 2 diabetes mellitus [25, 26] and obesity [23]. A mechanistic link has not been fully proven; however, there is evidence that treatment of hyperglycaemia with phlorizin completely reverses the increased expression of hybrids seen in the skeletal muscle of diabetic rats [21]. More recently, elegant experiments by Sherajee et al. [65] demonstrated that treatment of vascular smooth muscle cells (VSMCs) with the mineralocorticoid hormone aldosterone leads to a threefold increase in expression of IGF-1R and a twofold increase in hybrid receptor expression in comparison to untreated cells; concurrent administration of aldosterone and eplerenone (an aldosterone antagonist) negated this. In vivo, insulin signalling in murine aorta was blunted by treatment with aldosterone and salt; in the same tissue, treatment with aldosterone and salt was associated with increased IGF-1R and hybrid expression as was seen in the VSMCs [65]. Treatment with aldosterone and salt rendered the mice hypertensive; interestingly, treatment with the antihypertensive hydralazine normalised blood pressure but did not have a favourable impact on hybrid receptor expression. This suggests that the increased IGF-1R and hybrid receptor expression was related to the aldosterone itself, rather than the effect on systemic blood pressure. Data derived from experiments using murine mammary epithelial cells show an increased level of hybrid expression in late pregnancy [62], suggesting that hybrid receptor expression is, to some degree, developmentally regulated. Contrary to this, hybrid receptor abundance has been shown to decrease with early development (7–26 days) in skeletal muscle of suckling pigs [75]. What happens to the expression of hybrids beyond this early stage of development is unclear. There is evidence of a link between longevity and reduced signalling though the IGF-1 axis [33, 73, 82]. Whether this is via an alteration in hybrid expression or another mechanism is not known. Expression of hybrids in placental tissue has been found to be down-regulated by tumour necrosis factor-α which is of importance when considering the role which IGF-1 plays in mutagenesis [32]. Further exploration of this aspect of IGF-R regulation is beyond the scope of this article.

IGF-1R and disease Although the link between increased hybrid receptors and endothelial cell dysfunction is beginning to be unravelled, it is currently unclear whether expression and function of IGF1R and hybrid receptors may play in the development of cardiovascular disease. As is described above, there is increased expression of the hybrid form of the IR/IGF-1R in patients with known risk factors for cardiovascular disease; in subjects with diabetes,

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obesity and systemic insulin resistance [23, 25, 26, 81], hybrid expression was shown to be inversely proportional to insulin sensitivity. Studies by Federici et al. [21, 22] suggest that increased hybrid receptor expression occurs secondary to hyperinsulinaemia and hyperglycaemia; treatment of hyperglycaemia leads to reduction in number of hybrid receptors. This would seem to imply that a vicious circle of hyperinsulinaemia, leading to increased hybrid receptor expression, reduced insulin sensitivity and further compensatory increases in insulin levels which are set in motion early on in the development of type 2 diabetes and may well precede the onset of symptoms or the development of abnormal blood glucose. As yet, there are no data to show whether the expression of hybrid receptors on the endothelium is altered in various disease states. If hybrid receptor expression on the endothelium is increased in response to hyperglycaemia and hyperinsulinaemia, as has been demonstrated in adipose, skeletal muscle and placental tissue, it could provide part of the explanation for the vascular insulin insensitivity and endothelial dysfunction seen in seemingly healthy South Asian males [13, 51]. It is unclear whether treatment of hyperinsulinaemia per se is sufficient to reduce hybrid numbers in tissues. In women with polycystic ovarian syndrome (PCOS), associated with both endothelial dysfunction and insulin resistance, treatment with metformin reduces endothelial dysfunction [3]. Interestingly, this study showed that endothelial function improved despite no change in insulin resistance. This suggests that this improvement was independent of an effect on insulin sensitivity. Although the above study did not measure insulin levels, metformin has previously been shown to reduce insulin levels in women with PCOS [83]. Whether metformin alters the expression and distribution of IGF-1R/IR hybrids is unknown, but this is a potential therapeutic avenue which requires further investigation.

Conclusion and future direction We have shown that manipulating numbers of IGF-1R both on a whole body level and specific to the endothelium have the potential to restore endothelial insulin sensitivity and improve endothelial function [1, 36]. Approaches to targeting the IGF1R (reviewed in [92]) including IGF1-R antibodies which lead to receptor degradation; antisense molecules which inhibit IGF-1R expression and apatmers which reduce IGF-1R numbers have shown promise in the treatment of malignancy; they require study in the field of endothelial biology. There have been significant improvements over the last decade in understanding the relationship between the insulin and IGF-1R and endothelial function. The function and regulation of hybrid receptors are beginning to be unravelled, and advances in this field may help to understand the development of accelerated atherosclerosis in patients with type 2 diabetes.

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