The major finding of this study is that PKC mediates endothelial insulin resistance and vascular smooth muscle hypersensitivity to insulin in DH rats. PKC, however, does not seem to be involved in acetylcholine-mediated vasodilation. The implications and limitations of these findings are elaborated.
Endothelial dysfunction (impaired endothelium-dependent vasorelaxation in response to blood shear stress or acetylcholine) is a salient feature of patients with metabolic syndrome, type 2 diabetes, and hypertension [2, 3]. The epidemiologic observations that smaller size at birth is associated with increased rates of coronary heart disease, stroke, type 2 diabetes, adiposity, and metabolic syndrome in adult life have been extensively replicated . IUPD rat is widely used as an animal model of developmentally programmed metabolic syndrome . Endothelial dysfunction has also been described in the IUPD rat model . In the current study, we used the rat model that combined IUPD and postnatal exposure to high salt and have extended observations of endothelial dysfunction to these conditions [10, 11, 18]. Moreover, we demonstrated that this endothelial dysfunction due to impaired acetylcholine-dependent vasodilation was accompanied by endothelial insulin resistance and SMC hyper contractility in response to both phenylephrine and insulin.
From vascular function point of view, endothelial insulin resistance is manifested by paradoxical contraction of the artery in response to insulin stimulation. Intracellular insulin signaling is mediated by two major pathways, Phosphoinositide (PI)3-kinase/Akt and mitogen-activated protein kinase (MAPK). The former induces NO production and, therefore, leads to vasorelaxation . The latter, however, is associated with the downstream release of endothelin 1 (ET1) that is consistent with vasoconstriction. In the insulin-sensitive endothelial cells, these pathways are well balanced, and insulin stimulation is usually associated with weak vasodilation. Insulin resistance is known to be pathway-selective; i.e., it affects primarily PI3-kinase signaling and either spares or even enhances MAPK/ET1 signaling . As a result, the net effect of insulin is swayed towards vasoconstriction.
It has been demonstrated that endothelial insulin resistance in other animal models of metabolic syndrome, e.g., Zucker fatty rats, diabetic mice, and neonatal rats with high glucose, was mediated by PKC-β [4, 18–20]. Most likely, PKC induces insulin resistance by serine phosphorylation of insulin receptor substrate(s) that prevents insulin-induced tyrosine phosphorylation and thus blocks insulin signaling . RBX treatment restored Akt phosphorylation and NO production in response to insulin in the Zucker fatty rats . Our functional data based on pharmacological inhibition of PKC by RBX indicate that PKC activation is also critical for endothelial insulin resistance in the femoral artery of DH rat.
It is unclear how PKC is activated in the arteries of DH rats. Angiotensin II (AngII) type 1 receptor signaling might be the most pertinent in this context [12, 22, 23]. AngII intracellular signaling involves both calcium and diacylglycerol generation that is consistent with downstream activation of conventional and novel PKC isoforms . AngII is known to induce vascular insulin resistance in a PKC-dependent manner . Our data demonstrated that both Capto and RBX independently conferred vascular benefits. It is unlikely though that angiotensin II signaling is solely mediated by PKC. It is equally unlikely that angiotensin II is the only upstream signal that activates PKC in the vasculature of DH rats. Alternatively, it is plausible that elevated blood glucose and fatty acids are metabolized into diacylglycerol in the vascular wall, and metabolically produced diacylglycerol stimulates PKC . In the current study, we used Capto as a positive control treatment for the blood pressure portion of the study rather than a true mechanistic tool. Future mechanistic studies are necessary to understand possible relationships between AngII-dependent and independent mechanisms of vascular PKC activation.
Surprisingly, we have observed clear dichotomy between drug effects on endothelial insulin resistance and endothelial dysfunction due to impaired acetylcholine-dependent vasodilation. While endothelial insulin resistance was completely ameliorated by Capto and RBX alike, neither compound was able to restore arterial response to acetylcholine. Arguably, this phenomenon may reflect differential effects of complex DH rat milieu on NO production and release. The IUPD rat was diabetic and therefore more complicated than spontaneously hypertensive (SH) rat . It was reported that Capto prevented impaired endothelial response to acetylcholine in SH rats, while antihypertensive treatment did not restore endothelial function in rats with hypertension and diabetes [26–28]. We have also recently shown that restoration of endothelial response to acetylcholine in Zucker diabetic fatty rats required activation of peroxisome proliferation-activated receptor γ, thereby suggesting that this part of endothelial function in diabetic rats is regulated in an angiotensin-independent manner . Future studies are required for deeper mechanistic understanding of this phenomenon.
It is also unclear why exogenous insulin improved endothelial response to acetylcholine in every group. It is possible that insulin may act at the level of NO production. Agonists, including acetylcholine and bradykinin, are known to induce endothelial NO release from eNOS activation mediated in a calcium-dependent manner [30, 31]. In diabetes where eNOS activity is impaired, there is less NO to be released in response to a given dose of acetylcholine [1, 6]. Insulin stimulates Akt phosphorylation and thereby activates eNOS [1, 3]. Therefore, less acetylcholine may be needed to induce vasodilation in the presence of insulin. Phenomenon of insulin-dependent improvement of acetylcholine-induced vasodilation is also reported in the clinic [32, 33]. It would be intriguing to study effects of RBX on endothelial insulin sensitivity in the clinic to complement existing data solely focusing on the endothelial dysfunction due to impaired acetylcholine-dependent vasodilation [7–9]. This approach becomes even more appealing in light of new data demonstrating, that in isolated, genetically induced endothelial insulin resistance (mediated by endothelial-specific insulin receptor knock-out), dramatically enhanced development of atherosclerosis in an ApoE-deficient mice model .
The current study is the first attempt to address the role of PKC in a very complex non-genomic model related to metabolic syndrome. In follow-up studies, it is necessary to unravel relative contribution of individual risk factors (IUPD, high salt and age) that are known to have direct and indirect effects on vascular function as well as specific PKC isoforms involved in differential vascular response to various stimuli [12, 17, 34–36]. It is equally important to identify biochemical mechanisms of the observed functional phenomena. Nevertheless, defining PKC as a key regulator of vascular functional response to insulin provides important directions for future experimental and, potentially, clinical studies aiming at preventing vascular complications of metabolic disorders.