The term prediabetes was coined in the 1970s and describes individuals whose fasting glycemia does not meet the criteria for type 2 Diabetes Mellitus (DM2) diagnosis but is high enough (≥100 and ≤125 mg/dL) to be considered as normal (American Diabetes Association, 2021; Gaitán-González et al., 2021). Prediabetes constitutes a reliable indicator of high risk to develop DM2 and macrovascular disease (Rett and Gottwald-Hostalek, 2019); however, in the clinical environment, its diagnosis has been complicated because of the lack of performing the oral glucose tolerance test on a regular basis. The National Diabetes Data Group defined prediabetes as a condition characterized by impaired glucose tolerance (IGT) after 2 h of post oral glucose tolerance test (with blood glucose values between 140 and 199 mg/dL) (American Diabetes Association, 2021).

By the end of the 1990s, the American Diabetes Association and the World Health Organization (WHO) included impaired fasting glycemia (IFG) by showing fasting blood glucose levels (in mg/dL) from 100 to 125 or from 110 to 125, respectively, as a key parameter for prediabetes diagnosis. Finally, the American Diabetes Association incorporated the glycated hemoglobin A1c (HbA1c) criterion for the prediabetes definition. Currently, prediabetes is defined as IFG with blood glucose levels between 100 and 125 mg/dL, the presence of IGT, and/or HbA1c in a range of 5.7%–6.4% (American Diabetes Association, 2021). Nowadays, prediabetes is also associated with obesity, high levels of total cholesterol and/or triglycerides, and low levels of HDL-cholesterol (HDL-C), which are also common alterations in MetS (Punthakee et al., 2018).

MetS is defined as an association of diverse physiological and biochemical disturbances that constitute a major risk factor for the development of DM2 and cardiovascular disease (CVD) (Alberti et al., 2009). MetS is also associated with a wide range of pathologies like non-alcoholic fatty liver disease, polycystic ovary syndrome, obstructive sleep apnea, sexual dysfunction, and even breast, colon, and prostate cancers (Gaitán-González et al., 2021). The incidence of MetS has been rising concomitantly with insufficient physical exercise and unhealthy dietary habits in both young and adult populations worldwide. MetS prevalence depends on diverse factors such as age, gender, and ethnicity, with different studies indicating that about a quarter of the adult worldwide population has been diagnosed with MetS, and its incidence emulates the obesity and DM2 rates (Gupta and Gupta, 2010; Saklayen, 2018). For instance, in Mexico, the prevalence of MetS in individuals without incident DM2 is 43.9%, and abdominal obesity is 78.1% (Arellano-Campos et al., 2019). However, there is a three-fold higher risk of developing DM2 in subjects who had MetS compared to those who did not (Arellano-Campos et al., 2019). Therefore, there is an urgent need to control and prevent prediabetes and MetS to diminish the occurrence of DM2 and its adverse consequences on the cardiovascular system.

While the existence of MetS is not in doubt, a worldwide agreement concerning the main corporal and biochemical parameters for MetS diagnosis has been hard to achieve. Over the past years, several clinical organizations have tried to establish the criteria to best describe the signs and symptoms that characterize the metabolic risk of MetS. Different organizations, such as the WHO, the Joint Interim Statement by the International Diabetes Federation, the National Heart, Lung, and Blood Institute (NHLBI), the American Heart Association (AHA), the World Heart Federation, the International Atherosclerosis Society, and the International Association for the Study of Obesity have established a definition as well as a standard for MetS diagnosis (Table 1). The harmonized definition of MetS establishes that a person must fulfill at least three of the following criteria: central obesity, hypertriglyceridemia, high blood pressure, impaired fasting glycemia (a common feature with prediabetes), and low levels of HDL-C to be diagnosed with MetS (Alberti et al., 2009; Neeland et al., 2024). It is also important to prevent the progression of MetS to more advanced stages, including end-organ damage such as the kidney, to avoid the emergence of the cardiovascular-kidney-metabolic syndrome (Neeland et al., 2024).

Due to its high probability of leading to the development of DM2, it has long been debated whether MetS should be considered as a prediabetic state or not, independently of the presence of IFG. For instance, MetS is not necessarily a prediabetic state because it may occur without the presence of IR (Meigs et al., 2007). This notion is associated with the AHA/NHLBI definition for the MetS, which was structured to simplify the criteria for MetS to standard blood test parameters for facilitating its diagnosis and its focus on obesity as a requirement (Grundy, 2005).

On the other hand, an insulin blood test is not a frequently requested exam by clinicians for prediabetes diagnosis, and it does not lend itself as well to large epidemiological studies, where a quick and simple insulin assessment is important (Huang, 2009). Several experts have considered that MetS definitions that exclude hyperinsulinemia or IR as part of the MetS components fail to provide a proper MetS diagnosis (Reaven, 2005). In fact, when the euglycemic clamp test is carried out in people with high visceral fat, there exists a close relationship with the presence of IR (Ruderman et al., 1998). IR is an entity where the insulin signaling pathway is altered at several signal transduction points and favors the development of dyslipidemia, obesity, and arterial hypertension (Ruderman et al., 1998; Eckel et al., 2005); then, IR should be considered as part of MetS pathophysiology. Moreover, experimental MetS models develop hyperinsulinemia and IR in organs with a critical metabolic role, such as the liver and the heart (Özcan et al., 2004; Landa-Galvan et al., 2020). Although prediabetes and MetS are different clinical entities, both share IFG in their criteria for diagnosis. IFG underlies hyperinsulinemia and IR, indirectly determined by the IGT test. Also, both MetS and prediabetes can trigger the development of DM2 (Reaven, 1988; Salazar et al., 2017) (Figure 1).

MetS and prediabetes are involved in several microvascular and macrovascular diseases (Palladino et al., 2020). For instance, the Atherosclerosis Risk in Communities study determined that prediabetic patients were 30% more prone to be hospitalized (Schneider et al., 2016). An increase of 1% in the HbA1c was related to an augmented risk in coronary heart disease and 10-year cardiovascular mortality (Khaw et al., 2004; Wasserman et al., 2018). Likewise, the three fundamental microvascular complications: retinopathy, nephropathy, and neuropathy, have been associated with prediabetes in several studies (Brannick et al., 2016).

In patients with IFG, the in vivo vasodilatory response to intra-arterial infusion of ACh, an endothelium-dependent vasodilator, was reduced compared to healthy subjects. In contrast, the vasodilatory response to sodium nitroprusside (SNP), which is endothelium-independent, was similar between the two groups, indicating that mainly endothelium-dependent vasodilation was impaired in individuals with prediabetes (Vehkavaara et al., 1999). Another study evaluated both endothelium-dependent and -independent vasodilatory responses with the same vasodilators, reporting impairment of both mechanisms compared to healthy subjects (Caballero et al., 1999). These findings suggest that vascular dysfunction in prediabetes can be heterogeneous, with some individuals exhibiting isolated endothelial impairment, while others present vascular abnormalities that affect both ECs and VSMCs function.

Some studies have suggested that the mechanisms underlying vascular dysfunction in prediabetes may differ from diabetes-associated vasculopathy. Instead of the advanced vascular damage induced by chronic hyperglycemia in DM2, IR in prediabetes may contribute to endothelial and VSMCs dysfunction (Jiang et al., 1999b). In the aorta and microvessels of obese Zucker rats (OZR), insulin signaling is impaired, as evidenced by a reduced tyrosine phosphorylation of the insulin receptor substrates IRS-1 and IRS-2, decreased phosphatidylinositol 3-kinase (PI3K) activity, and diminished serine phosphorylation of Akt upon insulin stimulation (Jiang et al., 1999a).

Although prediabetes and MetS share a common pathway to induce vascular dysfunction through hyperinsulinemia, some studies indicate that the pathophysiology of prediabetes could be related to early pancreatic β-cells dysfunction. This situation leads to a progressive decline in insulin secretion, with no IR establishment, unlike the pathophysiology of DM2 (Abdul-Ghani and DeFronzo, 2009; Kanat et al., 2012; Kabadi, 2017). Therefore, hyperinsulinemia is not always a factor participating in the vascular dysfunction in prediabetes.

MetS correlates well with the development of several cardiac diseases, including diastolic dysfunction, heart attacks, and arrhythmia (Dincer, 2012; Tune et al., 2017), underlying macrovascular and microvascular diseases. Several studies have shown that MetS is a risk factor for coronary artery (CA) disease (Carr and Brunzell, 2004) and arterial thickness and stiffness, which lead to disturbances in blood pressure (Scuteri et al., 2004; Czernichow et al., 2005). Also, MetS impairs microvascular peripheral cerebral perfusion (Nazzaro et al., 2013) and increases stroke events (Ford, 2004).

Although hyperglycemia is a crucial factor of vascular complications in diabetes, vascular dysfunction has also been reported in MetS patients even in the absence of an overt hyperglycemic condition (Galassi et al., 2006; Rundek et al., 2010); therefore, more attention is needed to understand the effects of dyslipidemia, hyperinsulinemia, and IR as key factors of vascular complications.

While in prediabetes endothelial dysfunction is a precursor to more severe vascular issues, in MetS the inflammation and the oxidative stress are more pronounced, particularly involving tumor necrosis factor-α (TNF-α). Small arteries of OZR have impaired endothelium-dependent vasodilation in response to ACh, whereas endothelium-independent vasodilation in response to SNP was comparable with control animals, along with decreased endothelial nitric oxide synthase (eNOS) expression, increased both mRNA and protein expression of TNF-α, and reactive oxygen species (ROS) (Picchi et al., 2006). Increased levels of TNF-α have also been found in the obese (ob/ob) mouse model (Hotamisligil et al., 1993).

In vascular tissue, ROS are generated in both ECs and VSMCs, with their production being exacerbated in MetS (López-Acosta et al., 2023). For instance, VSMCs or ECs isolated from the aorta and cultured with a high concentration of the free fatty acid palmitate (200 µM), mimicking a dyslipidemic environment, exhibited increased ROS production, which was associated with augmented diacylglycerol synthesis and PKC activity (Inoguchi et al., 2000). Elevated ROS levels promote the transition of VSMCs from a contractile to a proliferative phenotype (Badran et al., 2020), contributing to increased tunica media thickness and impaired vasodilatory response.

The etiology of vasculopathy in prediabetes and MetS may share common mechanisms involving alterations in the insulin receptor (InsR) signaling pathway, which affect both ECs and VSMCs. However, specific vascular alterations in MetS, such as increased ROS production and a proinflammatory state, may contribute to distinct pathological features.

Nevertheless, we propose that alterations in intracellular Ca²⁺ handling, as well as changes in the expression or activity of the four proteins involved in regulating the vascular tone (the Ca_V1.2, the SERCA pump, the RyRs, and the BK_Ca channels) in VSMCs impair the vascular function. These mechanisms will be further discussed in the following sections.

Insulin is a 51-amino acid peptide hormone secreted by pancreatic β-cells, which plays a fundamental role in maintaining energy homeostasis by regulating glucose and lipid metabolism. To accomplish this, insulin triggers glucose uptake in the muscle and adipose tissues and promotes glycogenesis in the liver and muscle. Insulin also suppresses glyconeogenesis in the liver and exerts a significant effect on lipid metabolism, including fatty acid and triglyceride synthesis and inhibition of lipolysis (Gutiérrez-Rodelo et al., 2017). Insulin elicits critical biological effects in cardiovascular tissues, including VSMCs, such as vasorelaxation, by stimulating nitric oxide (NO) production, decreasing [Ca²⁺]_cyt, and enhancing myosin light chain (MLC) sensitization through autocrine and paracrine mechanisms (Sowers, 2004).

Insulin plays a critical role in the cardiovascular system, where it regulates cardiac contractility, vascular tone, lipid, glucose, and protein metabolism (Muniyappa et al., 2007; Landa-Galvan et al., 2020). The biological actions of this hormone begin when it binds to the InsR, a tetrameric membrane protein with intrinsic tyrosine kinase activity, which undergoes auto-phosphorylation and promotes the phosphorylation of several intracellular scaffolding substrates, such as the IRS on tyrosine residues (pY). This substrate subsequently functions as a docking protein for downstream signaling molecules, activating different signaling pathways (Saltiel, 2021). The primary signaling pathways activated in response to insulin are: 1) the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, which is responsible for most of their metabolic actions. IRS serves as a scaffold protein for PI3K, which favors the conversion of phosphatidylinositol diphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3), stimulating Akt activation via phosphorylation by the phosphoinositide-dependent protein kinases PDK1 and PDK2; and 2) the mitogen-activated kinase (MAPK) pathway, which regulates gene expression, cellular growth, and proliferation (Olivares-Reyes et al., 2009; Gutiérrez-Rodelo et al., 2017; White and Kahn, 2021). Subsequently, Akt regulates glucose uptake through the inhibitory phosphorylation of Akt substrate of 160 kDa (AS160), increasing the trafficking of glucose transporter 4 (GLUT4) storage vesicles to the cell membrane and allowing glucose uptake. GLUT4 is expressed in VSMCs and mediates both insulin-dependent and non-insulin-dependent glucose uptake (Figure 2) (Banz et al., 1996; Park et al., 2005).

InsRs are present in both ECs and VSMCs in blood vessels. Insulin promotes glucose uptake in these cells through a mechanism involving GLUT4 (Banz et al., 1996; Park et al., 2005). In ECs, insulin induces the production of NO through the PI3K/Akt pathway, which activates eNOS (Figure 2). Endothelial-derived NO diffuses into VSMCs to activate the guanylate cyclase (GC) enzyme, which increases cGMP levels to promote vascular relaxation by Ca²⁺-dependent and Ca²⁺-independent mechanisms. Interestingly, insulin treatment does not alter intracellular Ca²⁺ levels in ECs (Hartell et al., 2005), which suggests that insulin-stimulated NO production is Ca²⁺-independent and mediated by Akt activation. Intriguingly, the expression of both eNOS and inducible NOS has also been detected in VSMCs (Trovati et al., 1999; Muniyappa et al., 2007; Salt, 2013). In fact, insulin stimulation of isolated VSMCs increases NO synthesis by both NOS isoforms in a PI3K/Akt-dependent manner, leading to relaxation via activation of protein kinase G (PKG), which suggests that insulin directly regulates vascular tone (Lee and Ragolia, 2006; Muniyappa et al., 2007; Salt, 2013). PKG mediates vasorelaxation mainly by phosphorylating target proteins such as the myosin phosphatase–targeting subunit and the IP₃ receptor–associated cGMP kinase substrate (Krawutschke et al., 2015). PKG also phosphorylates the BK_Ca channel (Alioua et al., 1998; Thorpe et al., 2017; Moraes et al., 2024) and phospholamban, the negative regulator of the SERCA pump, increasing its activity (Sarcevic et al., 1989; Moraes et al., 2024) (Figure 2). However, alterations in these phosphorylation sites in the context of IR have not been shown. Moreover, it has been reported that PKG phosphorylates the cardiac isoform of RyRs (RyR2), thereby modulating its activity via the activation of the PI3K/Akt/NOS signaling pathway (Baine et al., 2020; Gonano et al., 2022); although it is still unknown whether this occurs in the vasculature. Insulin also inhibits activation of the small GTPase RhoA in a NO/cGMP-dependent manner, leading to increased MLC phosphatase activity. Thus, it is possible that these effects, together with the inhibition of Ca²⁺ influx and the increase in Ca²⁺ efflux, underlie the direct effects of insulin on vascular tone (Salt, 2013). These mechanisms increase blood flow and promote the use of glucose in the target tissues (Gutiérrez-Rodelo et al., 2017).

Although evidence suggests that insulin physiologically regulates vascular tone by decreasing [Ca²⁺]_cyt in VSMCs, the precise mechanisms by which this hormone exerts its effects remain to be completely elucidated. Pioneering research conducted in the 1990s demonstrated that insulin modulates agonist-induced increases in [Ca²⁺]_cyt through Ca_V1.2 and alters the activity of MLC phosphatases in VSMCs (Muniyappa et al., 2007). Insulin may attenuate VSMC contractile responses by diminishing agonist-mediated increases in [Ca²⁺]_cyt, partly by decreasing Ca²⁺ influx through both receptor- and voltage-gated channels (Standley et al., 1991). Although the identity of the insulin-regulated channels in VSMCs and the associated mechanisms are not fully elucidated at present, it is highly probable that Cav1.2 is the target of insulin regulation.

IR is a systemic disorder in which cells fail to respond to normal levels of circulating insulin. Under this condition, the highly critical metabolic functions of this hormone, mainly in hepatic, muscular, and adipose tissues, such as glucose uptake and synthesis of glycogen, lipids, and proteins, are altered (Olivares-Reyes et al., 2009; Gutiérrez-Rodelo et al., 2017; Vazquez-Jimenez et al., 2021).

IR is considered a condition associated with prediabetes, MetS, and DM2. It has been identified that the most common alterations that give rise to the IR condition are carried out at the level of the InsR itself and the IRS in the effector molecules downstream of the InsR, such as PI3K and Akt, or by changes in the InsR gene expression or proteins that participate in the pathway, such as GLUT4 transporters. It has been shown that the altered expression of GLUT4 transporters can influence vascular reactivity and thus contribute to vascular disease (Atkins et al., 2015).

At the molecular level, the most common alterations in IR are: 1) a decreased number of InsR and its catalytic activity; 2) an increase in InsR and IRS serine/threonine (Ser/Thr) phosphorylation, followed by increased activity of protein tyrosine phosphatase, and 3) decreased PI3K and Akt activity (Gutiérrez-Rodelo et al., 2017).

The detrimental effects of IR involve VSMCs’ proliferation, vasoconstriction, and proinflammatory activity (Ormazabal et al., 2018).

As a multifunctional intracellular messenger, Ca²⁺ can signal both the contraction and relaxation of VSMCs; the fine-tuning of these signals is a crucial feature of smooth muscles. Particularly, elementary local Ca²⁺ signals produced by RyRs participate in vasorelaxation. Under physiological conditions, the local Ca²⁺ signals, also known as Ca²⁺ sparks, activate nearby BK_Ca channels that generate STOCs. STOCs have a key role in the control of arterial tone by shifting plasma membrane potential toward less positive values, which, in turn, limits Ca²⁺ influx through Ca_V1.2 and diminishes [Ca²⁺]_cyt opposing vasoconstriction. Therefore, Ca²⁺ sparks and STOCs are a functional coupled unit that regulates arterial tone. The coordinated opening of RyR clusters produces the Ca²⁺ sparks, and their ignition is under the rapid and direct control of the Ca²⁺ content inside the SR Ca²⁺ stores (Essin et al., 2007). Cheranov and Jaggar (2002) demonstrated a positive relationship between the SR Ca²⁺ load and the Ca²⁺ spark frequency in VSMCs (Cheranov and Jaggar, 2002). To date, the most effective mechanism to trigger Ca²⁺ sparks in VSMCs appears to be the Ca²⁺ inside the SR, which is loaded primarily by the SERCA pump. Additional molecular mechanisms that control the frequency and spatio-temporal properties of Ca²⁺ sparks in VSMCs have also been shown; for instance, the removal of proteins such as phospholamban (PLN) (Wellman et al., 2001), RyR3 (Löhn et al., 2001), FK 506 Binding Protein of 12.6 kDa (FKBP12.6) (Ji et al., 2004), and sorcin (Rueda et al., 2006) increases the frequency of Ca²⁺ sparks in VSMCs.

Most recent evidence has further supported the participation of RyR2 in global and local SR Ca²⁺ release with the help of the first conditional knockout of RyR2 in VSMCs (SM-RyR2-KO mouse) (Kaßmann et al., 2019). Studies using the SM-RyR2-KO mouse showed that the RyR2 isoform plays a significant role in global caffeine-induced Ca²⁺ release within VSMCs, and that RyR2 expression is also a prerequisite for the formation of Ca²⁺ sparks, which limit arterial myogenic constriction to pressure (Kaßmann et al., 2019). Therefore, Ca_V1.2, SERCA pump, RyRs, and BK_Ca channels form a functional unit that regulates the vascular tone; any alteration of these proteins would lead to changes in vasoconstriction or vasorelaxation. In the next section, we summarize and discuss the studies that report alterations in the expression or the activity of these proteins in experimental models with characteristics of prediabetes or MetS.

In the settings of DM1 and DM2, vascular dysfunction has been extensively investigated and summarized in compelling reviews (Feener and King, 1997; Creager et al., 2003; Fernández-Velasco et al., 2014; Nieves-Cintrón et al., 2018; Li et al., 2023). For instance, when hyperglycemia is established in DM2, there is a downregulation in K_v and BK_Ca channel activity by mechanisms associated with oxidation, and an increase in Ca_V1.2 activity via protein kinase A phosphorylation, hence disrupting Ca²⁺ signaling and resting membrane potential, leading to impaired myogenic tone and vascular reactivity (Nieves-Cintrón et al., 2021; Pereira Da Silva et al., 2022). Likewise, in the diabetic environment, the SERCA pump has been reported to be altered. For instance, in CA smooth muscle cells from diabetic dyslipidemic pigs, SERCA activity and protein expression were increased, which may induced SR Ca²⁺ overload (Hill et al., 2001). Conversely, a decrease in SERCA2 expression was found in the aorta of the diabetic (db/db) mouse without assessing its activity (Kimura et al., 2022); therefore, the data are still not conclusive. Also, in the diabetic (db/db) mouse, along with lower RyRs expression, a concomitant diminution of Ca²⁺ spark properties was reported in cerebral artery VSMCs, though Ca²⁺ spark frequency remained similar. Nevertheless, STOC frequency was depressed, perhaps due to a decrease in the BK channel β1/α subunit ratio found in db/db vascular tissues (Rueda et al., 2013). These results suggest that alterations in the functional unit that regulates vascular tone participate in diabetic vasculopathy.

Little information is available about the effects of prediabetes and MetS on this set of proteins (the Ca_V1.2, the SERCA pump, the RyRs, and the BK_Ca channels) in the human vasculature. In this regard, an increased open probability of BK_Ca channels has been reported in human CA of atherosclerotic plaques with respect to BK_Ca channels from non-atherosclerotic CAs (Wiecha et al., 1997), which contrasts with the existence of BK_Ca channels with shorter open time and lower Ca²⁺ sensitivity in atherosclerotic human aorta, similar to the activity of BK_Ca channels in aorta with proliferative phenotype (Volotina et al., 1991). The lack of conclusive results may be related to the type of blood vessel studied (resistance vs cursive conductance artery).

In this review, we focus on the available information reporting changes in the expression and/or the activity of the above-mentioned proteins in experimental models with characteristics of prediabetes and MetS (Table 2).

The SERCA pump is the most studied protein of the functional unit, it links IR and ER stress with the Ca²⁺ handling alterations. A dysfunctional SERCA was reported in CA smooth muscle cells of Ossabaw pigs fed with an atherogenic high-fat diet (HFD) for 6 months (Neeb et al., 2010; Dineen et al., 2015). In VSMCs of this animal model, the Endothelin-1-induced Ca²⁺ peak was decreased similarly to that of control cells in which the SERCA pump was inhibited by thapsigargin (Neeb et al., 2010; Dineen et al., 2015). Therefore, it was suggested that the SERCA pump contributes significantly to buffering the Endothelin-1 associated Ca²⁺ response, and its dysfunction impairs VSMC relaxation from Ossabaw pig CA (Neeb et al., 2010; Dineen et al., 2015). Similarly, after an atherogenic diet for 11 months, a MetS young swine model exhibited SR Ca²⁺ store dysregulation in CA, evidenced by the attenuated caffeine-induced SERCA activity (Badin et al., 2018). However, another study showed increased expression and activity of the SERCA pump in CA of male Yucatan pigs, as a compensatory mechanism (Hill et al., 2003), but SERCA2b protein expression levels was reported without changes in CA of male Yucatan swine subjected to a high-fat and high-cholesterol diet; though SERCA pump activity was not assessed (Witczak et al., 2006).

The OZR is also a relevant animal model for MetS in humans, which develops alterations in vascular reactivity. An increase in the expression of Ca_V1.2 and the β1 subunit of BK_Ca channels has been consistently shown in CA from OZR, without changes in the expression of the BK_Caα subunit, nor in RyRs or the SERCA pump (Climent et al., 2017; Climent et al., 2020). While an increase in the expression of Ca_V1.2 will favor more Ca²⁺ influx, the concomitant increase in BK_Caβ1 subunit, which enhances the Ca²⁺ sensitivity of the BK_Ca channels, will counterbalance the otherwise abnormal Ca²⁺ influx, leading to a preserved basal tone despite obesity alterations (Climent et al., 2017; Climent et al., 2020). Another report also showed that cerebral arteries from Sprague Dawley rats fed with a high-cholesterol diet had increased protein levels of the BK_Caβ1 subunit with augmented open probability of BK_Ca channels (Bukiya et al., 2021). Therefore, it was hypothesized that VSMCs would be hyperpolarized, which would decrease Ca²⁺ entry and contraction. However, cerebral arteries from OZ rats that exhibited IR and features of MetS, such as impaired glucose tolerance, hyperinsulinemia, hypertriglyceridemia, and hypercholesterolemia, showed a diminished generation of Ca²⁺ sparks and reduced vasodilation (Katakam et al., 2014). In addition, cerebral arteries from Sprague-Dawley rats fed with a fructose-rich diet for 4 weeks showed decreased functional activity of BK_Ca channels without changes in the BK_Ca protein expression (Erdös et al., 2002a; Erdös et al., 2002b; Erdös et al., 2004), which contrasts with no changes in BK_Ca activity but increased expression of BK_Caβ1 subunit in cerebral arteries from Sprague-Dawley rats fed with HFD (Howitt et al., 2011). Moreover, a reduction in SERCA2a protein expression was demonstrated in mesenteric lymphatic vessels of Sprague-Dawley rats fed with HFD (Lee et al., 2020), suggesting that metabolic alterations impair SERCA expression or activity in other vessels besides arteries. Notably, the impairment of BK_Ca channel activity was associated with higher IR-induced oxidative stress because the loss of function of BK_Ca channels was prevented by scavenging ROS. Dysfunctional RyRs and BK_Ca channels would lead to a reduction in the control of vascular tone in the setting of fructose-induced IR in rats.

MetS features participate in altering the reactivity and wall mechanics of cerebral arteries in OZR (Brooks et al., 2015). Mesenteric arteries of adult OZR (8–10 weeks of age) that developed obesity, mild hyperglycemia, and hyperinsulinemia showed increased levels of RyRs, although with reduced activity. The expression of Ca_V1.2 was also increased compared to lean Zucker Rats, and therefore, OZR showed augmented vasocontraction (Sánchez et al., 2018).

Alterations in the proteins of interest have also been reported in the JCR:LA-cp rat. This animal model was developed in 1978 with an autosomal recessive corpulent (cp) trait resulting from a premature stop codon in the extracellular domain of the leptin receptor (Diane et al., 2016). Homozygous JCR:LA-cp rats display the pathophysiology of obesity with a MetS-like phenotype. The aortas of JCR:LA-cp rats presented increased Ca_V1.2 expression levels, which likely contributed to the increase in mean arterial blood pressure (Howitt et al., 2011). The consumption of high-sucrose during the pre-natal stage is also important for the physiological activity of VSMCs, because the offspring of rats that consumed high-sucrose during gestation showed decreased expression of the Ca_V1.2, and the α and β subunits of the BK_Ca channel, together with the impairment of their functional activity (Feng et al., 2019).

Additional studies in experimental models have reported alterations in other important proteins for proper vascular function. For instance, the reduction in the bioavailability of vascular-derived NO, together with an increase in the systemic proinflammatory condition in the settings of MetS, augmented TNF-α levels inducing the abnormal remodeling of the resistance blood vessels, a condition that was alleviated by the acute treatment with the antioxidant tempol (Brooks et al., 2015). In the CA of the MetS Ossabaw pig model, it was reported a decreased expression and a loss of function of K_V7 channels, which hyperpolarize VSMCs to induce relaxation (Chen et al., 2016). The reduced K_V7 channel expression may lead to sustained histamine-induced contractions and reduced endothelium-dependent relaxation, both risk factors for coronary spasm (Chen et al., 2016).

It is also important to consider that the transition of VSMCs from the contractile phenotype in the healthy swine to the proliferative phenotype in mild atherosclerosis has been associated with increases in the SERCA activity, SR Ca²⁺ load, and the L-type Ca²⁺ channel function (Badin et al., 2022), and it deserves future studies. Interestingly, the dysregulation in the intracellular Ca²⁺ handling is similar in CA of Ossabaw miniature swine with respect to what has been found in coronary disease of humans (Badin et al., 2022), suggesting similar underlying mechanisms.