Impaired glucose tolerance and type 2 diabetes mellitus (T2DM) are associated with increased risk of cardiovascular disease (CVD; [1
]) Accumulating evidence indicates that elevated postprandial hyperglycemia is an independent risk factor for CVD and cardiovascular mortality in individuals with, and without, T2DM [2
]. The etiology of elevated postprandial hyperglycemia and increased cardiovascular risk has not been firmly established [3
] but endothelial dysfunction is hypothesized to be the major mechanistic link [4
]. Specifically, studies have shown that acute glucose infusion [5
] or ingestion [6
] can cause impairment in flow-mediated dilation (FMD) of the brachial artery in humans. Such impairment in endothelial function caused by acute glucose excursions appears to be exacerbated in conditions of glucose dysregulation, such that individuals with impaired glucose tolerance or T2DM experience a greater decline in FMD in response to glucose ingestion [7
]. Experimental studies in humans [6
] and mechanistic studies in cell culture [8
] suggest that glycemic fluctuations may impair endothelial function by increasing oxidative stress and promoting an inflammatory response. As such, it is hypothesized that over time, repeated exposure to elevated postprandial hyperglycemia results in cumulative endothelial damage that contributes to increased risk of CVD.
Although FMD is a well-established indicator of peripheral vascular function that is linked to CVD risk [9
], it does not provide details into the cellular or molecular responses of endothelial cells. Endothelial microparticles (EMPs) are small (~100–1000 nm in diameter) vesicles shed from the plasma membrane of endothelial cells in response to activation, apoptosis, or damage. Circulating levels of EMPs are elevated in atherosclerosis, hypertension, T2DM, and metabolic syndrome [10
], and as such are regarded as biomarkers for endothelial damage and dysfunction. EMPs can be characterized by the surface proteins associated with events triggering their release. CD31+/CD42b- EMPs are believed to be shed from apoptotic endothelial cells, whereas CD62E+ (E-selectin) EMPs indicate inflammatory activation of the endothelial cell of origin. Thus, measurement of circulating CD31+/CD42b- and CD62E+ EMPs can provide direct insight into damage and inflammation of the vascular endothelium [11
The primary purpose of this exploratory investigation was to determine whether acute glucose ingestion would increase EMP release and impair FMD in humans. In order to perturb glucose tolerance, we studied the impact of a 75-gram oral glucose tolerance test (OGTT) drink before and after a 7-day low-carbohydrate high-fat diet (HFD) in young healthy male participants. Short-term HFDs have previously been shown to promote relative glucose intolerance in healthy human participants [12
], and therefore allowed us to determine whether glucose ingestion impacted EMP release in the context of relative increase in postprandial hyperglycemia using a within-subjects design. This approach has the advantage of limiting the influence of baseline vascular dysfunction and endothelial damage that would have confounded a cross-sectional study comparing individuals of differing glucose tolerance status. Since high-fat feeding in animal models has been linked to endothelial damage and dysfunction [14
], this design also provided the opportunity to determine the impact of short-term HFD on basal EMP levels and FMD. In addition, cerebral blood flow (CBF) is altered in response to HFDs in animals [15
], however, data on extracranial CBF in humans is lacking. For this reason, we took this opportunity to assess CBF as an exploratory outcome.
The main findings of the present study are that the one-week low-carbohydrate high-fat diet, which causes relative glucose intolerance (as we reported previously; [13
]), coincides with a reduction in FMD in the fasting state and following ingestion of glucose. Furthermore, the consumption of a HFD for one week led to increased levels of endothelial damage markers (CD31+/CD42b- and CD62E+ EMPs) during a physiological excursion into hyperglycemia. These findings indicate that a short-term HFD in young healthy men (i) can reduce FMD; and (ii) may render the endothelium susceptible to hyperglycemia-induced damage. We also report findings on the impact of glucose ingestion on extracranial CBF, with the main findings indicating that an acute excursion into hyperglycemia induced increases in ICA, VA, and CCA diameter with a corresponding reduction in velocity in ICA but no statistically significant changes in flow in any of these vessels.
Our results indicate that FMD is attenuated in healthy young men after both an excursion into hyperglycemia and following consumption of the HFD for one week. However, despite the induction of relative glucose intolerance with the HFD, there were no synergistic (i.e., interactive) effects between the two, as the consumption of 75 g of glucose led to a similar reduction in FMD pre- and post-HFD. It is well established that FMD is reduced after consumption of an OGTT drink [7
] and a single high-fat meal in humans [30
]. Furthermore, short-term high-fat diets are often used in animal studies to induce endothelial dysfunction [31
]. However, research examining FMD following short-term low-carbohydrate high-fat diet interventions in young healthy human populations is lacking. Longer duration low-carbohydrate high-fat diet interventions have demonstrated increases [32
], reductions [33
], and no change [34
] in FMD, but these studies contain confounding factors, most importantly co-existing weight loss and caloric restriction. This inconsistency in the literature makes it difficult to interpret the effect of repeated high-fat feeding on FMD in the absence of weight loss. Our findings demonstrate that a short-term HFD leads to a reduction in FMD, similar to the effect on FMD following a single high-fat meal. It has previously been suggested that the impairment in FMD following the consumption of a high-fat meal could be attributed to heightened oxidative stress and reduced nitric oxide (NO) bioavailability [35
It is well established that acute hyperglycemia impairs endothelial function [7
], which is also suggested to be mediated by increased oxidative stress and reduced NO bioavailability [36
]. A recent meta-analysis investigating the acute effects of meal consumption on FMD reported an average reduction of ~2% in postprandial FMD [37
]. Our findings, although demonstrating a smaller decrease in postprandial FMD (0.58%), are similar to the literature in that respect. Following the HFD, glucose levels were higher after the OGTT, but the hyperglycemia-induced reduction in FMD was not exacerbated (i.e., no interaction effects). This is perhaps suggestive of separate mechanisms leading to the fasting reduction in FMD following the HFD, and the acute hyperglycemia-induced depression in FMD. While the clinical relevance of these small reductions in FMD cannot be determined by the present study in young healthy males, it is possible that the increase in risk would be even greater for an already at-risk population. It is important to note that baseline brachial artery diameter and SRAUC were unchanged throughout the study, indicating that the changes observed in FMD were not due to these variables.
Microparticles (MP) are defined as submicron vesicles that are shed from the plasma membranes of various cell types in response to activation, injury, and/or apoptosis [10
]. Although initially regarded as cellular debris [38
], MPs are now recognized to play important physiological roles such as signalling molecules (reviewed in [39
]). MPs, including EMPs, can contain various biologically active molecules such as proteins, cytokines, mRNAs, or microRNAs [40
]. Expressed at the surface of MPs are most of the membrane-associated proteins of their parent cells, making flow cytometry a viable method for the detection and differentiation of these particles. It has been suggested that EMPs are markers of damage, with CD62E+ EMPs being indicative of inflammatory activation and CD31+/CD42b- EMPs indicative of apoptosis [10
]. There seems to be a link between hyperglycemia and circulating EMPs, given the elevated levels reported in T2DM patients [41
]. Animal models have demonstrated that EMPs generated under high-glucose conditions can induce vascular inflammation and impair endothelial function, whereas EMPs generated from healthy endothelial cells do not [42
]. For this reason, we hypothesized that EMPs would increase following an excursion into hyperglycemia in humans and that this might be related to endothelial function as measured by FMD. While we did observe a reduction in FMD and an increase in circulating EMPs following consumption of a glucose load both pre- and post-HFD, there was no correlation between EMPs and FMD. In addition, high-fat meals have also been shown to increase circulating EMPs [43
]; however, we did not observe an increase in basal EMPs following the HFD, despite the reduction in FMD. This would indicate that the EMPs were not directly responsible for the impairment in FMD, but rather both are mediated by other, possibly separate, mechanisms. It also appears that the impact of the HFD, in terms of endothelial damage markers, was only revealed when combined with hyperglycemia. This suggests that consuming a HFD over the short-term predisposes the endothelium to hyperglycemia-induced damage.
We observed a significant increase in diameter in the ICA, VA, and CCA following consumption of a 75-gram glucose drink, with no significant increase in flow in any of these vessels. It has previously been reported that ICA diameter has been shown to increase with elevations in circulating insulin concentration [44
]. Indeed, there was a 10-fold increase in insulin concentration during the OGTT when the increases in diameter were measured. A reduction in velocity was detected in the ICA, which explains the consistent ICA flow. However, we did not detect statistically significant reductions of velocity in the VA or CCA or in their respective blood flow. This is likely due to the relatively small sample size and resultant insufficient power to detect such changes. Nonetheless, there appeared to be no direct effects of the short-term HFD on basal CBF in young healthy men. The implications of more chronic changes in glucose and insulin on cerebrovascular health and remodelling require future study.
We also observed a modest reduction in mean arterial pressure 60 minutes post glucose consumption and a reduction in the fasting state following the HFD. It is possible that the reduction following glucose consumption is related to splanchnic blood pooling, which is commonly seen in the postprandial state [45
]. The lower mean-arterial pressure seen post-HFD could be due to a reduction in sympathetic tone. The finding of reduced peripheral PWV after the HFD supports this view, however, without a direct measure of sympathetic tone in this, this remains speculative.