Let's keep it under control: a novel method to study the impact of acute hyperglycaemia on vasculature

Vascular complications are the major cause of both morbidity and mortality in diabetes mellitus (DM) patients. These complications may be the result of interactions from the multiple abnormalities that occur in DM, including dyslipidaemia, hyperglycaemia, and both genetic and epigenetic aberrations in numerous cell types. A major hallmark of DM, chronic hyperglycaemia, leads to vascular complications that adversely impact the macro- and microvascular systems of multiple organs. Previous research has linked the diabetic complications within the vasculature to abnormalities in cell signalling and gene expression (Barrett et al. 2017). In the absence of DM, acute hyperglycaemia (AH), defined as exposure to hyperglycaemia for hours to days, has been shown to cause vascular injury similar to that of DM. This was demonstrated in the DECODE (Diabetes Epidemiology: Collaborative Analysis of Diagnostic Criteria in Europe) study, where vascular function was compromised by AH regardless of diabetic status (DECODE study group, 1999). However, due to the complex pathophysiology of DM, it is difficult to investigate the direct impact of AH on vascular function. One such confounding factor is the body's response to meal ingestion. Studies on the impact of AH on the vasculature have not effectively controlled for autonomic nervous system activation (Lipsitz et al. 1993). Furthermore, the impact of meal-induced insulin release cannot be understated with respect to the study of both macro- and microvascular function. A lack of control for such factors is a likely cause for disparities in previous literature on vascular function in response to AH, and no studies have hitherto examined the impact of AH with such controls in place, in healthy or diseased subjects. In a recent article published in The Journal of Physiology, Horton et al. (2022) examined the impact of AH on both macro- and microvascular function by employing a novel methodology that controls for insulin release. The group utilized classic euglycaemic (EU) and AH clamping procedures, but infused participants with octreotide (OCT) (30 ng/kg/min) to prevent endogenous insulin release. Participants aged between 18 and 35 years were recruited and screened to qualify for this study. Once recruited, subjects were randomized to one of two study protocols. The first protocol was designed to model EU. In this protocol, saline infusion took place for 90 min to determine baseline vascular function. Then, OCT (30 ng/kg/min) with insulin replacement (0.15 mU/kg/min) was infused for 240 min. In this protocol, plasma glucose was sampled every 10 min and plasma insulin was sampled every 30 min. Additionally, intercellular adhesion molecule-1 (ICAM-1), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) and protein carbonyls were sampled at baseline and at end-study. The second protocol was designed to model AH. In this protocol, OCT (30 ng/kg/min) with insulin replacement (0.15 mU/kg/min) was infused continuously for 90 min. Baseline vascular function measurements were collected in the final 30 min of infusion. After this, a 20% dextrose infusion was used to raise plasma glucose and obtain AH. Plasma glucose was raised and maintained at approximately 200 mg/dl. In the second protocol, plasma glucose was sampled every 5 min and plasma insulin was sampled every 30 min. ICAM-1, IL-6, TNF-α and protein carbonyls were also sampled at baseline and at end-study. Concentrations of plasma insulin, glucose and inflammatory biomarker ICAM-1 were measured between EU and AH. Plasma insulin concentrations in both EU and AH groups were maintained at baseline following 4 h of euglycaemic or 20% dextrose infusion, validating the authors’ methodology of assessing vascular function independent of insulin, which is crucial to the novelty of this study. To control for potential confounding effects of endothelial dysfunction inflicted by AH, the authors measured ICAM-1 levels in both groups before and after clamp procedures. ICAM-1 did not differ between EU and AH groups either before or after infusion, demonstrating that neither protocol induced endothelial inflammation nor dysfunction. The authors clearly demonstrated the effectiveness of their protocol in eliminating confounding effects of an altered insulin milieu, as well as inflammatory responses; to add to the robustness of their strategy, it may be useful to measure additional classical markers for inflammation-induced endothelial dysfunction such as Von Willebrand factor. Following the validation of how effectively they were able to control insulin and endothelial inflammatory responses, the authors first examined the impact of 4 h of AH on macrovascular function. Measured by cardiovascular ultrasound, they identified an increase in flow-mediated dilatation (FMD) in the left brachial artery of subjects in the AH group. FMD provides an excellent non-invasive metric for testing endothelial function and has been shown to correlate well with invasive testing of endothelial function, and was thus appropriately used in this study (Raitakari & Celermajer, 2000). The authors did not detect changes in carotid–femoral pulse wave velocity or post-ischaemic flow velocity in either intervention. The observed increase in FMD in response to AH had not hitherto been demonstrated and signifies the importance of controlling for factors such as plasma insulin, as well as autonomic nervous system output that would otherwise be a complication of high carbohydrate and oral glucose administration. Contributing to the comprehensiveness of their study, Horton et al. evaluated microvascular function in cardiac and skeletal muscle with ultrasound enhanced by Definity microbubble infusion. The authors measured an increase in their primary outcome for function and microvascular blood volume, in both skeletal and cardiac muscle following 4 h of AH and not EU. Importantly, Horton et al. demonstrated that OCT infusion alone did not alter any of microvascular blood volume, microvascular flow velocity or microvascular blood flow, which further validated their control group and substantiates these novel findings as those which are truly independent of factors that would otherwise impact vascular function. Finally, whilst not a focus of their study, the authors also reported no interaction between biological sex and vascular function in response to AH, which is an interesting preliminary divergence that would be of tremendous physiological value, especially given the demonstrated effectiveness of this protocol in controlling for factors that have previously been shown to influence both macro- and microvascular function. The novel findings published by Horton et al. have shown that in the context of young, healthy adults, AH does not decrease macro- and microvascular flow. Importantly, this is the first study to control for endogenous factors such as plasma insulin which may have affected the outcomes of previous studies. As well, the study protocol induced AH using i.v. glucose, bypassing the autonomic nervous system response, whereas other studies induced AH orally. Other outcomes of this study have helped to elucidate the effect of OCT on vascular function during both EU and AH, which was previously unknown. As it was shown that OCT does not alter vascular function, it can now be used in the protocol of similar future studies. It is important to consider the demographics of the participants in this study, as DM and its accompanying pathologies are significantly linked to obesity and ageing. While using young adults with no comorbidities is important to develop a baseline measurement of the effect of AH on FMD, older adults with a higher body mass index would provide further clinical relevance. Although the authors listed this as a limitation, it does provide immense impetus for future studies to examine whether older adults with obesity and reduced vascular function may react differently to AH induced by i.v. glucose. It is also important to consider the decision to use a 4 h duration of AH as opposed to a shorter time period, but the authors clearly described the reason for this choice. In previous studies, 4 h of increased free fatty acids in plasma was shown to negatively impact vascular function, although, Horton et al.’s findings suggested the opposite. This again provides future direction for studies that examine these changes over a shorter duration of AH. These novel findings from Horton et al. suggest that the response to AH is dependent on both the study population and the mode of glucose delivery. This is a landmark study with the potential to alter the methodology of studies focusing on vascular function in the setting of DM. It is therefore likely that in the future, with more experimentation, this may be increasingly generalizable to a larger population. No competing interests declared. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None.

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