Effect of hypoglycaemia on measures of myocardial blood flow and myocardial injury in adults with and without type 1 diabetes: A prospective, randomised, open‐label, blinded endpoint, cross‐over study

Abstract Aims This study examined the effect of experimentally‐induced hypoglycaemia on measures of myocardial blood flow and myocardial injury in adults with, and without, type 1 diabetes. Methods In a prospective, randomised, open‐label, blinded, endpoint cross‐over study, 17 young adults with type 1 diabetes with no cardiovascular risk factors, and 10 healthy non‐diabetic volunteers, underwent hyperinsulinaemic‐euglycaemic (blood glucose 4.5–5.5 mmol/L) and hypoglycaemic (2.2–2.5 mmol/L) clamps. Myocardial blood flow was assessed using transthoracic echocardiography Doppler coronary flow reserve (CFR) and myocardial injury using plasma high‐sensitivity cardiac troponin I (hs‐cTnI) concentration. Results During hypoglycaemia, coronary flow reserve trended non‐significantly lower in those with type 1 diabetes than in the non‐diabetic participants (3.54 ± 0.47 vs. 3.89 ± 0.89). A generalised linear mixed‐model analysis examined diabetes status and euglycaemia or hypoglycaemia as factors affecting CFR. No statistically significant difference in CFR was observed for diabetes status (p = .23) or between euglycaemia and hypoglycaemia (p = .31). No changes in hs‐cTnI occurred during hypoglycaemia or in the recovery period (p = .86). Conclusions A small change in CFR was not statistically significant in this study, implying hypoglycaemia may require more than coronary vasomotor dysfunction to cause harm. Further larger studies are required to investigate this putative problem.


| INTRODUC TI ON
Diabetes is now a global pandemic 1 in which the principal cause of death is cardiovascular disease. 2 While strict control improves some outcomes, clinical trials in people with type 2 diabetes who have cardiovascular disease have shown that intensive glucose-lowering therapy is associated with higher mortality. 3,4 It has been suggested that exposure to hypoglycaemia may be contributing to this greater morbidity and mortality. 5,6 Hypoglycaemia is a common occurrence in people with insulintreated diabetes. It causes profound autonomic stimulation leading to activation of the sympatho-adrenal system with profuse catecholamine release. This has pronounced systemic and regional haemodynamic effects, 7 including large increments in stroke volume and cardiac output, 8 caused by increased left ventricular ejection fraction and myocardial contractility. 9 This augmented cardiac workload may provoke myocardial ischaemia or cardiac failure in people who have established cardiac disease.
Mechanisms that could promote adverse effects of hypoglycaemia on cardiovascular function have been proposed. 10 Endothelial dysfunction is a key pathophysiological pathway underlying macrovascular complications in type 1 and type 2 diabetes. A detrimental effect of hypoglycaemia on peripheral endothelial function has been reported. 11 However, several differences exist between peripheral and coronary arterial endothelium, such as the presence of shunt vessels and the microvascular architecture. 12 Direct measurement of coronary vasomotor function may therefore provide a more valid measure of any cardiovascular impairment resulting from limited vascular responsiveness. Measurement of coronary microvascular dysfunction by calculation of coronary flow reserve (CFR) is the preferred investigative technique. 13 Coronary flow reserve measures the capacity of the coronary circulation to increase flow during maximal resistance vessel vasodilatation. Maximal hyperaemia is achieved by intravenous infusion of adenosine 14 and coronary flow velocity can be measured non-invasively by transthoracic Doppler echocardiography. 15,16 CFR is reduced in people with type 1 diabetes during euglycaemia 17 and in individuals with type 1 diabetes with evidence of microvascular disease in the form of retinopathy. 18 CFR has also been shown to correlate well with long term hard outcomes in coronary heart disease. 19,20 It is unclear how hypoglycaemia affects CFR.
Cardiac troponins are a biomarker of myocardial injury. In recent years, the introduction of a highly sensitive cardiac troponin I (hs-cTnI) assay allows the detection of small degrees of myocardial damage.
Lowering the diagnostic threshold for myocardial injury has improved outcomes for people with type 1 myocardial infarction. 21 Hs-cTNI also provides prognostic information regarding long term outcomes in cohorts of people with underlying coronary heart disease. 22 People with type 2 diabetes often have multiple cardiovascular risk factors that can confound attempts to investigate hypoglycaemia as a causative mechanism for cardiovascular morbidity and mortality. Younger people with type 1 diabetes of relatively short duration are less likely to have acquired these additional risk factors. To examine whether hypoglycaemia might adversely affect myocardial blood flow and cause myocardial injury, coronary flow reserve and cardiac troponins were therefore measured during experimentally-induced hypoglycaemia in people with type 1 diabetes who had no confounding factors.

| Study participants
Young healthy male adults with type 1 diabetes with no microvascular complications or cardiovascular risk factors were recruited from diabetes out-patient clinics in Lothian, Scotland. Men without diabetes, matched for age, were recruited by poster advertisements and from a database of volunteers. Only male subjects were studied to avoid the confounding effect of the variability of coronary flow reserve that occurs during the menstrual cycle. 23 In addition, because the counterregulatory hormonal responses to hypoglycaemia differ between men and women, 24 the study was confined to men to avoid a potential effect of gender on the magnitude of the sympathoadrenal stimulus during hypoglycaemia.
Exclusion criteria included co-existent systemic disease, malignancy, chronic alcoholism, psychiatric disorder, any history of cardiac conduction abnormality, impaired awareness of hypoglycaemia (as assessed by the method of Gold et al. 25 ), past history of severe hypoglycaemia, and any evidence of overt microvascular complications including retinopathy and neuropathy or the presence of microalbuminuria. The participants did not have any overt evidence of autonomic neuropathy. The use of any medications apart from insulin therapy, including beta-blockers and angiotensin-converting enzyme inhibitors, was an exclusion criterion.
A total of 17 male adults with type 1 diabetes and 10 age-matched individuals without diabetes were studied (Table 1). Participants with type 1 diabetes had reasonable glycaemic control, (average glycated haemoglobin (HbA1c): 8.0 ± 1.1%; 64 + 11 mmol/mol), with a median duration of diabetes of 15 years (range 2-35 years), which is consistent with average quality of glycaemic control recorded in the adult population with type 1 diabetes in Scotland. The two groups of participants did not differ in age or body-mass index ( Table 1). The participants performed more frequent glucose testing 24 h before each of the glucose clamp studies to ensure that they had not been exposed to antecedent hypoglycaemia before the study.
The study was conducted with the informed written consent of all subjects, the approval of the Lothian Medical Research Ethics committee, and in accordance with the Declaration of Helsinki.

| Study design
Participants attended two study visits, performed on separate days at least 2 weeks apart to avoid any potential carry-over effects. Two  Figure S1). This study design has been employed previously by our group, 26 with modifications for the cardiovascular investigations.
Participants attended in a fasting state, having abstained from the consumption of caffeine-containing food and beverages for 24 h.
Venous cannulae were inserted for intravenous infusion of dextrose and insulin, and blood sampling. A modified version of the hyperinsulinaemic glucose clamp was employed. 27 To arterialise blood samples, the non-dominant arm was wrapped in a heated blanket with a retrograde intravenous cannula inserted into the forearm. An additional cannula was inserted into the non-dominant antecubital fossa to infuse insulin (human Actrapid; Novo Nordisk, Crawley, U.K.) and 20% dextrose. Insulin was infused at a constant rate of 1.5 mU/kg/ min with a Gemini PCI pump (Alaris Medical Systems). Blood samples were taken at 5-min intervals and analysed by a glucose oxidase method (2300 STAT; YSI, Yellow Springs, OH). The dextrose infusion rate was adjusted to maintain the appropriate arterialised blood glucose concentration. During a run-in period, arterialised blood glucose was maintained at 4.5 mmol/L for 20 min. Blood glucose was then either maintained at 4.5 mmol/L throughout (the euglycaemia condition), or lowered over 20 min to 2.5 mmol/L (the hypoglycaemia condition), and maintained at this level for 30 min before restoration of euglycaemia. During the glucose clamp, the participants underwent an ultrasound examination by a trained ultrasound operator, using a well-described technique. 16,23 The timepoints were labelled as baseline, experimental (either euglycaemia or hypoglycaemiablinded to the sonographer), and recovery (Supplemental Figure S1).
Continuous electrocardiographic monitoring and regular blood pressure monitoring were performed throughout the study.

| Coronary flow velocity measurements
During each study condition, the left anterior descending coro- to record spectral Doppler signals during hyperaemic conditions. 14 Coronary velocities were measured at baseline and peak hyperaemic conditions from the Doppler signal recordings. Measurements were averaged over three cardiac cycles. CFR was defined as the ratio of hyperaemic to basal velocities, using maximum velocity (Vmax) parameters (Supplemental Figure S2). Blood pressure was recorded at baseline, during adenosine infusion and at recovery. CFR was calculated at baseline, during the experimental phase (0-20 min) and in the recovery phase.

| High-sensitivity cardiac troponin I concentration
Blood samples were taken prior to the assessment of CFR, during the experimental hyperinsulinaemic clamp, and during the recovery period (Supplemental Figure S1). High-sensitivity cardiac troponin I concentrations were determined using the ARCHITECT STAT highsensitive troponin I assay (Abbott Laboratories). This is the first clinically approved high-sensitivity troponin I assay, which has excellent precision at very low concentrations. The limit of detection is 1.2 ng/L and precision profiling in our laboratory has demonstrated an inter-assay coefficient of variation (CV) of <10% at 5 ng/L. The upper reference limit or 99th centile is 16 ng/L for women and 34 ng/L for men. 28-30

| Statistical methods
A power calculation was performed using results from a previous study using a similar technique, 31 a sample size of 12 allows an 80%

| RE SULTS
Study participants were all healthy young men with normal a bodymass index (BMI). Baseline characteristics of both groups are provided in Table 1. During the hypoglycaemia session (Table 2), the mean glucose nadir was 2.34 ± 0.2 mmol/L; 42.12 + 3.8 mg/dL (p < .001 compared to baseline), which is sufficient to stimulate a brisk sympatho-adrenal counterregulatory response. Heart rate, systolic and mean blood pressure did not change during euglycaemia in either group. During hypoglycaemia, a trend was apparent towards an increase in heart rate and systolic blood pressure (BP), and a decrease in diastolic BP in both groups (Table 2). In the type 1 diabetes group, the increments in heart rate and systolic blood pressure did not reach statistical significance. In the non-diabetic group, the heart rate increased from 71 ± 9 beats per minute (bpm) to 78 ± 8 bpm (p = .02) while the systolic BP increased from 116 ± 11 to 124 ± 12 mmHg (p = .001).

| Coronary flow velocities and reserve
No differences were observed between the coronary flow velocities of the group with type 1 diabetes and the non-diabetic group at baseline (Supplemental Figure S3). During hyperaemia after adenosine infusion, a trend for lower coronary flow velocities was observed in people with type 1 diabetes, but this did not reach statistical significance (Supplemental Figure S3).

| High-sensitivity cardiac troponin I
The hs-cTnI values were in a skewed distribution and were, there-   The main limitation of the study is the small sample size. While this was in part a consequence of the robust exclusion criteria, the demanding study design also limited recruitment of potential participants, specifically because of the use of adenosine to induce maximal hyperaemia of the coronary blood vessels. The rationale for using adenosine was its short half-life, which meant that the CFR values from one measurement to the next would not be confounded by residual effects of adenosine. The disadvantage of this approach is that adenosine is often poorly tolerated because it induces unpleasant side effects of chest tightness and facial flushing, which diminishes the willingness of volunteers to participate.
In addition, the glucose clamp procedure is onerous and had to be repeated at least 2 weeks apart, which was a further limitation to recruitment.
In retrospect, the power calculation may have benefited from In the present study, no significant change in CFR was observed during hypoglycaemia. While a non-significant trend towards a lower CFR was observed in the participants with type 1 diabetes during euglycaemia, a trend towards a decline in CFR was also observed during hypoglycaemia, consistent with previous observations. 17 The lowest CFR values during hypoglycaemia were observed in the participants with type 1 diabetes. The increments in heart rate and systolic blood pressure in the group with type 1 diabetes did not achieve statistical significance ( Recent randomised controlled trials (RCTs) that did not target strict glycaemic control but used drugs with a low risk of hypoglycaemia have shown beneficial cardiovascular outcomes. 38,39 This is in direct contrast to previous RCTs, in which very strict glycaemic control was pursued and the incidence of severe hypoglycaemia was high. 10 These findings suggest that avoidance of hypoglycaemia is important to achieve cardiovascular benefit.

| CON CLUS ION
Although in the present study hypoglycaemia had no effect on markers of ischaemia, a small reduction in CFR was apparent during hypoglycaemia, with the lowest numerical value occurring in young adults with type 1 diabetes during hypoglycaemia. Further larger studies that include female participants are required to confirm or refute this observation. If the present observation of a lower CFR is confirmed, this would raise concern that hypoglycaemia may promote myocardial ischaemia in older people with diabetes who have established coronary heart disease. While coronary blood flow should be examined during hypoglycaemia in people with type 2 diabetes who have a greater risk of cardiovascular disease, this was not undertaken because of the potential cardiac risk to such participants.
The present study should be repeated in a larger number of people with type 1 diabetes, using a more tolerable form of investigation and a real time biomarker of cardiac injury. As population studies have shown an association between severe hypoglycaemia and cardiovascular risk 5,40 more mechanistic studies are required to elucidate potential reasons for this association.

ACK N OWLED G EM ENTS
We express our gratitude to the volunteers and to the nursing and support team at the Wellcome Trust Clinical Research Facility at the Royal Infirmary of Edinburgh for their help in the study. In addition, we acknowledge the invaluable assistance of the Scottish Diabetes Research Network and the clerical staff at the Department of Diabetes at the Royal Infirmary of Edinburgh.

CO N FLI C T O F I NTE R E S T
The authors disclose no conflicts of interest in this study.