Recruitment and Study Population
Recruitment for this study was achieved by community advertisement and direct advertisement to healthcare clinics both within and outside the University of Virginia (UVA) Health System. Healthy young adults met inclusion criteria if they were ≥18 and ≤35 years old, had normal body mass index (18-25 kg/m2), did not have DM, and had fasting plasma glucose <100 mg/dL and blood pressure <140/90 mmHg at time of screening. Subjects were excluded if they were current smokers or quit smoking <5 years ago, had a first-degree relative with type 2 DM, were taking vasoactive medications (e.g., anti-hypertensives, diuretics, statins, etc.), were pregnant (i.e., positive pregnancy test) or nursing, had history of allergy or prior adverse reaction to octreotide, or significant premorbid disease that could, in the investigator’s opinion, affect outcome measures or subject safety.
Clinical Assessment and Initial Screening
All screening visits and infusion studies were conducted at the UVA Clinical Research Unit (CRU). Each subject gave written informed consent at their initial visit prior to being carefully screened to verify inclusion/exclusion criteria and certify overall good health. Screening included a detailed medical history and physical examination along with fasting measures of complete blood count, comprehensive metabolic panel, lipid panel, plasma glucose, and serum pregnancy test.
Experimental Protocols
Randomization was conducted by study personnel using a 1:1:1:1 allocation with a computer-generated sequence program [26]. After randomization, study personnel were blinded to subject and protocol when evaluating outcome measures. Subjects underwent four infusion protocols (Figure 1) designed to test the effects of euglycemia, hyperglycemia, euglycemic-hyperinsulinemia, and hyperglycemic-hyperinsulinemia on arterial stiffness. All protocols were approved by the UVA Institutional Review Board (#19948), with each protocol being performed ≥2 but ≤4 weeks apart for individual subjects. For each protocol, we measured cfPWV, AI, SEVR, systolic blood pressure, diastolic blood pressure, pulse pressure, mean arterial pressure, and heart rate immediately before (i.e., baseline) and at the end of the infusion period (Figure 1). Study participants were instructed to avoid alcohol, exercise, and caffeine for 24 hours and fast overnight prior to admission to the CRU. Infusion studies began with placement of intravenous catheters in the right wrist for blood sampling and in the right antecubital fossa for administration of insulin, glucose, and octreotide (OCT). Studies began with simultaneous infusion of regular insulin and OCT to maintain plasma insulin near basal levels. We did not replace glucagon or growth hormone, as there is currently no evidence that acutely suppressing basal levels of either hormone affects vascular function.
Protocol A (Euglycemia): A 90-minute saline infusion was initiated, with baseline vascular function measurements obtained during the final 30 minutes (Figure 1A). Then, OCT (30 ng/kg/min) with basal insulin replacement (0.15 mU/min/kg) was infused for 240 minutes. Blood glucose (BG) was sampled every 10 minutes and plasma insulin every 30 minutes. Euglycemia (EU) was maintained by a variable-rate glucose infusion using the negative feedback principle [27]. We then repeated vascular measurements over the final 30 minutes of study.
Protocol B (Hyperglycemia): Octreotide and basal insulin replacement were continuously infused for 90 minutes, with baseline vascular measurements obtained over the final 30 minutes (Figure 1B). Then, a primed, continuous variable-rate 20% dextrose infusion began to acutely raise and maintain BG at ~200 mg/dL using the hyperglycemic clamp method [27]. BG was sampled every 5 minutes and plasma insulin every 30 minutes, with repeat vascular measurements obtained over the final 30 minutes of hyperglycemia.
Protocol C (Euglycemic-Hyperinsulinemia): Euglycemia was maintained throughout this protocol by a variable-rate 20% dextrose infusion using the negative feedback principle [27]. Baseline arterial stiffness measurements were obtained during the final 30 minutes of an OCT (30 ng/kg/min) plus basal insulin (0.15 mU/min/kg) infusion (Figure 1C). Then, hyperinsulinemia was initiated with a primed (2 mU/kg/min x 10 min), continuous (1 mU/kg/min x 110 min) infusion and OCT continued for 120 minutes. Blood glucose (BG) was sampled every 5 minutes and plasma insulin every 30 minutes, with repeat arterial stiffness, SEVR, and hemodynamic measurements obtained during the final 30 minutes of the insulin clamp.
Protocol D (Hyperglycemic-Hyperinsulinemia): As in Protocol C, a variable-rate 20% dextrose infusion maintained euglycemia while OCT (30 ng/kg/min) and basal insulin (0.15 mU/min/kg) were simultaneously infused for the first 90 minutes of this study (Figure 1D). Then, a primed, variable-rate 20% dextrose infusion began to acutely raise and subsequently maintain BG at ~200 mg/dL using the hyperglycemic clamp method [27]. BG was then sampled every 5 minutes and plasma insulin every 30 minutes, with baseline arterial stiffness measurements obtained over the final 30 minutes of the 120-minute hyperglycemic period (Figure 1B). Subsequently, hyperinsulinemia was initiated with a primed (2 mU/kg/min x 10 min), continuous (1 mU/kg/min x 110 min) infusion with OCT and hyperglycemia maintained for 120 minutes. BG was sampled every 5 minutes with plasma insulin every 30 minutes, and repeat arterial stiffness, SEVR, and hemodynamic measurements were again obtained during the final 30 minutes of the insulin clamp.
Hemodynamics: Clinical hemodynamic assessments were obtained at two time points during each protocol (Figure 1). Blood pressure, pulse pressure, mean arterial pressure, and heart rate were measured and/or calculated with a Sphygmacor tonometer (ATCOR USA; Napierville, IL).
Carotid-Femoral Pulse Wave Velocity (cfPWV): To assess central aortic stiffness, cfPWV was measured per expert recommendations [28] using a Sphygmacor tonometer by the same trained operator. To minimize the effects of sympathetic activity on cfPWV measurements, participants laid in the supine position in a temperature-controlled room for at least 15 minutes prior to measurement. We measured the distance from the suprasternal notch to the carotid pulse and from the suprasternal notch to the femoral pulse on the same side. For each cfPWV measure, 10 seconds of carotid and 10 seconds of femoral arterial waveforms were recorded. cfPWV measures were made in duplicate and the mean value was reported. Of note, the cfPWV data in this manuscript were included in a separate report examining macro- and microvascular functional responses to the two insulin clamp protocols [29].
Radial Artery Augmentation Index (AI): To assess peripheral arterial stiffness, we measured AI noninvasively with a Sphygmacor tonometer. As with cfPWV, radial AI measurements were obtained by the same trained operator after participants laid in the supine position in a temperature-controlled room for at least 15 minutes prior to measurement. Radial AI was calculated as the difference of the amplitude of the late systolic peak to the early systolic peak divided by the pulse pressure and expressed as a percentage. Radial AI values were determined for each pulse over a 30 second period and a mean value was calculated by the device for each patient and corrected for a heart rate of 75 beats per minute.
Subendocardial Viability Ratio (SEVR): Measurements used to calculate SEVR were obtained with a Sphygmacor tonometer. The area under the curve of the systolic and diastolic portions of the central aortic pulse wave were measured using pulse wave analysis. In the present study, the tonometric SEVR was as provided by the manufacturer; specifically, it was approximated automatically using the following equation: tonometric SEVR=diastolic aortic area/systolic aortic area [30, 31].
Biochemical Analyses
Complete blood count, comprehensive metabolic panel, lipid panel, fasting plasma glucose, and serum pregnancy tests were assayed at the UVA Clinical Chemistry Laboratory. Plasma glucose was measured with the YSI 2700 Biochemistry Analyzer (Yellow Springs Instrument Company; Yellow Springs, OH). Plasma insulin was measured with the ALPCO Insulin ELISA (ALPCO; Salem, NH). Insulin assays were read on a Synergy 2 microplate reader (BioTek; Winooski, VT).
Data Storage
Study data are stored in a Research Electronic Data Capture (REDCap) [32] project file repository hosted at UVA. The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Statistical Analyses
Sample Size: Our prior work has demonstrated that sample sizes of approximately 10-15 subjects were sufficient to identify significant within-study changes in macrovascular function under multiple metabolic conditions [33-36]. A crude sample size calculation using the Cohen’s d effect size from a prior study of changes in cfPWV during euglycemic-hyperinsulinemia [35] indicated that a sample size of 10 subjects would have ≥95% power to detect meaningful differences within each protocol.
Outcomes: The primary outcome for each protocol was change in cfPWV and secondary outcomes for each protocol included changes in AI, SEVR, systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse pressure, and plasma insulin.
Descriptive Summarization: Patient demographics were summarized using common descriptive statistics. The arithmetic mean and standard error of mean, standard deviation, median, and interquartile range were used to summarize continuous scaled outcome measures.
Statistical Analyses: Data are expressed as either mean ± SEM or as change within protocol. Within-protocol changes were analyzed using paired, two-tailed t-test and two sample, unequal variance t-test where appropriate. Between-protocol changes were analyzed using mixed modeling for repeated measures. Spearman’s correlation was used to evaluate the relationship between cfPWV and radial AI. All statistical analyses were performed with Excel (Microsoft; Redmond, WA) and GraphPad Prism 8 (GraphPad Software; San Diego, CA). In all cases a p-value of <0.05 was accepted as statistically significant.