Recruitment and Study Population
Recruitment for this study was achieved by public advertisement. Healthy young adults met inclusion criteria if they were ≥18 and ≤35 years old, had normal body mass index (18-25 kg/m2), and had a screening fasting plasma glucose <100 mg/dL and a screening blood pressure <140/90 mmHg. 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 morbidity 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 University of Virginia (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. 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.
Study Design
We followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines27 to analyze and report this randomized trial. Randomization of study sequence was conducted by study personnel using a 1:1:1:1 starting allocation from a computer-generated sequence program28. The protocol was designed to assess: (1) Protocol A followed by Protocol B (or vice-versa), with subsequent crossover to Protocols C and D (in randomized order); or (2) Protocol C followed by Protocol D (or vice-versa), with subsequent crossover to Protocols A and B (in randomized order). Subjects underwent four infusion protocols (Figure 1) to test the discrete 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 to allow a washout period between studies. Within each protocol, we measured cfPWV, AIx, Pf, Pb, RM, 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). All vascular assessments for this study were measured per expert recommendations by the same trained operator29. After randomization, study personnel were blinded to subject and protocol when evaluating outcome measures. Study participants were instructed to avoid alcohol, exercise, and caffeine for 24 hours and to fast overnight prior to admission to the CRU. We placed intravenous catheters in the right wrist for blood sampling and in the right antecubital fossa for administration of insulin, glucose, and octreotide (OCT). Studies then 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 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 principle30. We repeated vascular measurements over the final 30 minutes of OCT infusion.
Protocol B (Isolated Hyperglycemia): Octreotide with basal insulin replacement was continuously infused for 90 minutes while euglycemia was maintained. Baseline vascular measurements were obtained over the final 30 minutes (Figure 1B). Then, a primed, variable-rate 20% dextrose infusion began to acutely raise and subsequently maintain blood glucose at ~200 mg/dL using the hyperglycemic clamp method30. Blood glucose 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 principle30. 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 was sampled every 5 minutes and plasma insulin every 30 minutes, with repeat arterial stiffness 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 blood glucose at ~200 mg/dL. Blood glucose 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. Blood glucose was sampled every 5 minutes with plasma insulin every 30 minutes, and repeat arterial stiffness and hemodynamic measurements were again obtained during the final 30 minutes of the insulin clamp.
Hemodynamic Function: 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 SphygmoCor® tonometer (ATCOR USA; Napierville, IL).
Carotid-Femoral Pulse Wave Velocity (cfPWV): To assess central aortic stiffness, cfPWV was measured with a SphygmoCor tonometer. To minimize the effects of sympathetic activity on cfPWV measurements, participants laid supine 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. cfPWV intraobserver reliability was also assessed by having the operator record three serial cfPWV measurements on the same subject over a 4-hour period. The coefficient of variation for cfPWV was 3.63%, indicating good intraobserver reliability31. 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 protocols32.
Augmentation Index (AIx): To assess muscular conduit arterial stiffness, we measured AIx noninvasively with a SphygmoCor tonometer. AIx measurements were obtained at the radial artery by the same trained operator with participants lying in the supine position in a temperature-controlled room for at least 15 minutes prior to measurement. AIx 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. AIx 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.
Wave Separation Analyses (Pf, Pb, and RM): We derived the central aortic pressure waveform by recording radial artery pressure waves through aplanation tonometry using a SphygmoCor tonometer. Briefly, after 20 sequential waveforms were acquired and an ensemble was averaged, a validated general transfer function was used to synthesize the central aortic pressure wave noninvasively. Wave separation analysis then decomposed pressure and flow waveforms into their forward (Pf) and backward (Pb) components29. Reflection magnitude (RM) was calculated as the ratio of the amplitudes of the backward/forward pressure waves33.
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)34 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: A sample size calculation using the Cohen’s d effect size from a previous study of cfPWV changes during euglycemic-hyperinsulinemia35 in metabolic syndrome subjects indicated that ≥8 participants in each protocol would have ≥80% power to detect meaningful cfPWV changes in the current study.
Outcomes: The primary outcome for each protocol was change in cfPWV. Secondary outcomes for each protocol included changes in AI, Pf, Pb, RM, 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.
Data Transformation and Summarization: Given that the data distributions for some outcome measures were initially skewed, we rescaled the pre- and post-intervention outcome measures for each protocol to the natural logarithmic scale (i.e., loge). The analytical outcome data were then derived by subtracting the loge transformed pre-intervention outcome measures from the loge transformed post-intervention outcome variable measures. For all outcome measures, the point estimate for the mean pre-intervention outcome measures, the point estimate for the mean pre- to post-intervention outcome measure change, and the point estimate for the difference between the mean of the pre-intervention outcome measures and the mean pre- to post-intervention outcome measure change for each protocol were converted via natural logarithmic antilog transformation (i.e., ex) to a geometric mean ratio scale.
Outcome Measure Analyses: The pre- to post-intervention outcome variable change for each protocol was compared by linear mixed model (LMM) analysis of covariance. Significance was set at α= 0.05 (two-tailed test). All statistical analyses were performed with SAS Studio 3.8 (SAS; Cary, NC). LMM Specification: The analytical outcome data of each protocol served as the dependent variable measurements of the LMM. An indicator variable to identify the protocol (i.e., A, B, C, or D) served as one of the LMM independent variables, and the loge transformed pre-intervention outcome measurements of each protocol served as a second LMM independent variable. Note that the loge transformed pre-intervention measurements were included as part of the LMM so that the between-admission comparison of the mean pre- to post-intervention change in the outcome measure could be standardized to a common reference pre-intervention measurement value. Hypothesis testing: Primary hypotheses under the null tested whether the mean within-protocol change in each outcome measure was equal to zero.