Insulin increases central aortic stiffness in response to hyperglycemia in healthy humans: A randomized four-arm study

Abstract

Introduction:

Increasing arterial stiffness is a feature of vascular aging that is accelerated by conditions that enhance cardiovascular risk, including diabetes mellitus. Multiple studies demonstrate divergence of carotid-femoral pulse wave velocity and augmentation index in persons with diabetes mellitus, though mechanisms responsible for this are unclear.

Materials and methods:

We tested the effect of acutely and independently increasing plasma glucose, plasma insulin, or both on hemodynamic function and markers of arterial stiffness (including carotid-femoral pulse wave velocity, augmentation index, forward and backward wave reflection amplitude, and wave reflection magnitude) in a four-arm, randomized study of healthy young adults.

Results:

Carotid-femoral pulse wave velocity increased only during hyperglycemic-hyperinsulinemia (+0.36 m/s; p = 0.032), while other markers of arterial stiffness did not change (all p > 0.05). Heart rate (+3.62 bpm; p = 0.009), mean arterial pressure (+4.14 mmHg; p = 0.033), central diastolic blood pressure (+4.16 mmHg; p = 0.038), and peripheral diastolic blood pressure (+4.09 mmHg; p = 0.044) also significantly increased during hyperglycemic-hyperinsulinemia.

Conclusions:

Hyperglycemic-hyperinsulinemia acutely increased cfPWV, heart rate, mean arterial pressure, and diastolic blood pressure in healthy humans, perhaps reflecting enhanced sympathetic tone. Whether repeated bouts of hyperglycemia with hyperinsulinemia contribute to chronically-enhanced arterial stiffness remains unknown.

Keywords

Insulin hyperglycemia octreotide vascular stiffness

Introduction

Arterial stiffness develops from dynamic interactions between structural and cellular elements of the vessel wall influenced by both hemodynamic forces and extrinsic factors (including hormones, salt, and glucose).¹ Arterial stiffness increases with vascular aging due to gradual loss of arterial elasticity² and is accelerated by conditions that increase cardiovascular risk, including diabetes mellitus (DM).³ Notably, arterial stiffness often antedates and is itself a strong risk factor for many cardiovascular diseases (e.g. heart failure, myocardial infarction, etc.).^3,4 Data from numerous studies demonstrate that increased arterial stiffness (assessed by carotid-femoral pulse wave velocity (cfPWV),^5–11 augmentation index (AIx),^12,13 reflection magnitude (RM),^14,15 or backward wave amplitude (Pb)¹⁶) predicts cardiovascular events and/or mortality and may explain some of the notable residual cardiovascular risk associated with even well-controlled hypertension.¹⁷

Arterial stiffness increases with deteriorating glucose tolerance status¹⁸ and is associated with insulin resistance¹⁹; however, the independent and combined roles of acute hyperglycemia and/or hyperinsulinemia on arterial stiffness are not well-defined.²⁰ Moreover, prior studies have identified divergence of arterial stiffness markers (specifically increased cfPWV and reduced AIx) in persons with DM,^21–23 though mechanisms responsible for this are also unclear. A recent editorial by Currie and Delles encouraged investigation of healthy cohorts to better-understand mechanisms contributing to alterations in arterial stiffness.²⁴ As arterial stiffness is increased in even adolescents with DM,²⁵ healthy young adults without vascular dysfunction comprise the ideal population in which to investigate metabolic mechanisms linked to acute changes in arterial stiffness. With these points in mind, we sought to quantify the independent effects of elevated concentrations of insulin, glucose, and both on arterial stiffness in healthy humans. Greenfield et al.²⁶ previously showed that AIx decreases in response to the magnitude of postprandial glucose and insulin responses after low- and high-carbohydrate meal ingestion. To isolate the effects of insulin and glucose from those of incretins and the autonomic changes that occur with oral glucose or meal ingestion, we used intravenous glucose and insulin infusions with co-administration of octreotide (OCT) to block endogenous insulin secretion. We then measured cfPWV, AIx, RM, forward wave amplitude (Pf), Pb, and systemic hemodynamic function during euglycemia, hyperglycemia, euglycemic-hyperinsulinemia, and hyperglycemic-hyperinsulinemia.

Materials and methods

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 program.²⁸ 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 operator.²⁹ 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 h 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.

Figure 1.

Experimental protocols: (a) euglycemia, (b) hyperglycemia, (c) euglycemic-hyperinsulinemia, and (d) hyperglycemic-hyperinsulinemia.

Protocol A (Euglycemia)

A 90-min saline infusion was initiated, with baseline vascular function measurements obtained during the final 30 min (Figure 1(a)). Then, OCT (30 ng/kg/min) with basal insulin replacement (0.15 mU/min/kg) was infused for 240 min. Blood glucose was sampled every 10 min and plasma insulin every 30 min. Euglycemia (EU) was maintained by a variable-rate glucose infusion using the negative feedback principle.³⁰ We repeated vascular measurements over the final 30 min of OCT infusion.

Protocol B (Isolated Hyperglycemia)

Octreotide with basal insulin replacement was continuously infused for 90 min while euglycemia was maintained. Baseline vascular measurements were obtained over the final 30 min (Figure 1(b)). 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 method.³⁰ Blood glucose was sampled every 5 min and plasma insulin every 30 min, with repeat vascular measurements obtained over the final 30 min of hyperglycemia.

Protocol C (Euglycemic-Hyperinsulinemia)

Euglycemia was maintained throughout this protocol by a variable-rate 20% dextrose infusion using the negative feedback principle.³⁰ Baseline arterial stiffness measurements were obtained during the final 30 mins of an OCT (30 ng/kg/min) plus basal insulin (0.15 mU/min/kg) infusion (Figure 1(c)). Then, hyperinsulinemia was initiated with a primed (2 mU/kg/min × 10 min), continuous (1 mU/kg/min × 110 min) infusion and OCT continued for 120 min. Blood glucose was sampled every 5 min and plasma insulin every 30 min, with repeat arterial stiffness and hemodynamic measurements obtained during the final 30 min 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 min of this study (Figure 1(d)). 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 min and plasma insulin every 30 min, with baseline arterial stiffness measurements obtained over the final 30 min of the 120-min hyperglycemic period (Figure 1(b)). Subsequently, hyperinsulinemia was initiated with a primed (2 mU/kg/min × 10 min), continuous (1 mU/kg/min × 110 min) infusion with OCT and hyperglycemia maintained for 120 min. Blood glucose was sampled every 5 min with plasma insulin every 30 min, and repeat arterial stiffness and hemodynamic measurements were again obtained during the final 30 min 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 min 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 s of carotid and 10 s 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-h period. The coefficient of variation for cfPWV was 3.63%, indicating good intraobserver reliability.³¹ 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.³²

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 min 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 s 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) components.²⁹ Reflection magnitude (RM) was calculated as the ratio of the amplitudes of the backward/forward pressure waves.³³

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. log_e). The analytical outcome data were then derived by subtracting the log_e transformed pre-intervention outcome measures from the log_e 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. e^x) 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 log_e transformed pre-intervention outcome measurements of each protocol served as a second LMM independent variable. Note that the log_e 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.

Results

Baseline subject characteristics and demographics

Table 1 details baseline demographics of the 19 total study participants. All had normal BMI, blood pressure, and fasting plasma glucose. Notably, 13 subjects completed Protocol A, 10 subjects Protocol B, 14 subjects Protocol C, and 12 subjects Protocol D. Nine subjects completed all four protocols. Three subjects voluntarily withdrew after completing only Protocols C and D, two after completing only Protocol C, and two after completing only Protocol A. One subject moved out of the area after completing Protocols A and B, one experienced scheduling difficulty after completing only Protocols A and D, and one completed Protocol D but then experienced abdominal cramping (due to octreotide) that led to study termination during their second admission. An electrode wire malfunction resulted in no post-intervention assessment of cfPWV for one subject in Protocol B.

Table 1.

Baseline subject characteristics and demographics.

Variable	Mean ± SEM
Sex	12 Female; 7 Male
Age (years)	24.4 ± 1.0
Body mass index (kg/m²)	22.4 ± 0.4
Systolic blood pressure (mmHg)	114.9 ± 2.6
Diastolic blood pressure (mmHg)	66.5 ± 1.9
Fasting blood glucose (mg/dL)	87.8 ± 1.4
Total cholesterol (mg/dL)	162.4 ± 5.4
LDL cholesterol (mg/dL)	92.1 ± 5.1
HDL cholesterol (mg/dL)	59.9 ± 2.9
Triglycerides (mg/dL)	63.6 ± 5.0

SEM: standard error of mean.

Plasma insulin and glucose concentrations

Figure 2 shows the time course for mean plasma glucose (upper panel), mean glucose infusion rate (middle panel), and mean plasma insulin levels (lower panel) throughout each protocol. Plasma glucose levels rose significantly from baseline within Protocols B and D, and plasma insulin concentrations rose significantly from baseline within Protocols C and D. These increases did not differ between respective protocol pairs.

Figure 2.

Time course for mean plasma glucose (Panel a), mean glucose infusion rate (Panel b), and mean plasma insulin (Panel c) throughout each infusion protocol.

Arterial Stiffness

Table 2 shows the summary statistics for pre- and post-intervention measures of arterial stiffness within each protocol. cfPWV did not change during euglycemia, hyperglycemia, or euglycemic-hyperinsulinemia (all p > 0.05), but significantly increased after hyperinsulinemia was added to hyperglycemia (+0.36 m/s; p = 0.032). AIx significantly decreased during euglycemia (Protocol A), but did not significantly change within any other protocol. There were no significant changes in Pf, Pb, or RM within any protocol.

Table 2.

Summary statistics for pre- and post-intervention cfPWV, AI, Pf, Pb, and RM.

Vascular parameter	Protocol	Assessment	n	Mean ± SD	p-Value
cfPWV (m/sec)	EG	Pre	13	5.25 ± 0.47	0.635
	EG	Post	13	5.21 ± 0.64	0.635
	HG	Pre	9	4.81 ± 0.70	0.830
	HG	Post	9	4.79 ± 0.79	0.830
	EH	Pre	14	5.19 ± 0.95	0.495
	EH	Post	14	5.06 ± 0.76	0.495
	HH	Pre	11	4.71 ± 0.41	0.032
	HH	Post	11	5.07 ± 0.67	0.032
AI (%)	EG	Pre	13	0.08 ± 12.82	0.046
	EG	Post	13	−4.15 ± 11.72	0.046
	HG	Pre	10	−2.80 ± 13.36	0.205
	HG	Post	10	−4.80 ± 12.13	0.205
	EH	Pre	14	0.15 ± 8.49	0.143
	EH	Post	14	−2.23 ± 11.81	0.143
	HH	Pre	11	−5.09 ± 9.19	0.062
	HH	Post	11	−8.45 ± 9.16	0.062
Pf (mmHg)	EG	Pre	13	26.30 ± 3.53	0.918
	EG	Post	13	26.20 ± 3.55	0.918
	HG	Pre	10	25.40 ± 3.98	0.075
	HG	Post	10	27.30 ± 5.03	0.075
	EH	Pre	14	26.54 ± 4.72	0.390
	EH	Post	14	28.00 ± 5.12	0.390
	HH	Pre	11	28.20 ± 3.26	0.729
	HH	Post	11	28.90 ± 3.35	0.729
Pb (mmHg)	EG	Pre	13	12.90 ± 3.81	0.243
	EG	Post	13	11.60 ± 2.27	0.243
	HG	Pre	10	12.10 ± 3.11	0.541
	HG	Post	10	12.50 ± 2.99	0.541
	EH	Pre	14	12.69 ± 3.61	0.716
	EH	Post	14	12.77 ± 2.55	0.716
	HH	Pre	11	13.00 ± 3.32	0.446
	HH	Post	11	12.09 ± 2.81	0.446
RM (%)	EG	Pre	13	47.60 ± 14.39	0.375
	EG	Post	13	44.10 ± 8.61	0.375
	HG	Pre	10	47.70 ± 14.45	0.775
	HG	Post	10	47.90 ± 12.23	0.775
	EH	Pre	14	44.15 ± 8.05	0.645
	EH	Post	14	44.85 ± 5.01	0.645
	HH	Pre	11	43.55 ± 9.27	0.230
	HH	Post	11	41.09 ± 8.04	0.230

cfPWV: carotid-femoral pulse wave velocity; AI: augmentation index; Pf: forward wave amplitude; Pb: backward wave amplitude; RM: reflection magnitude; EG: euglycemia; HG: hyperglycemia; EH: euglycemic-hyperinsulinemia; HH: hyperglycemic-hyperinsulinemia; SD: standard deviation.

Hemodynamic function

Table 3 details the within-protocol changes for all hemodynamic parameters. Heart rate (+3.62 bpm; p = 0.009), mean arterial pressure (+4.14 mmHg; p = 0.033), central diastolic blood pressure (+4.16 mmHg; p = 0.038), and peripheral diastolic blood pressure (+4.09 mmHg; p = 0.044) significantly increased during only hyperglycemic-hyperinsulinemia. There were no significant changes in aortic or peripheral pulse pressure or systolic blood pressure within any protocol (all p > 0.05).

Table 3.

Summary statistics for pre- and post-intervention hemodynamic parameters.

Hemodynamic parameter	Protocol	Assessment	n	Mean ± SD	p-Value
Central sBP	EG	Pre	13	95.54 ± 7.86	0.768
	EG	Post	13	95.00 ± 9.21	0.768
	HG	Pre	10	95.20 ± 10.71	0.512
	HG	Post	10	93.80 ± 8.97	0.512
	EH	Pre	14	96.43 ± 11.22	0.252
	EH	Post	14	98.57 ± 7.66	0.252
	HH	Pre	12	92.75 ± 9.77	0.243
	HH	Post	12	95.64 ± 7.84	0.243
Central dBP	EG	Pre	13	66.23 ± 6.57	0.442
	EG	Post	13	67.69 ± 6.87	0.442
	HG	Pre	10	66.10 ± 7.88	0.384
	HG	Post	10	64.40 ± 7.65	0.384
	EH	Pre	14	67.29 ± 11.03	0.080
	EH	Post	14	70.29 ± 8.33	0.080
	HH	Pre	12	61.75 ± 11.25	0.038
	HH	Post	12	65.91 ± 8.29	0.038
Central PP	EG	Pre	13	30.00 ± 5.40	0.124
	EG	Post	13	27.31 ± 4.11	0.124
	HG	Pre	10	29.10 ± 4.61	0.889
	HG	Post	10	29.40 ± 5.52	0.889
	EH	Pre	14	29.14 ± 6.61	0.597
	EH	Post	14	28.29 ± 5.58	0.597
	HH	Pre	12	31.00 ± 4.97	0.367
	HH	Post	12	29.73 ± 4.29	0.367
Peripheral sBP	EG	Pre	13	110.15 ± 8.81	0.630
	EG	Post	13	111.23 ± 10.83	0.630
	HG	Pre	10	110.10 ± 11.32	0.783
	HG	Post	10	110.80 ± 10.74	0.783
	EH	Pre	14	111.86 ± 6.61	0.051
	EH	Post	14	116.00 ± 5.58	0.051
	HH	Pre	12	110.25 ± 9.37	0.161
	HH	Post	12	114.27 ± 8.87	0.161
Peripheral dBP	EG	Pre	13	65.62 ± 6.56	0.522
	EG	Post	13	66.85 ± 6.99	0.522
	HG	Pre	10	65.40 ± 7.81	0.386
	HG	Post	10	63.70 ± 7.56	0.386
	EH	Pre	14	66.36 ± 10.67	0.089
	EH	Post	14	69.29 ± 7.95	0.089
	HH	Pre	12	61.00 ± 11.07	0.044
	HH	Post	12	65.09 ± 8.43	0.044
Peripheral PP	EG	Pre	13	46.46 ± 7.47	0.981
	EG	Post	13	46.46 ± 8.38	0.981
	HG	Pre	10	44.70 ± 6.27	0.379
	HG	Post	10	47.10 ± 8.27	0.379
	EH	Pre	14	45.50 ± 7.86	0.412
	EH	Post	14	47.43 ± 9.52	0.412
	HH	Pre	12	49.25 ± 5.72	0.704
	HH	Post	12	49.18 ± 5.79	0.704
MAP	EG	Pre	13	79.85 ± 6.53	0.804
		Post	13	80.31 ± 7.39	0.804
	HG	Pre	10	78.90 ± 8.76	0.381
		Post	10	77.30 ± 8.21	0.381
	EH	Pre	14	80.50 ± 10.83	0.089
		Post	14	83.21 ± 8.05	0.089
	HH	Pre	12	75.50 ± 10.60	0.033
		Post	12	79.64 ± 7.98	0.033
HR	EG	Pre	13	61.92 ± 10.78	0.588
	EG	Post	13	62.67 ± 9.89	0.588
	HG	Pre	10	55.40 ± 8.22	0.917
	HG	Post	10	55.10 ± 6.42	0.917
	EH	Pre	14	59.64 ± 9.14	0.120
	EH	Post	14	61.93 ± 13.59	0.120
	HH	Pre	12	58.83 ± 6.98	0.009
	HH	Post	12	62.45 ± 7.17	0.009

sBP: systolic blood pressure; dBP: diastolic blood pressure; PP: pulse pressure; MAP: mean arterial pressure; HR: heart rate; EG: euglycemia; HG: hyperglycemia; EH: euglycemic-hyperinsulinemia; HH: hyperglycemic-hyperinsulinemia; SD: standard deviation.

Discussion

To our knowledge, this study is the first to investigate the discrete effects of acute hyperglycemia and hyperinsulinemia on cfPWV, AIx, RM, Pf, and Pb in the same subjects, with several novel observations warranting discussion. First, the combination of hyperglycemia and hyperinsulinemia increased cfPWV, while isolated hyperglycemia or hyperinsulinemia did not. Second, hyperglycemic-hyperinsulinemia acutely increased heart rate, mean arterial pressure, and diastolic blood pressure. Finally, no significant changes were seen in AIx, RM, Pf, and Pb during hyperglycemic-hyperinsulinemia.

Prior work from Puzantian et al.²⁰ found that acute hyperglycemia (using pancreatic clamping methodology in healthy subjects) did not alter cfPWV, but they noted that further studies were needed to determine the independent and combined roles of glucose and insulin on cfPWV. To our knowledge, the current study is the first to investigate this question. Infusion of OCT allowed us to isolate the effects of insulin and glucose and provide the first evidence that moderate hyperglycemia unmasks an action of physiologic hyperinsulinemia to increase central aortic stiffness in healthy humans. Here we must note that heart rate, mean arterial pressure, and diastolic blood pressure also rose significantly during hyperglycemic-hyperinsulinemia. A positive association between heart rate and cfPWV has been demonstrated in recent studies.^36,37 Bikia et al.³⁸ recently performed an in-silico evaluation of the impact of heart rate on cfPWV and confirmed that small heart rate changes only slightly alter cfPWV, but they suggested that a more clinically significant impact on cfPWV should be considered in cases wherein heart rate might vary to a greater extent. The mean increase in heart rate and cfPWV during hyperglycemic-hyperinsulinemia in the current study were 3.62 bpm and 0.4 m/s, respectively. Prior reports show that the effect of heart rate on cfPWV is small and on the order of 0.02 m/s per 1 bpm change,^36,37 thus increased heart rate alone is unlikely to explain the increase in cfPWV observed during hyperglycemic-hyperinsulinemia. We also note that acute hyperglycemic-hyperinsulinemia significantly increased mean arterial pressure and diastolic blood pressure without altering systolic blood pressure. These findings mirror prior reports demonstrating that increases in heart rate^39–41 and arterial stiffness^42,43 precede the development of systolic hypertension. Similarly, acute stiffening of the thoracic aorta and its branches (i.e. the arterial distribution represented by cfPWV) corresponds to increases in mean arterial pressure.⁴⁴ Increased transmural pressure at the level of the central aorta causes the elastic lamellae of this large artery to stretch and become stiffer, resulting in higher cfPWV.⁴⁴ The increase of mean arterial pressure observed in the current study corresponded to an acute increase in cfPWV. Such increases of vascular tone, occurring repetitively, may contribute to vascular remodeling.⁴⁵ Further studies are needed to investigate and test this hypothesis.

We also found that AIx, RM, Pf, and Pb did not significantly change during hyperglycemic-hyperinsulinemia. This result aligns with previous studies reporting that increased cfPWV is not associated with elevated AIx in persons with DM.^21–23 Our prior work has also shown that insulin has opposing effects on cfPWV and AIx in various metabolic conditions. Specifically, insulin (during euglycemia) acutely reduced AIx in both healthy and metabolic syndrome subjects, but increased cfPWV in metabolic syndrome subjects only.³⁵ In that study, metabolic syndrome subjects were insulin-resistant and had chronically higher fasting plasma glucose and insulin concentrations (i.e. the milieu of metabolic syndrome and type 2 DM). Lacy et al.²¹ hypothesized that dissipation of the energy of the incident pressure wave in persons with DM blunting wave reflections might explain this phenomenon. In other words, it is reduced wave reflections, not the increased outgoing pressure wave, that result in the attenuation of AIx in persons with DM.^21,22 In contrast, Khoshdel et al.²³ theorized that AIx underestimates arterial stiffness in persons with DM because of their wider pulse pressure compared to healthy controls. In this hypothesis, the main determinant of AIx is pulse pressure and not the reflected wave.²² The current study provides some clarification for this debate. In our healthy subjects with intact vascular function, the acute combination of hyperglycemia with hyperinsulinemia increased heart rate, mean arterial pressure, diastolic blood pressure, and cfPWV without significantly altering pulse pressure. We note that cfPWV is a direct measure of arterial stiffness, while AIx and pulse pressure are surrogate indicators of arterial stiffness.⁴⁶ Pulse pressure did not significantly change within the hyperglycemic-hyperinsulinemia protocol (or within any protocol) of the current study. These results suggest blunted wave reflections may better explain why AIx likely underestimates arterial stiffness in type 2 DM,²² and that the acute changes in cfPWV are independent of pulse pressure.

Taken together, our findings suggest a potential mechanism whereby acute, repetitive metabolic alterations could impact hemodynamic function and aortic stiffness. These results may have implications for the accelerated vascular aging seen in persons with both types 1 and 2 DM who regularly experience the acute combination of hyperglycemia and hyperinsulinemia. We caution, however, that the chronic impact of glucose and insulin per se on arterial stiffness includes a more complex vascular remodeling not seen with the acute metabolic interventions studied here. For example, widened pulse pressure has been associated with DM in many studies,^47–49 but we did not see a change in pulse pressure within any protocol of the current study. Acute alterations in arterial stiffness result from changes in endothelial function, vascular tone, heart rate, and other causes, whereas chronic arterial stiffening is mainly due to vascular remodeling, fibrosis, and calcification.⁵⁰ Whether repeated, acute measurable episodes of arterial stiffening contribute to long-term remodeling, fibrosis, and vascular injury remains a point of speculation. Recent work has shown that hyperglycemic-hyperinsulinemia significantly increases circulating norepinephrine (but not epinephrine) in healthy humans.⁵¹ The increases in cfPWV, heart rate, mean arterial pressure, and diastolic blood pressure observed during hyperglycemic-hyperinsulinemia in the current study mimic the enhanced sympathetic nervous system activity seen in insulin-resistant states like obesity and type 2 DM.^52,53 Our results, combined with these prior reports, point to sympathetic stimulation as a potential explanation for the hemodynamic changes seen during acute hyperglycemic-hyperinsulinemia. Unfortunately, we did not measure sympathetic nervous system activity in our study. Future work will focus on investigating this question and establishing a mechanistic basis for the hemodynamic effects observed during hyperglycemic-hyperinsulinemia.

While the current study provides novel insight into acute vascular changes during hyperglycemic-hyperinsulinemia, several study limitations should be mentioned. First, we studied a small number of healthy young adults and the study was powered to detect within-protocol responses to glucose and insulin. Thus, we did not examine between-protocol differences. Second, persons with DM or those who are older and/or less healthy might respond differently to our study protocols. Third, while AIx is widely used to evaluate arterial stiffness, conflicting data exist in regards to its validity.^54,55 A recent scientific statement from the American Heart Association recommended using wave separation analysis, as opposed to aortic AIx alone, when investigations are focused on the role of wave reflection.²⁹ These considerations led us to examine Pf, Pb, and RM in the current study. Fourth, mean cfPWV baseline values varied between protocols, though these differences were not statistically significant. This raises the issue of whether the significant within-protocol change seen during hyperglycemic-hyperinsulinemia was influenced by some “regression to the mean.” We considered this, but the concomitant changes seen in heart rate, diastolic blood pressure, and mean arterial pressure rather suggest the cfPWV change is legitimate and possibly due to the sympathetic mechanisms discussed above. Finally, we cannot rule out that OCT has in some unknown manner skewed the observed vascular responses, and this possibility should not be discounted. We do note, however, that no vasoactive effects have been identified in previous studies using a similar dose of OCT^51,56–58 and that OCT infusion does not alter the hemodynamic effects of acute hyperglycemia.⁵⁹

Conclusion

Hyperglycemic-hyperinsulinemia acutely increased cfPWV, heart rate, mean arterial pressure, and diastolic blood pressure in healthy humans, perhaps reflecting enhanced sympathetic tone. Whether repeated bouts of hyperglycemia with hyperinsulinemia (as occur regularly during the postprandial period in persons with DM and insulin-resistant syndromes) contribute to chronically-enhanced arterial stiffness remains unknown.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by National Institutes of Health research grants F32HL14230401, KL2TR003016, and UL1TR003015 (to WBH), and RO1DK073059 and RO1HL142250 (to EJB).

Ethics approval and consent to participate

All protocols were approved by the University of Virginia Institutional Review Board (#19948). Each subject gave written informed consent at their initial screening visit prior to study participation.

Clinical trial information

ClinicalTrials.gov identifier NCT03520569 (registered 9 May 2018).

ORCID iD

William B Horton

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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