Hyperglycemia Without Hyperinsulinemia Produces Both Sympathetic Neural Activation and Vasodilation in Normal Humans

Hyperglycemia Without Hyperinsulinemia Produces Both Sympathetic Neural Activation and Vasodilation in Normal Humans Robert P. Hoffman Martin Hausberg Christine A. Sinkey Erling A. Anderson

ABSTRACT To explore the effects of the acute induction of hyperglycemia on sympathetic activity and vascular function we studied eight normal control subjects (28 6 3 years of age). Muscle sympathetic nerve activity (MSNA) and forearm vascular resistance (FVR) were measured before (5.4 6 0.2 mmol/L) and during systemic infusion of 20% dextrose with octreotide (250 mg/h) and low dose insulin (4 mU·m22·min21) with 60 min of hyperglycemia (venous plasma glucose, 12.5 6 0.6 mmol/L). To control for the effects of hyperosmolarity and volume infusion subjects returned for two control studies with equal volume 20% mannitol and 0.2% saline infusions instead of dextrose infusion. The increase in MSNA during hyperglycemia (178 6 48 units)

INTRODUCTION

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linical evidence suggests significant pathophysiologic relationships between diabetic autonomic neuropathy, and macrovascular disease. They are frequently present in com-

Departments of Pediatrics (R.P.H.), Internal Medicine (M.H.), and Anesthesia (C.A.S., E.A.A.), and the Clinical Research and Cardiovascular Centers, University of Iowa College of Medicine, Iowa City, Iowa, USA Reprint requests to be sent to: Dr. Robert P. Hoffman, Department of Pediatrics, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242, USA. Journal of Diabetes and Its Complications 1999; 13:17–22  1999 Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

was significantly greater than the increase during mannitol (69 6 46 units, p , 0.001) or during 0.2% saline (28 6 28 units, p , 0.001). The decreases in FVR after 60 min of hyperglycemia (20 6 4 units, p 5 0.002) and mannitol (13 6 4 units, p 5 0.033) were significantly greater than the decrease during saline (0.1 6 4 units). The changes in FVR during hyperglycemia and mannitol did not differ. Acute hyperglycemia causes sympathoexcitation and peripheral vasodilation. The vascular effect may be mediated by increased osmolar load. (Journal of Diabetes and Its Complications 13; 1: 17–22, 1999.)  1999 Elsevier Science Inc.

bination in individual patients. Chronic hyperglycemia clearly plays a major role in the development of each of these diabetic complications.1 Yet, the short-term direct effects of hyperglycemia on autonomic and vascular function are poorly understood. What little is known has been learned through use of the hyperglycemic glucose clamp technique or oral glucose tolerance test in normal subjects.2–5 These studies have demonstrated both increases5 and decreases6 in limb vascular resistance and possible increases in sympathetic activity.6 Because hyperinsulinemia is present simultaneously with hyperglycemia in these studies, the results may not reflect the actual pathophysiology in 1056-8727/99/$–see front matter PII S1056-8727(98)00019-1

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type I diabetes as hyperglycemia is usually due to insulin deficiency. Hyperinsulinemia has been shown to cause sympathoexcitation and vasodilation without changes in plasma glucose concentration.7 Therefore, to determine the short-term effect of hyperglycemia, we measured muscle sympathetic nerve activity (MSNA) and forearm vascular resistance (FVR) during hyperglycemia with octreotide induced suppression of insulin secretion. To control for the effects of hyperosmolarity and volume, each subject was studied on two additional occasions using mannitol and 0.2% saline infusions. We have previously shown that MSNA is lower in type I diabetes subjects than in control subjects8 and that intensive diabetes therapy with improved glycemic control is associated with higher levels of MSNA.9 One possible reason for the lower MSNA in type I diabetes subjects is cardiovascular changes secondary to hyperglycemic induced fluid shifts. METHODS Subjects. Eight healthy subjects (five men and three women) on no medications were studied. Their mean age was 28.0 6 3.2 years and their mean body-mass index was 24.3 6 5.4 kg/m2 (mean 6 SD). Approval of the Institutional Review Board on Human Investigation and informed consent were obtained. Hemodynamic Measurements. Blood pressure was measured by automated sphygmomanometer and heart rate by electrocardiography. Mean arterial pressure (MAP) was calculated as one-third of the systolic pressure plus two-thirds the diastolic pressure. Forearm blood flow (FBF) was measured by venous occlusion plethysmography using an air filled plethysmographic cuff.7 Forearm vascular resistance (FVR) was calculated as mean arterial pressure divided by FBF. Central venous pressure (CVP) was measured via a small cannula inserted into antecubital vein using local anesthesia and advanced into an intrathoracic vein. Microneurography. Intraneural recording techniques (microneurography) were used to obtain post-ganglionic muscle sympathetic nerve activity from a muscle fascicle of the peroneal nerve posterior to the fibular head as previously described.10–12 Sympathetic bursts were identified by visual inspection of the records. We have previously shown blinded interobserver and intraobserver variability to be low (8.4% and 5.2% respectively).8,9 Protocol. Each subject was admitted to the Clinical Research Center of the University of Iowa College of Medicine the night before study. At 7:30 a.m. intravenous catheters were placed in the antecubital fossa of both arms and infusions of octreotide (250 mg/min), insulin (4 mU·m22·min21), and 20% dextrose as needed were started. The catheter in the left arm was used to

draw blood for the measurement of plasma glucose and insulin levels every 10 min. The plasma glucose level during this initial control period was clamped at fasting levels. The infusions of somatostatin and insulin were given for 90 min to control for the effects of somatostatin on MSNA and forearm vascular resistance (unpublished data), prior to taking the subjects to the Human Cardiovascular Physiology Laboratory and starting the search for a nerve recording site. The infusions were continued throughout the remainder of the study. Once a site was found, baseline measurements were collected over two 5-min intervals separated by 5 min. After completion of baseline measurements the dextrose infusion rate was adjusted to acutely raise the plasma glucose concentration to 11.1 mmol/L and to maintain this level for the next 60 min. Data for blood pressure, MSNA, and FBF were then collected for 5-min intervals every 10 min throughout the hyperglycemic period. After completion of the study, subjects returned within the next 3 months for the two control studies. During these the 20% dextrose infusion rate was adjusted to maintain plasma glucose concentration at fasting levels throughout the study and either 20% mannitol or 0.2% saline were given over the last 60 min to control for increased osmolarity during hyperglycemia and for volume infusion. The rates of the mannitol and saline infusions were matched to the 20% dextrose infusion rate in the first study over the last 60 min. Assays. Plasma glucose concentration was measured using the YSI 2300 Stat Glucose Analyzer. Plasma insulin concentrations were measured by radioimmunoassay. Statistical Analysis. FBF, FVR, MAP, CVP, and MSNA were measured and averaged over 5-min intervals every 10 min at baseline and beginning 15 min into the infusions. Analysis of variance of repeated measures with study session as a grouping factor was used to assess statistical significance between the responses for hyperglycemia, mannitol, and 0.2% saline. Planned contrasts were used to study differences between variables at baseline just prior to the infusions and at the 55–60 min interval. Statistical significance was set at p , 0.05. Results are reported as mean 6 standard error. RESULTS Plasma Glucose and Insulin Concentrations. Figure 1 shows the mean plasma glucose levels and infusion rate for 20% dextrose during the study. Mean fasting plasma glucose concentrations did not differ for the three studies. During hyperglycemia, the mean plasma glucose concentration increased to 12.7 6 0.5 mmol/L by 25 min, and remained at that level throughout the rest of the study. Plasma glucose levels did not change during the

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just prior to glucose infusion was 61 6 9 pmol/L and, at the end of 60 min of hyperglycemia, the mean plasma insulin level was 76 6 10 pmol/L. Baseline levels of plasma osmolality for the three sessions did not differ (hyperglycemia, 286 6 1; mannitol, 287 6 2; 0.2% saline, 287 6 2 mosm/L). As expected, there were significant differences in osmolarity changes between the three study sessions (time by infusion interaction, p 5 0.005). Osmolality increased at the end of hyperglycemia (291 6 1 mosm/L) and mannitol (291 6 1 mosm/L) (p , 0.001) by similar amounts, but did not change during 0.2% saline (285 6 2 mosm/L). MSNA. Integrated MSNA responses varied significantly (see Table 1 and Figure 1) between infusions (time by infusion interaction, p 5 0.009). MSNA was significantly increased after 60 min of hyperglycemia (time effect, p , 0.001). No significant change was seen after 60 min of mannitol or 0.2% saline. The increase in MSNA during hyperglycemia was greater than the change during either mannitol (p 5 0.002) or 0.2% saline (p 5 0.001). Similar results were found if MSNA was analyzed as bursts/min although the time by infusion interaction did not quite reach statistical significance (p 5 0.076). FIGURE 1 Mean plasma glucose levels for all three sessions (top) and infusion rate for either 20% dextrose, mannitol, or 0.2% saline (bottom).

mannitol or 0.2% saline studies. Fasting plasma insulin concentrations did not differ between the studies (hyperglycemia, 63 6 21; mannitol, 122 6 69; 0.2% saline 59 6 32 pmol/L). Mean plasma insulin levels did not change after 90 min of somatostatin and insulin infusion or during the remainder of any of the studies. Specifically, for the hyperglycemia study the mean plasma insulin level

Hemodynamic Measurements. Heart rate increased with time (time effect, p 5 0.001). A near significant difference in heart responses (see Table 1 and Figure 2) between infusions was present (time by infusion interaction, p 5 0.056). A slight increase was seen after 60 min of hyperglycemia (p 5 0.029) and during 0.2% saline (p 5 0.012). No change in heart rate was seen during mannitol infusion. Mean arterial pressure did not change during any of the three infusions. Central venous pressure significantly changed with time over all three study infusions (p 5 0.010) but did not differ between infusions (time by infusion interaction, NS).

TABLE 1. SYMPATHETIC AND VASCULAR RESPONSES TO HYPERGLYCEMIA, MANNITOL, AND 0.2% SALINE INFUSION IN EIGHT HEALTHY CONTROL SUBJECTS Hyperglycemia

MSNA (bursts/min) Integrated MSNA Heart rate (beats min) CVP (mm Hg) MAP (mm Hg) FBF (mL/min per 100 mL tissue) FVR (units)

Mannitol

0.2% Saline

Baseline

60 min

Baseline

60 min

Baseline

60 min

14.6 6 1.6 170 6 23 57 6 3 4.6 6 0.3 84 6 3

23.0 6 3.2* 362 6 63† 62 6 5* 4.1 6 0.6 83 6 4

16.9 6 3.5 192 6 38 58 6 3 4.4 6 0.4 84 6 4

19.2 6 3.4 264 6 48 59 6 4 4.5 6 1.0 84 6 4

15.6 6 2.8 220 6 47 57 6 4 4.7 6 0.9 84 6 3

18.7 6 4.1 266 6 63 62 6 4* 4.0 6 1.3 83 6 4

1.7 6 0.3 60 6 8

2.6 6 0.5 41 6 7†

2.5 6 0.5 45 6 8

2.4 6 0.3 42 6 7

2.9 6 0.7 41 6 9

3.8 6 1.2* 34 6 6†

MSNA, muscle sympathetic nerve activity; CVP, central venous pressure; MAP, mean arterial pressure; FBF, forearm blood flow; FVR, forearm vascular resistance. * p , 0.05 versus baseline, † p , 0.01 versus baseline.

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FIGURE 2 Changes in muscle sympathetic nerve activity (integrated activity, top; burst/min, middle) and forearm vascular resistance (bottom) during dextrose, mannitol, and 0.2% saline infusions. (MSNA, muscle sympathetic nerve activity; FVR, forearm vascular resistance.)

None of the changes within a single infusion group reached statistical significance. In all subjects FBF varied significantly with time (p , 0.001) but not did differ between infusions (time by infusion interaction, NS). FVR changes, however, did differ between infusions (time by infusion interaction, p , 0.001). FVR was significantly lower after 60 min of hyperglycemia (p , 0.001) and mannitol (p 5 0.004), but not 0.2% saline. The falls in FVR during hyperglycemia (p 5 0.002) and mannitol (p 5 0.033) were significantly different from the change during saline but did not differ from each other. DISCUSSION These results demonstrate that hyperglycemia decreases FVR and increases MSNA even when plasma

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insulin concentrations are maintained at fasting levels. The vasodilatory effect may be mediated by increases in plasma osmolarity since similar changes occur during mannitol but not volume infusion. The same cannot be said for the sympathoexcitatory effect of hyperglycemia since MSNA increased only during hyperglycemia. Multiple studies have explored the effects of acute hyperinsulinemia on cardiovascular and autonomic function7,13,14 but only a few studies have examined the effect of systemic hyperglycemia. These studies have yielded conflicting results. Green and MacDonald6 performed studies very similar to ours except that hyperglycemia was maintained for only 20 min and no insulin inhibition was used. They found that glucose and mannitol infusion both caused increases in calf blood flow and plasma norepinephrine levels. Baron et al.2 found a progressive increase in leg blood flow with increasing plasma glucose concentrations in control, but not type I diabetes, subjects. Plasma insulin levels were clamped in a supraphysiologic range using somatostatin and insulin infusions. Vollenweider et al.3 found less vasodilation and less sympathoexcitation during hyperglycemic clamp with endogenous hyperinsulinemia than during euglycemic, hyperinsulinemic clamp with identical glucose infusion rates. Plasma insulin levels were lower during the hyperglycemic clamp, however. Fagius and Berne4 have reported increases in MSNA following oral glucose ingestion. These equivocal results may be attributable to differences in plasma insulin levels accompanying hyperglycemia. We are able to clearly state that our findings are due to hyperglycemia and not due to the effect of hyperinsulinemia.7 Furthermore, hyperglycemia with low plasma insulin concentrations more closely reflects the physiologic situation in type I diabetes. We found that 60 min of hyperglycemia causes peripheral vasodilation without affecting blood pressure. During matched volume infusions of 20% mannitol and 0.2% saline vasodilation occurred over the first 15 min of all three study sessions probably due to the high fluid infusion rates necessary to raise the plasma glucose concentration to the target level. However, the vasodilation persisted after 60 min of hyperglycemia and mannitol, but not 0.2% saline. Thus, the fall in vascular resistance seen after 60 min of hyperglycemia does not appear to be due to experimental design or volume infusion. Our results are in contrast to those of Marfella et al.15 and Giugliano et al.5 Marfella et al. reported that acute hyperglycemia increased blood pressure and plasma norepinephrine levels with or without octreotide-induced suppression of insulin secretion. Limb blood flow was not measured. Giugliano et al. reported decreased leg blood flow and increased mean arterial pressure during hyperglycemic clamp. There are several potential reasons for the differing results between our results and those of Marfella et al. In their study, octreotide was not started until 5 min before starting

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hyperglycemia thus plasma insulin levels increased over the first 20 min of hyperglycemia and then gradually decreased. In addition, we have unpublished data which indicates that octreotide infusion by itself acutely causes pronounced changes in hemodynamic function which resolve over 60 min. Last, the largest increases in blood pressure occurred after 60 min of hyperglycemia. We did not extend our study beyond 60 min because of concerns regarding subjects experiencing the urge to void due to hyperosmolar diuresis. Fagius and Karhurvaara have clearly demonstrated that bladder filling increases blood pressure.16 In the study by Giugeliano et al. there was no suppression of glucose induced insulin secretion and thus the subjects were hyperinsulinemic. In this study, even larger fluid volumes would be necessary to maintain hyperglycemia because of the hyperinsulinemia which would lead to even more rapid bladder filling. The hyperglycemic induced vasodilation we found is likely due to osmolar induced changes in plasma volume6 because mannitol and glucose infusion had similar effects. Studies using direct intra-arterial infusions of saline, dextrose and mannitol have yielded differing results regarding this possibility. Overbeck et al.17 found that intraarterial infusion of hypertonic saline and dextrose caused similar decreases in local vascular resistance. They attributed the fall in vascular resistance to increased limb osmolarity and interstial fluid. Houben et al.18 studied the effects of intraarterial infusions of 5% dextrose, 20% dextrose, and 20% mannitol on local vascular resistance. They found that limb vascular resistance fell only during 20% dextrose infusions even when higher limb glucose concentrations were achieved with 5% dextrose. They attributed the vasodilation during 20% dextrose infusion to hyperglycemia at the catheter tip. Whether these results apply to the situation in our experiment is unclear. We used intravenous, rather than intraarterial, dextrose and mannitol infusions and had systemic hyperglycemia present. We expected that increased osmolality might increase plasma volume19 and central venous pressure and thus stimulate cardiopulmonary baroreflex sympathetic suppression,20 which would then cause peripheral vasodilation. This was not the case. Central venous pressure was decreased after 60 min of hyperglycemia but was also similarly decreased after 0.2% saline. Furthermore, MSNA did not decrease at any time during the three infusions. Thus, the fall in vascular resistance during hyperglycemia and mannitol is the result of some unknown mechanism and not sympathoinhibition. One possible mechanism is hyperglycemic impairment of endothelin-1 induced vasoconstriction.21,22 Interestingly, as with insulin infusion,7 the hyperglycemic induced vasodilation was associated with an increase in sympathetic activity. No change occurred during similar length and volume mannitol and 0.2%

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saline infusion. During hyperinsulinemia it is thought that the increase in MSNA may act as counterbalance to prevent excessive vasodilation.23,24 This may be the case during hyperglycemia, as well. In any event, it is clear that the diminished MSNA levels in type I diabetes subjects we reported in our previous studies8,9 are not attributable due to acute hyperglycemic effects. Limitations. The major limitation of our study is the differing baseline FBF and FVR levels for the three studies. These differences are likely due to slightly different arm or cuff positioning for each session. A second possibility is increased apprehension during the hyperglycemia session. It was always the first study performed so that we could match infusion rates during the mannitol and 0.2% saline infusions. This is unlikely however since baseline heart rate and mean arterial pressure were nearly identical for all three studies and since most of the subjects had participated in previous studies and were familiar with the equipment. A second limitation is that only the differences in integrated MSNA reached statistical significance between hyperglycemia and mannitol and saline reached statistical significance. However the changes in bursts/ min nearly reached statistical significance. In conclusion, these results indicate that systemic hyperglycemia causes peripheral vasodilation and sympathoexcitation in normal subjects apart from changes in plasma insulin. Further study will be needed to determine whether these acute effects of hyperglycemia may lead to development of the chronic alterations in autonomic and vascular function that are present in subjects with type I diabetes. ACKNOWLEDGMENTS This work was funded by grants from the Juvenile Diabetes Foundation and the National Institute of Health (Clinical Research Center Grant RR59). Dr. Hausberg was supported by grant from the German Research Association. We thank Elaine Paul, Nandiah Subbiah, and the nurses and laboratory technicians of the Clinical Research Center for their assistance.

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