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Research Paper
Effect of bilateral carotid body resection on the counterregulatory response to hypoglycaemia in humans Erica A. Wehrwein1 , Jacqueline K. Limberg2 , Jennifer L. Taylor2 , Simmi Dube3 , Ananda Basu3 , Rita Basu3 , Robert A. Rizza3 , Timothy B. Curry2 and Michael J. Joyner2 1
Department of Physiology, Michigan State University, East Lansing, MI, USA Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA 3 Department of Endocrinology, Mayo Clinic, Rochester, MN, USA
Experimental Physiology
2
New Findings r What is the central question of this study? Hyperoxia blunts hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in carotid body-resected patients; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. r What is the main finding and its importance? Our data indicate that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, a relatively normal counterregulatory response to hypoglycaemia in carotid body-resected patients highlights: (i) the potential for long-term adaptations after carotid body resection; and (ii) the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.
Hyperoxia reduces hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in patients with bilateral carotid body resection; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. Five patients (three male and two female) with bilateral carotid body resection for glomus tumours underwent two 180 min hyperinsulinaemic, hypoglycaemic (3.3 mmol l−1 ) clamps separated by a minimum of 1 week and randomized to either normoxia (21% fractional inspired O2 ) or hyperoxia (100% fractional inspired O2 ). Ten healthy adults (seven male and three female) served as control subjects. Hypoglycaemia counterregulation in carotid body-resected patients was not significantly altered by hyperoxia (area under the curve expressed as a percentage of the normoxic response: glucose infusion rate, 111 ± 10%; cortisol, 94 ± 6%; glucagon, 107 ± 7%; growth hormone, 92 ± 10%; adrenaline, 89 ± 26%; noradrenaline, 79 ± 15%; main effect of condition, P > 0.05). This is in contrast to previously published results from healthy adults. However, the counterregulatory responses to hypoglycaemia during normoxia were not impaired in carotid body-resected patients when compared with control subjects (main effect of group, P > 0.05). Our data provide further corroborative evidence that the effect of hyperoxia on
C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
DOI: 10.1113/expphysiol.2014.083154
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hypoglycaemic counterregulation is mediated by the carotid bodies. However, relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation. (Received 22 September 2014; accepted after revision 4 November 2014; first published online 13 November 2014) Corresponding author M. J. Joyner: Department of Anesthesiology, 200 1st Street SW, SMH Joseph 4–184, Rochester, MN 55905, USA. Email: [email protected]
Introduction
Subjects
The carotid body chemoreceptors are well known for their role in sensing arterial oxygen levels and evoking increases in ventilation and sympathetic nerve activity in response to hypoxia (Prabhakar, 2000). In addition to important contributions from the liver, gastrointestinal tract and brain, recent evidence from a number of experimental models indicates that the carotid bodies also respond to low glucose and participate in the counterregulatory response to hypoglycaemia (Alvarez-Buylla et al. 1997; ´ Koyama et al. 2000, 2001; Pardal & Lopez-Barneo, 2002; Wehrwein et al. 2010). For example, removal of the carotid bodies in dogs impairs hypoglycaemia counterregulation (Koyama et al. 2000). In this context, we recently demonstrated in healthy humans that systemic hyperoxia, a manoeuvre that can acutely suppress the carotid body chemoreceptors (Downes & Lambertsen, 1966; Fitzgerald & Lahiri, 1986), blunts counterregulatory hormone responses to hypoglycaemia (Wehrwein et al. 2010). Carotid body resection in humans (for bilateral removal of glomus tumours) essentially eliminates the ventilatory response to hypoxia, which provides a measure of peripheral chemoreflex sensitivity (Timmers et al. 2003). Therefore, we hypothesized that hyperoxia would have no impact on the counterregulatory response to hypoglycaemia in patients with bilateral carotid body resection. Based on data showing that 40–60% of the counterregulatory response to hypoglycaemia is mediated by the carotid chemoreceptors (Koyama et al. 2000; Wehrwein et al. 2010), we further hypothesized that hypoglycaemia counterregulation would be impaired in bilateral carotid body-resected patients.
Subjects were 21–55 years of age, non-smokers, non-pregnant/breast feeding, with body mass index 21–32 kg m−2 . Data from control subjects were included in previous publications (n = 7, Wehrwein et al. 2010; n = 10, Limberg et al. 2014). All subjects had a physical examination and detailed medical history performed. Exclusion criteria included the following: chronic diseases (diabetes, sleep apnoea, obesity, gastric bypass surgery, or metabolic syndrome); medications known to affect endocrine, cardiovascular or autonomic function; unstable cardiac, renal, pulmonary, hepatic or cerebrovascular disease; abnormal blood pressure (>140/90 mmHg), fasting plasma glucose (>5.5 mmol l−1 ), or fasting lipid panel (low-density lipoprotein 130 mg dl−1 , high-density lipoprotein < 40 mg dl−1 and triglycerides 200 mg dl−1 ). All carotid body-resected patients received treatment at Mayo Clinic, Rochester, MN, USA. Each patient underwent separate surgeries for right and left removal of glomus tumours (paraganglioma). The time since surgery for each patient is reported in Table 1. Owing to the unique study population, subjects resided across the USA and travelled to the Mayo Clinic specifically for participation in the present investigation. Subjects who engaged in regular physical exercise programmes or were actively losing weight were excluded. Dietary advice was provided by a research dietician to ensure that subjects maintained constant body weight for 2 weeks prior to study. Body composition was measured using dual energy X-ray absorptiometry (Lunar iDXA software version 6.10; GE Healthcare Technologies, Madison, WI, USA). During a screening visit, subjects completed a hypoxic ventilatory response (HVR) test to determine carotid body chemosensitivity. To complete the HVR assessment, subjects were exposed to hypoxia using inspired gas mixtures of 21, 16 and 10% oxygen. Each level was maintained for 2 min, and CO2 was added to the inspired air to maintain end-tidal CO2 at baseline levels (isocapnia at eucapnic partial pressures). Respiratory measures were collected using a metabolic cart (Ultima CardiO2 gas exchange analysis system; MCG Diagnostics, Saint Paul,
Methods Approval
All experiments and procedures were approved by the Institutional Review Board at Mayo Clinic. Informed consent was obtained in writing from all subjects prior to study enrolment and testing. Studies conformed to the Declaration of Helsinki.
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MN, USA), and data were averaged every five to seven breaths. The slope of the breath-by-breath regression line for minute ventilation (in litres per minute) versus arterial oxygen saturation (expressed as a percentage) was assessed and used as an index of chemosensitivity. Subjects were instructed to avoid exercise, caffeine, medications (except birth control) and alcohol on the day before and day of the study. Monitoring
A 20-gauge, 5 cm brachial artery catheter was placed under ultrasound guidance after local anaesthesia for blood sampling and blood pressure monitoring (TruWave Pressure Transducer; Edwards Lifesciences, Irvine, CA, USA). Two intravenous catheters were placed in the arm opposite the brachial arterial catheter for infusions. Heart rate was monitored with a five-lead electrocardiogram, respirations via a pneumobelt/capnograph, and oxygen saturation by a pulse oximeter (Cardiocap/5; Datex-Ohmeda Inc., Louisville, CO, USA).
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The insulin infusion was started at 09.00 h. Arterial blood was drawn every 5–10 min at the bedside for measurement of glucose using a glucose oxidase method (Analox Instruments USA Inc., Lunenberg, MA, USA). Arterial blood was also drawn at specific timepoints (T−30, −20, −10, 0, 150, 160, 170 and 180 min) for measures of blood gasses, noradrenaline, adrenaline, cortisol, glucagon, growth hormone and insulin. Arterial blood gas samples were analysed immediately using an automatic blood gas analyser (ABL700; Radiometer, Westlake, OH, USA). All additional blood samples were immediately placed on ice, centrifuged, and plasma was stored at −80 °C until analysis. Plasma insulin was assessed using two-site immunoenzymatic assay (DxI automated immunoassay system; Beckman Instruments, Chaska, MN, USA). Cortisol was measured by a competitive binding immunoenzymatic assay (DxI automated immunoassay system; Beckman Instruments). Glucagon was measured by radioimmunoassay (Linco Research, St Louis, MO, USA). Plasma catecholamines were measured with reverse-phase high-performance liquid chromatography, with electrochemical detection after extraction with activated alumina.
Hyperinsulinaemic, hypoglycaemic clamp R Intravenous regular insulin (Novolin ; Novo Nordisk Inc., Princeton, NJ, USA) was infused at a constant rate of 2.0 mU (kg fat-free mass)−1 min−1 from protocol time (T) 0 to T180 min, and exogenous glucose (50% dextrose solution; Hospira, Inc., Lake Forest, IL, USA) was infused in amounts sufficient to maintain glucose concentrations at hypoglycaemic levels (3.3 mmol l−1 ).
Hyperoxia
Normoxia and hyperoxia trials occurred on different days separated by a minimum of 1 week, in random order, with subjects blinded to condition. During the hyperoxia study day, subjects breathed 100% oxygen through a face mask connected to a non-rebreathing valve and a large meteorological balloon, which served as a volume reservoir from T0 until the end of the study. On the normoxia day, the identical set-up was used except that the bag was filled with medical air (21% oxygen). Protocol
Subjects were admitted to the Clinical Research Unit of the Mayo Clinic at 17.00 h on the evening prior to study. A standard 10 kcal kg−1 meal (55% carbohydrate, 30% fat and 15% protein) was eaten between 18.00 and 18.30 h, and the subject fasted thereafter until the end of the study except for water. In the morning, the brachial artery catheter and intravenous lines were placed.
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Data analysis and statistics
Main outcome variables were evaluated as a change from baseline ( = clamp − baseline). Where relevant, baseline values were calculated as an average of T−30, −20, −10 and 0 min and clamp values as an average of T150, 160, 170 and 180 min. Analyses were performed using mixed-effects linear models to take into account the repeated-measures study design. To examine the effect of systemic hyperoxia on hypoglycaemia counterregulation in carotid body-resected patients, main effects of condition (normoxia or hyperoxia) and time (baseline or clamp) were assessed. In addition, an area under the curve (AUC) was calculated for glucose infusion rate and each hormone over time and expressed as a percentage of the normoxic response (normoxia = 100%). To examine the effect of carotid body resection on normal hypoglycaemia counterregulation, main effects of group (control or patient) and time (baseline or clamp) were assessed. To explore potential relationships between carotid body resection, carotid chemoreceptor sensitivity and hypoglycaemia counterregulation, Pearson’s product–moment correlations were also assessed. In all cases, distributional assumptions were assessed and appropriate transformations were used if necessary. All tests were two tailed, with P values of 0.05). In contrast to results from healthy control subjects [Fig. 2A (n = 7, Wehrwein et al. 2010; and n = 10, Limberg et al. 2014)], the counterregulatory response to hypoglycaemia in carotid body-resected patients was not significantly altered by hyperoxia (Table 3). Consistent with our hypothesis, the steady-state glucose infusion rate during the clamp in carotid body-resected patients was not different during hyperoxia when compared with normoxia (AUC as a percentage of normoxia, 111 ± 10%, P = 0.36; Fig. 2A). Furthermore, AUC values for counterregulatory hormones during hypoglycaemia (expressed as a percentage of the normoxic response) were not significantly altered by hyperoxia (Fig. 2B; C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
noradrenaline, 79 ± 15%, P = 0.24; adrenaline, 89 ± 26%, P = 0.71; cortisol, 94 ± 6%, P = 0.36; glucagon, 107 ± 7%, P = 0.40; and growth hormone, 92 ± 10%, P = 1.00). Hypoglycaemia counterregulation in carotid body-resected patients and healthy control subjects
Contrary to our hypothesis, during normoxia the glucose infusion rate required to maintain steady-state hypoglycaemia was not different between control and resected groups (P = 0.46; Table 4). Hypoglycaemia resulted in a significant increase in plasma catecholamines (noradrenaline and adrenaline) and in the counterregulatory hormones cortisol, glucagon and growth hormone (main effect of time, P < 0.01) in both groups. The increases in adrenaline, cortisol, glucagon and growth hormone during normoxic hypoglycaemia were not different between control and resected groups (main effect of group, P > 0.05; interaction between group and time, P > 0.05; Table 4). Of interest, those patients with the lowest level of chemoreceptor sensitivity (HVR) tended to require the greatest amount of glucose infused during the hyperinsulinaemic, hypoglycaemic clamp (relationship between HVR and glucose infusion rate during normoxia, r = 0.798, P = 0.11; Fig. 3).
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Discussion
hypoxia (HVR; Table 2 and Fig. 1A and B). Interestingly, those patients who had undergone carotid body resection most recently exhibited the greatest impairments in chemoreceptor sensitivity, and the HVR observed in those furthest from surgery was slightly more robust (although still markedly impaired from control levels; Fig. 1C). A low level of ‘residual chemosensitivity’ (5–10% of the full hypoxic response) has been identified previously after carotid body resection, and it is thought that chemosensitivity remains at a low level for >20 years after surgery (reviewed by Honda, 1992). In contrast, robust recovery of hypoxic chemosensitivity has been observed in animal models [85% recovery of the full hypoxic response in cats (Smith & Mills, 1980); and 30% recovery in ponies (Bisgard et al. 1980)]. These data indicate that the long-term effect of carotid body resection on chemoreceptor-mediated responses is likely to differ between animals and humans.
Our novel findings show that carotid body resection blunts the effect of hyperoxia on hypoglycaemic counterregulation in humans; however, carotid body-resected patients have relatively normal counterregulatory responses to hypoglycaemia. These observations, combined with our previous findings (Wehrwein et al. 2010), are consistent with the idea that the carotid bodies play an important role in systemic glucoregulation in healthy adults. In this context, short-term interventions, including removal of the carotid bodies in dogs (Koyama et al. 2000) and hyperoxia-mediated desensitization of the carotid chemoreceptors in healthy humans (Wehrwein et al. 2010), result in a 40–60% reduction in the counterregulatory response to hypoglycaemia. In contrast, the relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.
Hyperoxia in carotid body-resected patients
Evidence that the carotid bodies play a critical role in the systemic counterregulatory response to hypoglycaemia comes from Koyama et al. (2000), who performed hypoglycaemic clamps in carotid body-denervated dogs 16 days after surgery. The glucose infusion
Carotid body resection
Patients who had undergone bilateral carotid body resection for removal of glomus tumours exhibited essentially no chemoreflex-driven ventilatory response to
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Area Under the Curve (% of Normoxia)
Area Under the Curve (% of Normoxia)
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Figure 2. Minimal effect of hyperoxia on hypoglycaemia counterregulation in carotid body-resected patients A, in contrast to healthy control subjects (Wehrwein et al. 2010; Limberg et al. 2014), the steady-state glucose infusion rate during the clamp in carotid body-resected patients was not different during hyperoxia when compared with normoxia in carotid body-resected patients (expressed as a percentage of the normoxic response, where normoxia = 100%). ∗ P < 0.01 versus normoxia (Student’s paired t test within groups, normoxia versus hyperoxia). B, area under the curve values for counterregulatory hormones during hypoglycaemia (expressed as a percentage of the normoxic response, where normoxia = 100%) were not significantly altered by hyperoxia in carotid body-resected patients (Student’s paired t test within groups, normoxia versus hyperoxia).
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Table 4. Hypoglycaemia counterregulation in carotid body-resected patients and healthy control subjects Parameter Insulin (μU Control Patient
Baseline
Clamp
(Clamp – baseline)
ml−1 ) 4 ± 1 5 ± 2
132 ± 8† 151 ± 20†
128 ± 8 146 ± 18
5.4 ± 0.1 5.4 ± 0.1
3.4 ± 0.1† 3.3 ± 0.1†
−2.0 ± 0.1 −2.1 ± 0.1
–– ––
28 ± 4 38 ± 11
–– ––
Cortisol (μg dl−1 ) Control Patient
9 ± 1 11 ± 2
19 ± 2† 17 ± 2†
10 ± 2 6 ± 2
Glucagon (pg ml−1 ) Control Patient
63 ± 3 84 ± 14
101 ± 11† 127 ± 28†
39 ± 9 43 ± 33
1 ± 1 1 ± 1
11 ± 2† 14 ± 5†
10 ± 2 14 ± 5
206 ± 20 274 ± 45∗
355 ± 40† 512 ± 54∗†
149 ± 29 237 ± 75
28 ± 4 27 ± 13
748 ± 101† 510 ± 162†
720 ± 99 483 ± 153
Glucose (μmol ml−1 ) Control Patient Glucose infusion rate (μmol kg−1 min−1 ) Control Patient
Growth hormone (ng ml−1 ) Control Patient Noradrenaline (pg ml−1 ) Control Patient Adrenaline (pg ml−1 ) Control Patient
Data are presented as means ± SEM. Effect of group: ∗ P < 0.05 versus control. Effect of time: † P < 0.05 versus baseline.
rate required to maintain a steady glucose level was much higher in the denervated dogs when compared with control animals and demonstrates that absence of afferent information from the carotid bodies results in a decreased counterregulatory response during hypoglycaemia (Koyama et al. 2000). This interpretation is also supported by the markedly lower counterregulatory hormone responses to hypoglycaemia in the denervated animals (Koyama et al. 2000). Along similar lines, we recently showed that acute hyperoxia-mediated desensitization of the carotid chemoreceptors in healthy humans results in a reduction in the counterregulatory response to hypoglycaemia (Wehrwein et al. 2010). More specifically, we observed an increase in the glucose infusion rate and blunted rise in hormones important to counterregulation of hypoglycaemia in healthy adults when carotid body afferent activity was presumably reduced with systemic hyperoxia. However, our previous findings may have been confounded by widespread effects of a 3 h systemic exposure to hyperoxia. By studying carotid body-resected patients, we were able to test rigorously whether the effect of hyperoxia on hypoglycaemia counterregulation was mediated via the carotid chemoreceptors or some other mechanism. C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
In contrast to our observations in healthy adults, we observed a limited effect of systemic hyperoxia on the counterregulatory hormone response to hypoglycaemia in the absence of the carotid bodies in resected patients (Table 3 and Fig. 2). These data strongly suggest that systemic hyperoxia does not impair counterregulation in a non-specific manner but rather works specifically by limiting carotid body afferent input. These findings are consistent with the literature showing that the carotid bodies mediate effects of hyperoxia on other stimuli (Downes & Lambertsen, 1966; Lahiri & DeLaney, 1975); however, whether this effect is through low-glucose sensing or some other mechanism [e.g. altered insulin signalling (Ribeiro et al. 2013); non-specific inhibition of afferent traffic] at the level of the carotid chemoreceptors is not known.
Carotid body resection and hypoglycaemic counterregulation
In contrast to the acute models used by Koyama and colleagues [16 days (Koyama et al. 2000)] and our group previously [3 h (Wehrwein et al. 2010)], we did not observe any deficit in the counterregulatory
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responses to hypoglycaemia in patients with long-term carotid body resection. Specifically, the glucose infusion rate and the neurohormonal response to hypoglycaemia were similar between resected patients and healthy control subjects (Table 4). These results may suggest the following possibilities: (i) long-term adaptations occur after carotid body resection that allow for redundant mechanisms to compensate for the loss of the carotid bodies; or (ii) humans do not rely solely on carotid body glucose sensing in hypoglycaemic conditions. Given that the patients studied in the present investigation were 13 ± 5 years beyond the initial unilateral resection procedures and 5 ± 2 years from bilateral carotid body resection (Table 1), it may not be surprising that responses after chronic denervation in humans differ from more acute procedures (16 days) in dogs. We believe that these data highlight the adaptive and redundant nature of the counterregulatory response to hypoglycaemia and/or potential for physiological adaptations over time. In support of this idea, we observed a relationship between residual hypoxia sensitivity (HVR) after chronic bilateral resection and the amount of glucose infusion necessary to maintain plasma glucose levels in resected patients during the hypoglycaemic clamp (glucose infusion rate; Fig. 3). Although these correlations were not statistically significant, our data suggest that residual peripheral chemoreceptor function at the level of the carotid bodies may play a minor role in the counterregulatory response to hypoglycaemia many years after surgery.
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Additionally, it is possible that humans do not rely solely on carotid body glucose sensing in hypoglycaemic conditions or that other regulatory sites might adapt to play a greater role in the sensing of and recovery from hypoglycaemia upon loss of afferent input from the carotid bodies after surgical resection. Although we did not examine any specific ‘redundant mechanisms’ and thus cannot rule out any novel mechanisms at play in the patient or control groups, there are numerous peripheral and central sites that are known to sense blood glucose concentrations and may participate in the physiological response to hypoglycaemia. In particular, the hepatic portal vein has been shown to be important in counterregulation via sympathoadrenal mechanisms (Adachi, 1981; Donovan et al. 1991). In addition, numerous neural sites, both peripheral and central (reviewed by Verberne et al. 2014), have been implicated; this includes sympathetic and vagal peripheral afferents from the liver and gastrointestinal tract and the brain mechanism (reviewed by Borg et al. 1997; Ritter et al. 2011). The brain also has important glucose sensors in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract (Adachi et al. 1995). Furthermore, activation of aortic chemoreceptors (which otherwise play a minimal role in the hypoxic ventilatory response) has been shown to occur after carotid body resection in animal models (Smith & Mills, 1980) and may contribute to hypoglycaemia counterregulation.
Glucose infusion rate (µmol kg−1 min−1)
Experimental considerations 90 r = 0.798 p = 0.11 60
30
0 0.00
-0.05 -0.10 Hypoxic ventilatory response (l min−1 %−1)
-0.15
Figure 3. Relationship between carotid body chemosensitivity and hypoglycaemia counterregulation Those patients with the lowest level of chemoreceptor sensitivity (hypoxic ventilatory response) required the greatest amount of glucose infused during the hyperinsulinaemic, hypoglycaemic clamp (r = 0.798, P = 0.11).
There are three main limitations to the present study. First, HVR constitutes a compensatory response to hypoxia rather than a direct measure of carotid body activity during hypoglycaemia; despite this limitation, it is one of the best measures of peripheral chemoreflex responsiveness available in humans, is closely related to afferent activity in animal models (Fidone & Gonzalez, 1986) and provides the opportunity to examine the potential for ‘residual chemosensitivity’ in the present research cohort. Second, given the unique patient population, a small sample size restricted us to within-group comparisons only and did not allow for rigorous statistical testing between groups. Third, our patient group was significantly older than our control group and was more heterogeneous (e.g. larger age range). With regard to group differences in age, it is important to note that counterregulatory responses to hypoglycaemia are, if anything, blunted with age (Ortiz-Alonso et al. 1994). Given that we observed relatively normal responses to hypoglycaemia in older carotid body-resected patients when compared with our younger control group, this would lead us to underestimate (rather than overestimate) our conclusions. Furthermore, these small sample sizes are C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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not uncommon when studying this unique population (Honda, 1992) and, despite these limitations, results clearly indicate the following: (i) carotid body-resected patients exhibit blunted chemosensitivity; (ii) the effect of hyperoxia differs between control subjects and patients with bilateral carotid body resection; and (iii) hypoglycaemia counterregulation is not significantly impaired in carotid body-resected patients. Thus, the present limitations should not detract from our novel observations.
Summary and conclusion
Previously, we showed a substantial contribution of the carotid bodies to hypoglycaemic counterregulation in healthy humans by demonstrating that desensitization of the carotid bodies with hyperoxia significantly blunted counterregulation by 50% (Wehrwein et al. 2010). Our present data provide supporting evidence that the effect of hyperoxia on hypoglycaemic counterregulation is likely to be mediated, at least in part, by the carotid bodies. This supports the use of hyperoxia to desensitize the carotid chemoreceptors and argues against the notion that systemic hyperoxia acted at other sites to blunt counterregulatory responses to hypoglycaemia in control subjects. Additionally, while acute desensitization of the carotid bodies shows a significant role of the carotid bodies in mediating counterregulatory response to hypoglycaemia, the present study shows that chronic removal of the carotid bodies in humans does not result in impaired hypoglycaemia counterregulation. This does not invalidate the acute studies, but rather emphasizes the importance of redundant mechanisms in mediating counterregulatory responses to hypoglycaemia in humans and identifies differences between chronic denervation in humans and the more acute interventions common in animal models. Given that the findings from acute dog and human studies support that 50% of the normal response is mediated by the carotid bodies, there are still substantial contributions from other mechanisms. Thus, we speculate that maintenance of counterregulation in chronically resected patients is the result of long-term upregulation of other control mechanisms (e.g. liver and/or brain). In summary, when combined with results from our previous study (Wehrwein et al. 2010), our present data (i) support a role of the carotid bodies in hypoglycaemic counterregulation in humans; (ii) demonstrate the use of hyperoxia as a valid method to desensitize the carotid bodies in humans; and (iii) underscore the importance of redundant regulatory mechanisms in glucose homeostasis.
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Additional information Competing interests None declared.
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Author contributions Conception and design of protocol: E.A.W., A.B., R.B., R.A.R., T.B.C. and M.J.J. All authors carried out data acquisition, analysed and interpreted the data, drafted the article, revised it critically for important intellectual content and gave final approval of the version to be published. Funding Funding sources: NIH DK090541 (M.J.J., R.B.), NIH NS32352 (M.J.J.), NIH T32 DK07352 (E.A.W., J.K.L.), NIH F32 DK84624 (E.A.W.), NIH 1 UL1 RR024150 (Mayo Clinic CTSA, M.J.J.) and NIH DK29953 (R.B.). Acknowledgements Our deepest appreciation and thanks to our research participants. Additional thanks to Dr William Young and his staff from the Mayo Clinic Division of Endocrinology for assistance in subject recruitment. The authors also wish to thank Dr John Eisenach (Department of Anesthesiology) for placement of brachial artery catheters. The authors further wish to acknowledge the contributions of the following nursing and technical staff: Cheryl Shonkwiler, Barbara Norby, Shelly Roberts, Karen Krucker, Sarah Wolhart, Jean Knutson, Brent McConahey, Pamela Reich, Nancy Meyer, Pam Engrav, Christopher Johnson and Mike Mozer of the Mayo Clinic. In addition, we thank the Clinical Research Unit staff at Mayo Clinic, the Immunochemical Core Laboratory at Mayo Clinic, in particular Hilary Blair.
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Research Paper
Effect of bilateral carotid body resection on the counterregulatory response to hypoglycaemia in humans Erica A. Wehrwein1 , Jacqueline K. Limberg2 , Jennifer L. Taylor2 , Simmi Dube3 , Ananda Basu3 , Rita Basu3 , Robert A. Rizza3 , Timothy B. Curry2 and Michael J. Joyner2 1
Department of Physiology, Michigan State University, East Lansing, MI, USA Department of Anesthesiology, Mayo Clinic, Rochester, MN, USA 3 Department of Endocrinology, Mayo Clinic, Rochester, MN, USA
Experimental Physiology
2
New Findings r What is the central question of this study? Hyperoxia blunts hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in carotid body-resected patients; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. r What is the main finding and its importance? Our data indicate that the effect of hyperoxia on hypoglycaemic counterregulation is mediated by the carotid bodies. However, a relatively normal counterregulatory response to hypoglycaemia in carotid body-resected patients highlights: (i) the potential for long-term adaptations after carotid body resection; and (ii) the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.
Hyperoxia reduces hypoglycaemia counterregulation in healthy adults. We hypothesized that this effect is mediated by the carotid bodies and that: (i) hyperoxia would have no effect on hypoglycaemia counterregulation in patients with bilateral carotid body resection; and (ii) carotid body-resected patients would exhibit an impaired counterregulatory response to hypoglycaemia. Five patients (three male and two female) with bilateral carotid body resection for glomus tumours underwent two 180 min hyperinsulinaemic, hypoglycaemic (3.3 mmol l−1 ) clamps separated by a minimum of 1 week and randomized to either normoxia (21% fractional inspired O2 ) or hyperoxia (100% fractional inspired O2 ). Ten healthy adults (seven male and three female) served as control subjects. Hypoglycaemia counterregulation in carotid body-resected patients was not significantly altered by hyperoxia (area under the curve expressed as a percentage of the normoxic response: glucose infusion rate, 111 ± 10%; cortisol, 94 ± 6%; glucagon, 107 ± 7%; growth hormone, 92 ± 10%; adrenaline, 89 ± 26%; noradrenaline, 79 ± 15%; main effect of condition, P > 0.05). This is in contrast to previously published results from healthy adults. However, the counterregulatory responses to hypoglycaemia during normoxia were not impaired in carotid body-resected patients when compared with control subjects (main effect of group, P > 0.05). Our data provide further corroborative evidence that the effect of hyperoxia on
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DOI: 10.1113/expphysiol.2014.083154
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hypoglycaemic counterregulation is mediated by the carotid bodies. However, relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation. (Received 22 September 2014; accepted after revision 4 November 2014; first published online 13 November 2014) Corresponding author M. J. Joyner: Department of Anesthesiology, 200 1st Street SW, SMH Joseph 4–184, Rochester, MN 55905, USA. Email: [email protected]
Introduction
Subjects
The carotid body chemoreceptors are well known for their role in sensing arterial oxygen levels and evoking increases in ventilation and sympathetic nerve activity in response to hypoxia (Prabhakar, 2000). In addition to important contributions from the liver, gastrointestinal tract and brain, recent evidence from a number of experimental models indicates that the carotid bodies also respond to low glucose and participate in the counterregulatory response to hypoglycaemia (Alvarez-Buylla et al. 1997; ´ Koyama et al. 2000, 2001; Pardal & Lopez-Barneo, 2002; Wehrwein et al. 2010). For example, removal of the carotid bodies in dogs impairs hypoglycaemia counterregulation (Koyama et al. 2000). In this context, we recently demonstrated in healthy humans that systemic hyperoxia, a manoeuvre that can acutely suppress the carotid body chemoreceptors (Downes & Lambertsen, 1966; Fitzgerald & Lahiri, 1986), blunts counterregulatory hormone responses to hypoglycaemia (Wehrwein et al. 2010). Carotid body resection in humans (for bilateral removal of glomus tumours) essentially eliminates the ventilatory response to hypoxia, which provides a measure of peripheral chemoreflex sensitivity (Timmers et al. 2003). Therefore, we hypothesized that hyperoxia would have no impact on the counterregulatory response to hypoglycaemia in patients with bilateral carotid body resection. Based on data showing that 40–60% of the counterregulatory response to hypoglycaemia is mediated by the carotid chemoreceptors (Koyama et al. 2000; Wehrwein et al. 2010), we further hypothesized that hypoglycaemia counterregulation would be impaired in bilateral carotid body-resected patients.
Subjects were 21–55 years of age, non-smokers, non-pregnant/breast feeding, with body mass index 21–32 kg m−2 . Data from control subjects were included in previous publications (n = 7, Wehrwein et al. 2010; n = 10, Limberg et al. 2014). All subjects had a physical examination and detailed medical history performed. Exclusion criteria included the following: chronic diseases (diabetes, sleep apnoea, obesity, gastric bypass surgery, or metabolic syndrome); medications known to affect endocrine, cardiovascular or autonomic function; unstable cardiac, renal, pulmonary, hepatic or cerebrovascular disease; abnormal blood pressure (>140/90 mmHg), fasting plasma glucose (>5.5 mmol l−1 ), or fasting lipid panel (low-density lipoprotein 130 mg dl−1 , high-density lipoprotein < 40 mg dl−1 and triglycerides 200 mg dl−1 ). All carotid body-resected patients received treatment at Mayo Clinic, Rochester, MN, USA. Each patient underwent separate surgeries for right and left removal of glomus tumours (paraganglioma). The time since surgery for each patient is reported in Table 1. Owing to the unique study population, subjects resided across the USA and travelled to the Mayo Clinic specifically for participation in the present investigation. Subjects who engaged in regular physical exercise programmes or were actively losing weight were excluded. Dietary advice was provided by a research dietician to ensure that subjects maintained constant body weight for 2 weeks prior to study. Body composition was measured using dual energy X-ray absorptiometry (Lunar iDXA software version 6.10; GE Healthcare Technologies, Madison, WI, USA). During a screening visit, subjects completed a hypoxic ventilatory response (HVR) test to determine carotid body chemosensitivity. To complete the HVR assessment, subjects were exposed to hypoxia using inspired gas mixtures of 21, 16 and 10% oxygen. Each level was maintained for 2 min, and CO2 was added to the inspired air to maintain end-tidal CO2 at baseline levels (isocapnia at eucapnic partial pressures). Respiratory measures were collected using a metabolic cart (Ultima CardiO2 gas exchange analysis system; MCG Diagnostics, Saint Paul,
Methods Approval
All experiments and procedures were approved by the Institutional Review Board at Mayo Clinic. Informed consent was obtained in writing from all subjects prior to study enrolment and testing. Studies conformed to the Declaration of Helsinki.
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MN, USA), and data were averaged every five to seven breaths. The slope of the breath-by-breath regression line for minute ventilation (in litres per minute) versus arterial oxygen saturation (expressed as a percentage) was assessed and used as an index of chemosensitivity. Subjects were instructed to avoid exercise, caffeine, medications (except birth control) and alcohol on the day before and day of the study. Monitoring
A 20-gauge, 5 cm brachial artery catheter was placed under ultrasound guidance after local anaesthesia for blood sampling and blood pressure monitoring (TruWave Pressure Transducer; Edwards Lifesciences, Irvine, CA, USA). Two intravenous catheters were placed in the arm opposite the brachial arterial catheter for infusions. Heart rate was monitored with a five-lead electrocardiogram, respirations via a pneumobelt/capnograph, and oxygen saturation by a pulse oximeter (Cardiocap/5; Datex-Ohmeda Inc., Louisville, CO, USA).
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The insulin infusion was started at 09.00 h. Arterial blood was drawn every 5–10 min at the bedside for measurement of glucose using a glucose oxidase method (Analox Instruments USA Inc., Lunenberg, MA, USA). Arterial blood was also drawn at specific timepoints (T−30, −20, −10, 0, 150, 160, 170 and 180 min) for measures of blood gasses, noradrenaline, adrenaline, cortisol, glucagon, growth hormone and insulin. Arterial blood gas samples were analysed immediately using an automatic blood gas analyser (ABL700; Radiometer, Westlake, OH, USA). All additional blood samples were immediately placed on ice, centrifuged, and plasma was stored at −80 °C until analysis. Plasma insulin was assessed using two-site immunoenzymatic assay (DxI automated immunoassay system; Beckman Instruments, Chaska, MN, USA). Cortisol was measured by a competitive binding immunoenzymatic assay (DxI automated immunoassay system; Beckman Instruments). Glucagon was measured by radioimmunoassay (Linco Research, St Louis, MO, USA). Plasma catecholamines were measured with reverse-phase high-performance liquid chromatography, with electrochemical detection after extraction with activated alumina.
Hyperinsulinaemic, hypoglycaemic clamp R Intravenous regular insulin (Novolin ; Novo Nordisk Inc., Princeton, NJ, USA) was infused at a constant rate of 2.0 mU (kg fat-free mass)−1 min−1 from protocol time (T) 0 to T180 min, and exogenous glucose (50% dextrose solution; Hospira, Inc., Lake Forest, IL, USA) was infused in amounts sufficient to maintain glucose concentrations at hypoglycaemic levels (3.3 mmol l−1 ).
Hyperoxia
Normoxia and hyperoxia trials occurred on different days separated by a minimum of 1 week, in random order, with subjects blinded to condition. During the hyperoxia study day, subjects breathed 100% oxygen through a face mask connected to a non-rebreathing valve and a large meteorological balloon, which served as a volume reservoir from T0 until the end of the study. On the normoxia day, the identical set-up was used except that the bag was filled with medical air (21% oxygen). Protocol
Subjects were admitted to the Clinical Research Unit of the Mayo Clinic at 17.00 h on the evening prior to study. A standard 10 kcal kg−1 meal (55% carbohydrate, 30% fat and 15% protein) was eaten between 18.00 and 18.30 h, and the subject fasted thereafter until the end of the study except for water. In the morning, the brachial artery catheter and intravenous lines were placed.
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Data analysis and statistics
Main outcome variables were evaluated as a change from baseline ( = clamp − baseline). Where relevant, baseline values were calculated as an average of T−30, −20, −10 and 0 min and clamp values as an average of T150, 160, 170 and 180 min. Analyses were performed using mixed-effects linear models to take into account the repeated-measures study design. To examine the effect of systemic hyperoxia on hypoglycaemia counterregulation in carotid body-resected patients, main effects of condition (normoxia or hyperoxia) and time (baseline or clamp) were assessed. In addition, an area under the curve (AUC) was calculated for glucose infusion rate and each hormone over time and expressed as a percentage of the normoxic response (normoxia = 100%). To examine the effect of carotid body resection on normal hypoglycaemia counterregulation, main effects of group (control or patient) and time (baseline or clamp) were assessed. To explore potential relationships between carotid body resection, carotid chemoreceptor sensitivity and hypoglycaemia counterregulation, Pearson’s product–moment correlations were also assessed. In all cases, distributional assumptions were assessed and appropriate transformations were used if necessary. All tests were two tailed, with P values of 0.05). In contrast to results from healthy control subjects [Fig. 2A (n = 7, Wehrwein et al. 2010; and n = 10, Limberg et al. 2014)], the counterregulatory response to hypoglycaemia in carotid body-resected patients was not significantly altered by hyperoxia (Table 3). Consistent with our hypothesis, the steady-state glucose infusion rate during the clamp in carotid body-resected patients was not different during hyperoxia when compared with normoxia (AUC as a percentage of normoxia, 111 ± 10%, P = 0.36; Fig. 2A). Furthermore, AUC values for counterregulatory hormones during hypoglycaemia (expressed as a percentage of the normoxic response) were not significantly altered by hyperoxia (Fig. 2B; C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
noradrenaline, 79 ± 15%, P = 0.24; adrenaline, 89 ± 26%, P = 0.71; cortisol, 94 ± 6%, P = 0.36; glucagon, 107 ± 7%, P = 0.40; and growth hormone, 92 ± 10%, P = 1.00). Hypoglycaemia counterregulation in carotid body-resected patients and healthy control subjects
Contrary to our hypothesis, during normoxia the glucose infusion rate required to maintain steady-state hypoglycaemia was not different between control and resected groups (P = 0.46; Table 4). Hypoglycaemia resulted in a significant increase in plasma catecholamines (noradrenaline and adrenaline) and in the counterregulatory hormones cortisol, glucagon and growth hormone (main effect of time, P < 0.01) in both groups. The increases in adrenaline, cortisol, glucagon and growth hormone during normoxic hypoglycaemia were not different between control and resected groups (main effect of group, P > 0.05; interaction between group and time, P > 0.05; Table 4). Of interest, those patients with the lowest level of chemoreceptor sensitivity (HVR) tended to require the greatest amount of glucose infused during the hyperinsulinaemic, hypoglycaemic clamp (relationship between HVR and glucose infusion rate during normoxia, r = 0.798, P = 0.11; Fig. 3).
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Discussion
hypoxia (HVR; Table 2 and Fig. 1A and B). Interestingly, those patients who had undergone carotid body resection most recently exhibited the greatest impairments in chemoreceptor sensitivity, and the HVR observed in those furthest from surgery was slightly more robust (although still markedly impaired from control levels; Fig. 1C). A low level of ‘residual chemosensitivity’ (5–10% of the full hypoxic response) has been identified previously after carotid body resection, and it is thought that chemosensitivity remains at a low level for >20 years after surgery (reviewed by Honda, 1992). In contrast, robust recovery of hypoxic chemosensitivity has been observed in animal models [85% recovery of the full hypoxic response in cats (Smith & Mills, 1980); and 30% recovery in ponies (Bisgard et al. 1980)]. These data indicate that the long-term effect of carotid body resection on chemoreceptor-mediated responses is likely to differ between animals and humans.
Our novel findings show that carotid body resection blunts the effect of hyperoxia on hypoglycaemic counterregulation in humans; however, carotid body-resected patients have relatively normal counterregulatory responses to hypoglycaemia. These observations, combined with our previous findings (Wehrwein et al. 2010), are consistent with the idea that the carotid bodies play an important role in systemic glucoregulation in healthy adults. In this context, short-term interventions, including removal of the carotid bodies in dogs (Koyama et al. 2000) and hyperoxia-mediated desensitization of the carotid chemoreceptors in healthy humans (Wehrwein et al. 2010), result in a 40–60% reduction in the counterregulatory response to hypoglycaemia. In contrast, the relatively normal counterregulatory responses to hypoglycaemia in carotid body-resected patients highlight the importance of redundant mechanisms in mediating hypoglycaemia counterregulation.
Hyperoxia in carotid body-resected patients
Evidence that the carotid bodies play a critical role in the systemic counterregulatory response to hypoglycaemia comes from Koyama et al. (2000), who performed hypoglycaemic clamps in carotid body-denervated dogs 16 days after surgery. The glucose infusion
Carotid body resection
Patients who had undergone bilateral carotid body resection for removal of glomus tumours exhibited essentially no chemoreflex-driven ventilatory response to
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Area Under the Curve (% of Normoxia)
Area Under the Curve (% of Normoxia)
A
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Figure 2. Minimal effect of hyperoxia on hypoglycaemia counterregulation in carotid body-resected patients A, in contrast to healthy control subjects (Wehrwein et al. 2010; Limberg et al. 2014), the steady-state glucose infusion rate during the clamp in carotid body-resected patients was not different during hyperoxia when compared with normoxia in carotid body-resected patients (expressed as a percentage of the normoxic response, where normoxia = 100%). ∗ P < 0.01 versus normoxia (Student’s paired t test within groups, normoxia versus hyperoxia). B, area under the curve values for counterregulatory hormones during hypoglycaemia (expressed as a percentage of the normoxic response, where normoxia = 100%) were not significantly altered by hyperoxia in carotid body-resected patients (Student’s paired t test within groups, normoxia versus hyperoxia).
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Table 4. Hypoglycaemia counterregulation in carotid body-resected patients and healthy control subjects Parameter Insulin (μU Control Patient
Baseline
Clamp
(Clamp – baseline)
ml−1 ) 4 ± 1 5 ± 2
132 ± 8† 151 ± 20†
128 ± 8 146 ± 18
5.4 ± 0.1 5.4 ± 0.1
3.4 ± 0.1† 3.3 ± 0.1†
−2.0 ± 0.1 −2.1 ± 0.1
–– ––
28 ± 4 38 ± 11
–– ––
Cortisol (μg dl−1 ) Control Patient
9 ± 1 11 ± 2
19 ± 2† 17 ± 2†
10 ± 2 6 ± 2
Glucagon (pg ml−1 ) Control Patient
63 ± 3 84 ± 14
101 ± 11† 127 ± 28†
39 ± 9 43 ± 33
1 ± 1 1 ± 1
11 ± 2† 14 ± 5†
10 ± 2 14 ± 5
206 ± 20 274 ± 45∗
355 ± 40† 512 ± 54∗†
149 ± 29 237 ± 75
28 ± 4 27 ± 13
748 ± 101† 510 ± 162†
720 ± 99 483 ± 153
Glucose (μmol ml−1 ) Control Patient Glucose infusion rate (μmol kg−1 min−1 ) Control Patient
Growth hormone (ng ml−1 ) Control Patient Noradrenaline (pg ml−1 ) Control Patient Adrenaline (pg ml−1 ) Control Patient
Data are presented as means ± SEM. Effect of group: ∗ P < 0.05 versus control. Effect of time: † P < 0.05 versus baseline.
rate required to maintain a steady glucose level was much higher in the denervated dogs when compared with control animals and demonstrates that absence of afferent information from the carotid bodies results in a decreased counterregulatory response during hypoglycaemia (Koyama et al. 2000). This interpretation is also supported by the markedly lower counterregulatory hormone responses to hypoglycaemia in the denervated animals (Koyama et al. 2000). Along similar lines, we recently showed that acute hyperoxia-mediated desensitization of the carotid chemoreceptors in healthy humans results in a reduction in the counterregulatory response to hypoglycaemia (Wehrwein et al. 2010). More specifically, we observed an increase in the glucose infusion rate and blunted rise in hormones important to counterregulation of hypoglycaemia in healthy adults when carotid body afferent activity was presumably reduced with systemic hyperoxia. However, our previous findings may have been confounded by widespread effects of a 3 h systemic exposure to hyperoxia. By studying carotid body-resected patients, we were able to test rigorously whether the effect of hyperoxia on hypoglycaemia counterregulation was mediated via the carotid chemoreceptors or some other mechanism. C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
In contrast to our observations in healthy adults, we observed a limited effect of systemic hyperoxia on the counterregulatory hormone response to hypoglycaemia in the absence of the carotid bodies in resected patients (Table 3 and Fig. 2). These data strongly suggest that systemic hyperoxia does not impair counterregulation in a non-specific manner but rather works specifically by limiting carotid body afferent input. These findings are consistent with the literature showing that the carotid bodies mediate effects of hyperoxia on other stimuli (Downes & Lambertsen, 1966; Lahiri & DeLaney, 1975); however, whether this effect is through low-glucose sensing or some other mechanism [e.g. altered insulin signalling (Ribeiro et al. 2013); non-specific inhibition of afferent traffic] at the level of the carotid chemoreceptors is not known.
Carotid body resection and hypoglycaemic counterregulation
In contrast to the acute models used by Koyama and colleagues [16 days (Koyama et al. 2000)] and our group previously [3 h (Wehrwein et al. 2010)], we did not observe any deficit in the counterregulatory
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responses to hypoglycaemia in patients with long-term carotid body resection. Specifically, the glucose infusion rate and the neurohormonal response to hypoglycaemia were similar between resected patients and healthy control subjects (Table 4). These results may suggest the following possibilities: (i) long-term adaptations occur after carotid body resection that allow for redundant mechanisms to compensate for the loss of the carotid bodies; or (ii) humans do not rely solely on carotid body glucose sensing in hypoglycaemic conditions. Given that the patients studied in the present investigation were 13 ± 5 years beyond the initial unilateral resection procedures and 5 ± 2 years from bilateral carotid body resection (Table 1), it may not be surprising that responses after chronic denervation in humans differ from more acute procedures (16 days) in dogs. We believe that these data highlight the adaptive and redundant nature of the counterregulatory response to hypoglycaemia and/or potential for physiological adaptations over time. In support of this idea, we observed a relationship between residual hypoxia sensitivity (HVR) after chronic bilateral resection and the amount of glucose infusion necessary to maintain plasma glucose levels in resected patients during the hypoglycaemic clamp (glucose infusion rate; Fig. 3). Although these correlations were not statistically significant, our data suggest that residual peripheral chemoreceptor function at the level of the carotid bodies may play a minor role in the counterregulatory response to hypoglycaemia many years after surgery.
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Additionally, it is possible that humans do not rely solely on carotid body glucose sensing in hypoglycaemic conditions or that other regulatory sites might adapt to play a greater role in the sensing of and recovery from hypoglycaemia upon loss of afferent input from the carotid bodies after surgical resection. Although we did not examine any specific ‘redundant mechanisms’ and thus cannot rule out any novel mechanisms at play in the patient or control groups, there are numerous peripheral and central sites that are known to sense blood glucose concentrations and may participate in the physiological response to hypoglycaemia. In particular, the hepatic portal vein has been shown to be important in counterregulation via sympathoadrenal mechanisms (Adachi, 1981; Donovan et al. 1991). In addition, numerous neural sites, both peripheral and central (reviewed by Verberne et al. 2014), have been implicated; this includes sympathetic and vagal peripheral afferents from the liver and gastrointestinal tract and the brain mechanism (reviewed by Borg et al. 1997; Ritter et al. 2011). The brain also has important glucose sensors in the dorsal motor nucleus of the vagus and the nucleus of the solitary tract (Adachi et al. 1995). Furthermore, activation of aortic chemoreceptors (which otherwise play a minimal role in the hypoxic ventilatory response) has been shown to occur after carotid body resection in animal models (Smith & Mills, 1980) and may contribute to hypoglycaemia counterregulation.
Glucose infusion rate (µmol kg−1 min−1)
Experimental considerations 90 r = 0.798 p = 0.11 60
30
0 0.00
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-0.15
Figure 3. Relationship between carotid body chemosensitivity and hypoglycaemia counterregulation Those patients with the lowest level of chemoreceptor sensitivity (hypoxic ventilatory response) required the greatest amount of glucose infused during the hyperinsulinaemic, hypoglycaemic clamp (r = 0.798, P = 0.11).
There are three main limitations to the present study. First, HVR constitutes a compensatory response to hypoxia rather than a direct measure of carotid body activity during hypoglycaemia; despite this limitation, it is one of the best measures of peripheral chemoreflex responsiveness available in humans, is closely related to afferent activity in animal models (Fidone & Gonzalez, 1986) and provides the opportunity to examine the potential for ‘residual chemosensitivity’ in the present research cohort. Second, given the unique patient population, a small sample size restricted us to within-group comparisons only and did not allow for rigorous statistical testing between groups. Third, our patient group was significantly older than our control group and was more heterogeneous (e.g. larger age range). With regard to group differences in age, it is important to note that counterregulatory responses to hypoglycaemia are, if anything, blunted with age (Ortiz-Alonso et al. 1994). Given that we observed relatively normal responses to hypoglycaemia in older carotid body-resected patients when compared with our younger control group, this would lead us to underestimate (rather than overestimate) our conclusions. Furthermore, these small sample sizes are C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
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not uncommon when studying this unique population (Honda, 1992) and, despite these limitations, results clearly indicate the following: (i) carotid body-resected patients exhibit blunted chemosensitivity; (ii) the effect of hyperoxia differs between control subjects and patients with bilateral carotid body resection; and (iii) hypoglycaemia counterregulation is not significantly impaired in carotid body-resected patients. Thus, the present limitations should not detract from our novel observations.
Summary and conclusion
Previously, we showed a substantial contribution of the carotid bodies to hypoglycaemic counterregulation in healthy humans by demonstrating that desensitization of the carotid bodies with hyperoxia significantly blunted counterregulation by 50% (Wehrwein et al. 2010). Our present data provide supporting evidence that the effect of hyperoxia on hypoglycaemic counterregulation is likely to be mediated, at least in part, by the carotid bodies. This supports the use of hyperoxia to desensitize the carotid chemoreceptors and argues against the notion that systemic hyperoxia acted at other sites to blunt counterregulatory responses to hypoglycaemia in control subjects. Additionally, while acute desensitization of the carotid bodies shows a significant role of the carotid bodies in mediating counterregulatory response to hypoglycaemia, the present study shows that chronic removal of the carotid bodies in humans does not result in impaired hypoglycaemia counterregulation. This does not invalidate the acute studies, but rather emphasizes the importance of redundant mechanisms in mediating counterregulatory responses to hypoglycaemia in humans and identifies differences between chronic denervation in humans and the more acute interventions common in animal models. Given that the findings from acute dog and human studies support that 50% of the normal response is mediated by the carotid bodies, there are still substantial contributions from other mechanisms. Thus, we speculate that maintenance of counterregulation in chronically resected patients is the result of long-term upregulation of other control mechanisms (e.g. liver and/or brain). In summary, when combined with results from our previous study (Wehrwein et al. 2010), our present data (i) support a role of the carotid bodies in hypoglycaemic counterregulation in humans; (ii) demonstrate the use of hyperoxia as a valid method to desensitize the carotid bodies in humans; and (iii) underscore the importance of redundant regulatory mechanisms in glucose homeostasis.
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Additional information Competing interests None declared.
Exp Physiol 100.1 (2015) pp 69–78
Author contributions Conception and design of protocol: E.A.W., A.B., R.B., R.A.R., T.B.C. and M.J.J. All authors carried out data acquisition, analysed and interpreted the data, drafted the article, revised it critically for important intellectual content and gave final approval of the version to be published. Funding Funding sources: NIH DK090541 (M.J.J., R.B.), NIH NS32352 (M.J.J.), NIH T32 DK07352 (E.A.W., J.K.L.), NIH F32 DK84624 (E.A.W.), NIH 1 UL1 RR024150 (Mayo Clinic CTSA, M.J.J.) and NIH DK29953 (R.B.). Acknowledgements Our deepest appreciation and thanks to our research participants. Additional thanks to Dr William Young and his staff from the Mayo Clinic Division of Endocrinology for assistance in subject recruitment. The authors also wish to thank Dr John Eisenach (Department of Anesthesiology) for placement of brachial artery catheters. The authors further wish to acknowledge the contributions of the following nursing and technical staff: Cheryl Shonkwiler, Barbara Norby, Shelly Roberts, Karen Krucker, Sarah Wolhart, Jean Knutson, Brent McConahey, Pamela Reich, Nancy Meyer, Pam Engrav, Christopher Johnson and Mike Mozer of the Mayo Clinic. In addition, we thank the Clinical Research Unit staff at Mayo Clinic, the Immunochemical Core Laboratory at Mayo Clinic, in particular Hilary Blair.
C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society
