Carotid body denervation prevents the development of insulin resistance and hypertension induced by hypercaloric diets

Abstract

Increased sympathetic activity is a well-known pathophysiological mechanism in insulin resistance (IR) and hypertension (HT). The carotid bodies (CB) are peripheral chemoreceptors that classically respond to hypoxia by increasing chemosensory activity in the carotid sinus nerve (CSN), causing hyperventilation and activation of the sympathoadrenal system. Besides its role in the control of ventilation, the CB has been proposed as a glucose sensor implicated in the control of energy homeostasis. However, to date no studies have anticipated its role in the development of IR. Herein, we propose that CB overstimulation is involved in the etiology of IR and HT, core metabolic and hemodynamic disturbances of highly prevalent diseases like the metabolic syndrome, type 2 diabetes, and obstructive sleep apnoea. We demonstrate that CB activity is increased in IR animal models and that CSN resection prevents CB overactivation and diet-induced IR and HT. Moreover, we show that insulin triggers CB, highlighting a new role for hyperinsulinemia as a stimulus for CB overactivation. We propose that CB is implicated in the pathogenesis of metabolic and hemodynamic disturbances through sympathoadrenal overactivation and may represent a novel therapeutic target in these diseases.

FIG. 1.

CB activity is increased in rat models of IR and HT. A and B: Typical recordings of respiratory rate (Resp. Rate) (bpm), tidal volume (mL), and blood pressure in basal conditions and in response to ischemic hypoxia, induced by occlusions of common carotid artery (CCO) in a control rat and in a rat submitted to an HF diet. C: Typical recording of ventilatory parameters after CSN cut in an HF rat. D: Mean minute ventilation (VE) (product of respiratory frequency and tidal volume) in control, HA, and HSu rats. E: Effect of common carotid occlusion of 5, 10, and 15 s on minute ventilation in control, HF, and HSu rats. F: Effect of hypercaloric diets on CB catecholamines (dopamine [DA] plus DOPAC) basal release (20% O₂ plus 5% O₂ balanced N₂) (n = 5). G: Effect of hypercaloric diets on the release of catecholamines from CB evoked by hypoxia (5% O₂ plus 5% CO₂ balanced N₂) (n = 5). H: Effect of HF and HSu diets in CB weight. Control n = 19, HF n = 27, HSu n = 24. I: Effect of HF and HSu diets on the inmmunoreactivity for tyrosine hydroxylase (TH) (60 KDa) expressed as the ratio tyrosine hydroxylase to β-actin (43 KDa) expression. Left panel: Representative immunoreactivity for tyrosine hydroxylase and β-actin in the CB in % of control, HF, and HSu animals. Bars (D–I) represent means ± SEM. One- and two-way ANOVA with Dunnett and Bonferroni multicomparison tests, respectively; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05 vs. values within the same group.

FIG. 2.

CSN bilateral resection prevents IR and HT in HF and HSu animal models. A: Typical recording of respiratory rate (bpm) and tidal volume (mL) in response to ischemic hypoxia, induced by occlusion of common carotid artery in a rat submitted to CSN bilateral resection. The absence of increment in the ventilatory responses confirms CSN resection. B: Representative glucose excursion curve for insulin tolerance test in a control rat. Details on K_ITT calculation are described in research design and methods. A and C: Effect of CSN resection on insulin sensitivity determined by the insulin tolerance test, expressed as K_ITT in control, HF, and HSu rats. D: Effect of CSN resection on MAP in control, HF, and HSu rats. E: Absolute weight before and after hypercaloric diet administration and chronic sinus nerve resection. F: Increment in body weight, calculated as total weight variation during the experimental period, in control, HF, and HSu rats with and without CSN resection. G: Visceral fat, weighed postmortem and corrected to body weight in control, HF, and HSu rats with and without CSN resection. Bars represent means ± SEM. One- and two-way ANOVA with Dunnett and Bonferroni multicomparison tests, respectively; **P < 0.01, ***P < 0.001 vs. control; #P < 0.05; ##P < 0.01, ###P < 0.001 comparing values with and without CSN resection.

FIG. 3.

CSN bilateral resection prevents sympathoadrenal overactivation in HF and HSu animal models. A and B: Effect of CSN resection on circulating catecholamines, norepinephrine and epinephrine, respectively. C and D: Effect of CSN resection on adrenal medulla norepinephrine and epinephrine content, respectively. Bars represent means ± SEM. Two-way ANOVA with Bonferroni multicomparison tests, respectively; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05; ##P < 0.01, ###P < 0.001 comparing values with and without CSN resection.

FIG. 4.

Insulin receptors are present in the CB, and their phosphorylation (Phosp) increases in response to insulin. A: Representative Western blot showing insulin receptor immunoreactivity in the CB and insulin receptor phosphorylation immunoreactivity in control CBs (CTR) and in response to 1 and 100 nmol/L insulin (30 min incubation), respectively, corresponding to the 97 KDa band. A reprobing of the membranes with an anti–β-actin antibody, corresponding to the 42 KDa band, is shown below the gels. B: Average insulin receptor phosphorylation in control and in CBs incubated with 1 and 100 nmol/L insulin in relation to β-actin immunoreactivity (n = 3–4). **P < 0.01, *P < 0.05. One-way ANOVA with Dunnett multicomparison test comparing the groups with the control. Data are means ± SEM.

FIG. 5.

Insulin increases the neurosecretory responses in the CBs. A: Microscope field of dissociated rat CB cell culture and the typical recording of intracellular cell Ca²⁺, measured as the ratio of the fluorescent emission at 340/380 nm of chemoreceptor cells in basal conditions, in response to hypoxia (N₂), to 1 nmol/L insulin, and to 35 mmol/L K⁺. B: Effect of insulin on intracellular cell Ca²⁺, measured as means of the ΔRI in 179 chemoreceptor cells. In every cell, the fluorescence signal was integrated as a function of time (running integral [RI]). C and D: Time course for the release of ATP from CB in response to insulin (10 nmol/L) and dose-response curve for insulin action on ATP release and its comparison with the effect of hypoxia (5% O₂ plus 5% CO₂ balanced N₂). Release protocol consisted of two incubations of CBs in normoxic solutions (20% O₂ plus 5%CO₂ balanced N₂, 10 min), followed by insulin application during 30 min in normoxia and two final normoxic incubations. E and F: Group of experiments identical to C and D but measuring catecholamine (dopamine plus DOPAC) release from CB instead of ATP. ATP and catecholamine quantification in the CB are means of 4–6 data. Bars represent means ± SEM. One- and two-way ANOVA with Dunnett and Bonferroni multicomparison tests, respectively; *P < 0.05, **P < 0.01, *** P < 0.001 vs. control. Controls in the release experiments correspond to the period prior to insulin application. ins, insulin.

FIG. 6.

Insulin increases ventilation through a CB-mediated effect. A: Respiratory frequency and tidal volume (TV) recordings before and after administration of an intracarotid insulin (100 mg/kg) bolus. B: Mean basal ventilatory parameters, respiratory frequency (RR), tidal volume, and minute ventilation (VE) before insulin administration. C: Dose-response curve for the effect of insulin (1, 5, 10, 50, 100, and 200 mU/kg) on minute ventilation. For avoidance of the effect of hypoglycemia, the study of insulin effect on ventilation was performed in euglycemic conditions. Insulin effects on ventilation are means of 5–7 data. D: Typical respiratory frequency and tidal volume recordings due to the administration of an intracarotid insulin (100 mg/kg) bolus before and after CSN cut. E: Graph depicting a typical glucose perfusion curve to maintain euglycemia after insulin bolus and the levels of glycemia throughout the experiment. F: Total glucose concentrations perfused to maintain euglycemic clamp in response to the insulin concentrations (1, 5, 10, 50, 100, and 200 mU/kg) tested. Values represent means ± SEM. One-way ANOVA with Dunnett multicomparison test; *P < 0.05, **P < 0.01 vs. basal values. AUC, area under the curve; PulmFl, pulmonary flow.