Body weight and energy intake
Animals were randomly allocated to the HSu and HF groups, submitted to the respective diet protocol and then randomly allocated to CSN transection or sham surgery in which the CSN was left intact. The sham procedure did not alter body weight and energy intake in comparison with the respective controls (data not shown). Bilateral CSN resection was confirmed by the absence of increased ventilatory responses to ischaemic-hypoxia during common carotid artery occlusion (data not shown). Bilateral CSN resection and the sham procedure did not significantly modify animal behaviour or energy intake, measured as the average energy intake per day during the 3 weeks after CSN denervation (data not shown).
Bilateral CSN transection restores fasting plasma glucose and insulin sensitivity in animals continuously submitted to high-energy diets
IR was confirmed before and after surgery by an ITT (data is presented as the constant of the rate of glucose disappearance [K
ITT]). Figure 1 illustrates the effect of CSN transection on fasting plasma glucose and insulin sensitivity in control (Fig. 1a, b), HSu (Fig. 1c, d) and HF (Fig. 1e, f) animals, 3 weeks after surgery. Bilateral CSN ablation did not modify fasting plasma glucose in either control or HF animals (Fig. 1a, e). However, the high-sucrose diet significantly increased fasting plasma glucose, by 49%, whilst physiological glucose levels were restored as early as 1 week after CSN resection (controls, 4.63 ± 0.1 mmol/l; HSu 1 week after CSN resection, 4.61 ± 0.2 mmol/l) and were sustained in the ensuing 3 weeks (Fig. 1c). Compared to controls, the high-sugar and high-fat diets also decreased insulin sensitivity by 47% (Fig. 1d) and 40% (Fig. 1f), respectively. One week after CSN transection, insulin sensitivity was restored in HSu animals and partially restored in HF animals. Two weeks after CSN transection, insulin sensitivity was totally restored in both animal models, an effect that was sustained until the third week after CSN transection (Fig. 1d, f). Insulin sensitivity in control animals was not affected by CSN transection (Fig. 1b).
To test whether unilateral CSN transection was sufficient to improve insulin sensitivity, unilateral surgical ablation was performed in an independent subgroup of HF rats. Unilateral transection did not completely restore insulin sensitivity to physiological values in HF rats (Fig. 1g). Additionally, insulin sensitivity was monitored in a subgroup of HSu rats for 11 weeks post-surgery to evaluate whether the insulin-sensitising effect induced by bilateral CSN surgical ablation would be maintained. At the end of the 11-week period, insulin sensitivity was maintained at values similar to those of controls (Fig. 1h).
CSN transection restores plasma insulin and C-peptide in animal models of impaired insulin sensitivity
Table 1 displays the effect of CSN transection on plasma insulin and C-peptide concentrations. Compared to control animals, HSu and HF animals showed increases in plasma insulin of 123% and 120%, respectively. C-peptide levels were significantly increased, by 66% and 82% in HSu and HF rats, respectively. CSN transection did not modify either plasma insulin or C-peptide levels in control animals. However, in HSu and HF rats, CSN transection restored plasma insulin and C-peptide levels to levels similar to control values, showing that insulin secretion was regulated by CSN ablation (Table 1).
CSN transection improves insulin signalling in skeletal muscle and adipose tissue in animal models of impaired insulin sensitivity
Figure 2 shows western blot results for key proteins involved in insulin signalling pathways in insulin-sensitive tissues. In the skeletal muscle of HSu and HF animals, insulin receptor levels decreased significantly by 49% and 48%, respectively (Fig. 2a). Levels were restored in HSu and HF rats 3 weeks after CSN transection (Fig. 2a). Insulin receptor activity, as assessed by insulin receptor Tyr1361 phosphorylation, also decreased in both HSu and HF groups, by 47% and 31%, respectively (Fig. 2b). Chronic CSN transection not only restored, but actually increased, insulin receptor phosphorylation in control, HSu and HF animals, by 98%, 56% and 98%, respectively, relative to controls (Fig. 2b). HSu rats were also observed to have reduced levels (46% decrease) of GLUT4, which were fully restored 3 weeks after CSN transection, to levels similar to healthy controls (Fig. 2c).
In HSu and HF rats, insulin receptor levels were significantly decreased in adipose tissue, by 26% (Fig. 2d). CSN resection completely restored insulin receptor levels in HSu animals, but not in the HF, group (Fig. 2d). Similarly, insulin receptor phosphorylation decreased by 35% and 38% in HSu and HF animals, respectively, and CSN transection completely restored insulin receptor activity (Fig. 2e). The high-sugar and high-fat diets significantly decreased GLUT4 levels in adipose tissue, by 21% and 16%, respectively (Fig. 2h), and Akt values by 47% and 36%, respectively (Fig. 2f). CSN transection overturned the effect of the high-energy diets on adipose tissue levels of both GLUT4 and Akt (Fig. 2f, h). Akt activity decreased significantly, by 45% and 21% in HSu and HF rats, respectively (Fig. 2g). CSN transection increased Akt phosphorylation by 44% and 39% in HSu and HF animals, respectively (Fig. 2g).
In liver, insulin receptor levels and activity were not altered by either high-energy diets or CSN transection (Fig. 2i, j). In contrast, CSN ablation increased GLUT2 levels in HF animals by 40%, compared with controls (Fig. 2k).
CSN transection restores whole-body glucose tolerance, and liver and visceral adipose tissue glucose uptake in HF animals
The effect of chronic bilateral CSN resection on glucose uptake was evaluated using 2-deoxy[3H]glucose IVGTT. For these experiments, the HF model was selected because of its obesity phenotype and was compared with controls. Figure 3 shows similar IVGTT results to those observed in Fig. 1 for the ITT, as shown by the significant increase in the area under the glucose excursion curve in this group (controls, 1.6 × 107 ± 6.5 × 105; HF rats, 2.1 × 107 ± 1.7 × 106, Fig. 3b). Three weeks after CSN transection, glucose tolerance was restored in HF animals, shown by a decrease in AUC to values similar to those of control rats that had undergone the sham procedure (AUC HF with transected CSN, 1.6 × 107 ± 7.1 × 105).
After 6 weeks of the high-fat diet, the glucose metabolic index (Rg′), which evaluates glucose uptake, decreased by 42% and by 43% for the liver and pancreas, respectively. Glucose uptake by the soleus and gastrocnemius muscles did not significantly change, compared with controls (Fig. 3c). CSN transection produced a significant increase of 43% in 2-deoxy[3H]glucose uptake by the liver compared with HFD animals with an intact CSN. However, no change in glucose uptake was observed for the pancreas, soleus or gastrocnemius tissues (Rg′ of liver: HF without CSN transection, 3.15 ± 0.33 mmol [mg tissue]−1 min−1; HF with CSN transection, 4.50 ± 0.45 mmol [mg tissue]−1 min−1; Fig. 3c). Finally, in visceral adipose tissue, 6 weeks of the high-fat diet decreased glucose uptake by 45% compared with the control group, and this effect was completely restored by CSN transection (Rg′ of perienteric adipose tissue: controls without CSN transection, 1.46 ± 0.13 mmol [mg tissue]−1 min−1; HF without CSN transection, 0.81 ± 0.16 mmol [mg tissue]−1 min−1; HF with CSN transection, 1.44 ± 0.33 mmol [mg tissue]−1 min−1; Fig. 3d). No significant results were observed for epididymal and perinephric adipose tissue (Fig. 3d).
CSN denervation ameliorates lipid profile in animal models of impaired insulin sensitivity
The HSu and HF animals showed no change in either total cholesterol or LDL-cholesterol (Table 2). In contrast, HDL-cholesterol was decreased in HF animals by 28% and was restored to control values by CSN transection (Table 2). Additionally, the HSu and HF rats had increased triacylglycerol levels by 75% and 47%, respectively; these values returned to control levels after CSN resection (Table 2).
CSN transection normalises mean arterial pressure, nitric oxide metabolites and sympathetic activity in animal models of impared insulin sensitivity
In the current study, HSu and HF rats had a 39% increase in mean arterial pressure. These results are in line with the values previously obtained in our laboratory [5, 13]. Three weeks after CSN resection, mean arterial pressure was fully restored to control levels in both HSu (96.7 ± 11.8 mmHg) and HF (92.1 ± 9.6 mmHg) animals (Fig. 4a).
The effect of CSN resection on serum nitric oxide and its metabolites (NO + NO3) is depicted in Fig. 4b. Although no significant effects were observed in HSu rats, the HF rats had significantly increased NO + NO3 levels compared with controls (control, 10.6 ± 1.1 μmol/l; HF, 21.3 ± 2.0 μmol/l), with this effect being completely restored by chronic CSN resection.
Since CB overactivity contributes to the development of IR and hypertension through sympathoadrenal overstimulation , we also analysed sympathetic nervous activity using spectral analysis of heart rate variability and via the measurement of circulating and adrenal medullary catecholamines. We observed that sympathetic activity was increased in the animals fed a high-energy diet and that this effect was normalised by CSN transection (see ESM Results and ESM Fig. 1).