Arcuate nucleus injection of an anti-insulin affibody prevents the sympathetic response to insulin

Abstract

Accumulating evidence suggests that insulin acts within the hypothalamus to alter sympathetic nerve activity (SNA) and baroreflex function. Although insulin receptors are widely expressed across the hypothalamus, recent evidence suggests that neurons of the arcuate nucleus (ARC) play an important role in the sympathoexcitatory response to insulin. The purpose of the present study was to determine whether circulating insulin acts directly in the ARC to elevate SNA. In anesthetized male Sprague-Dawley rats (275-425 g), the action of insulin was neutralized by microinjection of an anti-insulin affibody (1 ng/40 nl). To verify the efficacy of the affibody, ARC pretreatment with injection of the anti-insulin affibody completely prevented the increase in lumbar SNA produced by ARC injection of insulin. Next, ARC pretreatment with the anti-insulin affibody attenuated the lumbar sympathoexcitatory response to intracerebroventricular injection of insulin. Third, a hyperinsulinemic-euglycemic clamp increased lumbar, but not renal, SNA in animals that received ARC injection of a control affibody. However, this sympathoexcitatory response was absent in animals pretreated with the anti-insulin affibody in the ARC. Injection of the anti-insulin affibody in the adjacent ventromedial hypothalamus did not alter the sympathoexcitatory response to insulin. The ability of the anti-insulin affibody to prevent the sympathetic effects of insulin cannot be attributed to a general inactivation or nonspecific effect on ARC neurons as the affibody did not alter the sympathoexcitatory response to ARC disinhibition by gabazine. Collectively, these findings suggest that circulating insulin acts within the ARC to increase SNA.

Keywords: blood pressure; lumbar; obesity; renal; sympathetic nerve activity.

Fig. 1.

Anti-insulin affibody prevents the sympathoexcitatory response to insulin in the arcuate nucleus (ARC). A and B: arterial blood pressure (ABP) and mean ABP (gray line) (top), integrated (∫) lumbar sympathetic nerve activity (SNA; middle), and raw lumbar SNA (bottom; 1-s segments) of rats that received an injection of the control antibody (A; n = 6) or anti-insulin affibody (B; n = 4) into the ARC 10 min before ARC injection of insulin. C: mean ± SE values of mean ABP and lumbar SNA. *P < 0.05, control vs. anti-insulin affibody. Arrows denote ARC injection. D: schematic illustration and photomicrographs of ARC injection sites. Coordinates are rostrocaudal levels in reference to the bregma using the atlas of Paxinos and Watson. (36). The photomicrograph shows the injection site for red (i) and green (ii) beads for the same animal, although sections are 80 μm apart. Scale bars = 200 μm. DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; 3V, third ventricle.

Fig. 2.

A: mean ABP, integrated lumbar SNA, and integrated renal SNA of rats injected with gabazine 10 min after injection of control or anti-insulin affibodies in the ARC. Gabazine significantly increased all variables, but the magnitudes were not different between groups. Values are means ± SE. Arrows denote ARC injection. B: schematic illustration of ARC injection sites.

Fig. 3.

A: mean ABP, integrated and raw lumbar SNA, and integrated and raw renal SNA of rats injected with control or anti-insulin affibodies at 10 min before an intracerebroventricular injection of insulin. In animals injected with the control affibody, intracerebroventricular injection of insulin significantly increased lumbar, but not renal, SNA. This lumbar sympathoexcitatory effect was attenuated in animals pretreated with ARC anti-insulin affibody. B: mean ± SE values of mean ABP, lumbar SNA, and renal SNA in both groups. Arrows denote ARC injection. *P < 0.05, control vs. anti-insulin affibody.

Fig. 4.

A–C: examples of mean ABP, integrated and raw lumbar SNA, and integrated and raw renal SNA of rats that received a hyperinsulinemic-euglycemic clamp after pretreatment with control affibody in the ARC (A), anti-insulin affibody in the ARC (B), or anti-insulin affibody in the VMH (C). Summary data are shown in Fig. 5, and histology is shown in Fig. 6. Infusion of insulin increased lumbar SNA in rats that received a control antibody injection in the ARC or an anti-insulin injection in the VMH. However, the lumbar sympathoexcitatory response was absent in rats pretreated with the anti-insulin affibody in the ARC. Arrows denote ARC injection.

Fig. 5.

Mean ± SE values of mean ABP, lumbar SNA, renal SNA, heart rate [in beats/min (bpm)], and blood glucose of rats that received a hyperinsulinemic-euglycemic clamp after pretreatment with the control affibody in the ARC, anti-insulin affibody in the ARC, or anti-insulin affibody in the VMH. Injection sites are shown in Fig. 6. Infusion of insulin increased lumbar SNA in rats that received a control antibody injection in the ARC or anti-insulin injection in the VMH. However, the lumbar sympathoexcitatory response was absent in rats pretreated with the anti-insulin affibody in the ARC. Arrows denote ARC injection. *P < 0.05, ARC control affibody vs. ARC anti-insulin affibody; †P < 0.05, VMH anti-insulin vs. ARC anti-insulin.

Fig. 6.

A: photomicrograph of a bilateral ARC injection site with left (i) and right (ii) sides. In this animal, the injection sites were 120 μm apart in the rostral-caudal plane. Scale bars = 100 μm. B and C: schematic illustration of ARC or VMH injection sites for animals injected with intracerebroventricular insulin (B) or that received a hyperinsulinemic-euglycemic clamp (C).

Fig. 7.

Left: mean ± SE values of plasma insulin levels in rats treated with a hyperinsulinemic-euglycemic clamp plus pretreatment with a control or an anti-insulin affibody in the ARC or VMH. As expected, plasma insulin levels increased from baseline values (P < 0.01). Right: mean ± SE values of plasma insulin levels of low-fat (LF), obesity-resistant (OR), or obesity-prone (OP) rats. OP rats had a significantly higher plasma insulin values than LF or OR rats (n = 8 animals/group). *P < 0.05. Plasma insulin values of OP rats did not significantly differ from those of normal rats infused with insulin.