Leptin-sensitive sensory nerves innervate white fat

Abstract

Leptin, the primary white adipose tissue (WAT) adipokine, is thought to convey lipid reserve information to the brain via the circulation. Because WAT responds to environmental/internal signals in a fat pad-specific (FPS) manner, systemic signals such as leptin would fail to communicate such distinctive information. Saturation of brain leptin transport systems also would fail to convey increased lipid levels beyond that point. WAT possesses sensory innervation exemplified by proven sensory-associated peptides in nerves within the tissue and by viral sensory nerve-specific transneuronal tract tracer, H129 strain of herpes simplex virus 1 labeling of dorsal root ganglia (DRG) pseudounipolar neurons, spinal cord and central sensory circuits. Leptin as a paracrine factor activating WAT sensory innervation could supply the brain with FPS information. Therefore, we tested for and found the presence of the long form of the leptin receptor (Ob-Rb) on DRG pseudounipolar neurons immunohistochemically labeled after injections of Fluorogold, a retrograde tract tracer, into inguinal WAT (IWAT). Intra-IWAT leptin injections (300 ng) significantly elevated IWAT nerve spike rate within 5 min and persisted for at least 30 min. Intra-IWAT leptin injections also induced significant c-Fos immunoreactivity (ir), indicating neural activation across DRG pseudounipolar sensory neurons labeled with Fluorogold IWAT injections. Intraperitoneal leptin injection did not increase c-Fos-ir in DRG or the arcuate nucleus, nor did it increase arcuate signal transducer and activator of transcription 3 phosphorylation-ir. Collectively, these results strongly suggest that endogenous leptin secreted from white adipocytes functions as a paracrine factor to activate spinal sensory nerves innervating the tissue.

Keywords: dorsal root ganglia; electrophysiology; tract tracing; white adipose tissue.

Fig. 1.

Distribution of the long form of the leptin receptor (Ob-Rb) immunoreactive (ir) neurons in dorsal root ganglia (DRG) at T₁₁–L₃ vertebral levels innervating inguinal (I) white adipose tissue (WAT). Representative microphotographs showing Fluorogold (FG) labeling from IWAT (A) and Ob-Rb-ir (B). Colocalization (arrows) of FG and Ob-Rb-ir is shown in the inset. C: percentage of Ob-Rb-ir primary afferent neurons at vertebral levels T₁₁–L₃ innervating IWAT. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. T₁₂. Scale bar = 50 μm in A and B and 25 μm in the inset.

Fig. 2.

Test for diffusion of the 300-ng intra-IWAT leptin injection by injecting this dose ip. Microphotographs taken at the level of the arcuate nucleus. c-Fos-ir 1 h after saline (A) and 300 ng ip leptin (B) injection. Note no difference in c-Fos-ir between saline (A) and 300 ng leptin (B) treatment. Signal transducer and activator of transcription 3 phosphorylation (pSTAT3)-ir 30 min after saline (C), 300 ng leptin (D), 3,000 ng leptin (E), and 30,000 ng leptin (F). Note no difference in pSTAT3-ir between ip injection of saline (C) and 300 ng leptin (D). Bottom, 3-μg (E) and 30-μg (F) leptin injections are positive controls for the ability to detect leptin-induced increases in pSTAT3-ir. Arc, Arcuate nucleus; ME, median eminence; VMH, ventromedial hypothalamic area; 3V, third ventricle. Scale bar = 50 μm.

Fig. 3.

Leptin-induced activation of DRG neurons innervating IWAT at the vertebral level T₁₃. A and D: FG-labeled DRG neurons innervating IWAT. B and E: c-Fos-ir 1 h after intra-IWAT injection of saline (A–C) or leptin (300 ng, D–F). C and F: merged images showing c-Fos-ir + FG-labeled neurons (white arrows). Scale bars = 50 μm.

Fig. 4.

Percentage of FG + c-Fos-ir colocalized neurons across the T₁₁–L₃ DRG innervating IWAT following intra-IWAT saline (open bars) or 300 ng leptin (filled bars) injections. Total percentage of c-Fos-ir + FG colocalized neurons in collapsed T₁₁–L₃ DRG is shown in the inset. *P < 0.05 vs. saline.

Fig. 5.

A: mean ± SE spike activity/30 s in decentralized afferent nerve fibers arising from the right IWAT in Siberian hamsters immediately before (baseline) and at 5, 10, 20, and 30 min after IWAT injections of a total of 300 ng recombinant rat leptin or saline vehicle injections; n = 6 and 7/group, respectively. *P < 0.05 vs. leptin baseline. #P < 0.05 vs. leptin at all other time points. Inset, trace of neurophysiological response to IWAT leptin in the decentralized WAT nerve (top) and individual spike from the IWAT neurophysiological trace (bottom). Arrow indicates leptin injection. B: mean ± SE spike activity/30 s in decentralized afferent nerve fibers arising from the right IWAT after ip injections of 300 ng recombinant rat leptin or saline vehicle injections (n = 5/group).