Motivation White adipose tissue (WAT) is a major energy store, endocrine organ and is critical to metabolic homeostasis. Adipocytes release stored energy through lipolysis; the hydrolysis of stored triglycerides to liberate free fatty acids and glycerol. WAT is known to be innervated by sympathetic and sensory nerves [1], though the direct contribution of nerves in controlling lipolysis is currently unclear. It is well established that lipolysis is stimulated by catecholamines through activation of β-adrenergic receptors expressed by adipocytes [2]. Sympathetic nerves innervating WAT are a potential source of norepinephrine, but evidence of their involvement in lipolytic control is limited. Here we have developed a model to study the effects of nerve stimulation on lipolysis in WAT ex vivo.
Methods Adult male C57BL/6J mice (8-10 weeks) were sacrificed by CO2 asphyxiation. Inguinal fat pads were identified and Immunocytochemistry: whole-mount immunocytochemistry was performed on whole fat pads, following previously described methods [3] Paraformaldehyde-fixed tissue was imaged by confocal microscopy. Nerves were stained with a chicken polyclonal antibody against β3-tubulin (1:500; Abcam), vasculature was stained with isolectin B4 (1:500; Invitrogen) and BODIPY used to visualise adipocytes. Ex vivo lipolysis assay: Sections (11 – 25mg) of inguinal fat pad were added to Dulbecco’s Modified Eagle’s medium containing 5.5 mM glucose and 2% (w/v) fatty acid-free bovine serum albumin. Tissue was challenged with pharmacological agents for 3 hours at 37oC, 5% CO2 and 95% relative humidity. Media samples were removed to assay free glycerol as an indirect measurement of lipolysis. Glycerol was quantified by a colorimetric absorbance assay.
Results β3-tubulin immunoreactivity revealed nervous innervation throughout the inguinal fat pad (N=5), with nerve bundles commonly observed tracking the length of the tissue. Finer innervation was mostly restricted to innervating large/medium-sized blood vessels. Parenchymal nervous innervation of white adipose was also observed (N=3). In lipolysis assays, it was observed that glycerol was constitutively released over 3 hours (N=5). Statistical analysis was conducted by ANOVA with post-hoc Tukey tests.
Norepinephrine (2µM) stimulated glycerol production above levels of constitutive glycerol production (P<0.01; N=5). We next employed veratridine to stimulate nerves, a natural product that inhibits voltage-gated Na+ channel inactivation, increasing nerve excitability. In these experiments, veratridine (100µM) stimulated glycerol production to the same level of norepinephrine (P>0.05; N=5). The effect of veratridine was abolished by tetrodotoxin (1µM; 30 mins preincubation) (P<0.01; N=5), with no significant difference observed from constitutive glycerol production (P>0.05; N=5).
Conclusions Our data reveal that white adipocytes and blood vessels of the mouse inguinal fat pad are innervated by nerves. Norepinephrine or pharmacologically increasing nervous excitability both stimulate lipolysis ex vivo in inguinal fat. Glycerol was also produced constitutively, suggesting basal lipolysis occurs in inguinal fat. Application of TTX was able to abolish veratridine-induced lipolysis to basal levels.