Characterization of a novel melanocortin receptor-containing node in the SNS outflow circuitry to brown adipose tissue involved in thermogenesis

Abstract

The melanocortins (MC) can affect interscapular brown adipose tissue (IBAT) thermogenesis via its sympathetic nervous system (SNS) innervation. We chose a site of high MC4-receptor (MC4-R) mRNA co-localization with SNS outflow neurons to IBAT, the subzona incerta (subZI) to test whether IBAT thermogenesis could be increased or decreased. We first performed immunohistochemical characterization of the subZI and found neurons and/or fibers in this area positive for melanin concentrating hormone, oxytocin, arginine vasopressin, agouti-related protein and alpha-melanocyte stimulating hormone. Functional characterization of the subZI was tested via site-specific microinjections. The MC3/4-R agonist, melanotan II [MTII (0.025, 0.05 and 0.075nmol)], and specific MC4-R agonist (cyclo [ß-Ala-His-D-Phe-Arg-Trp-Glu]-NH2; 0.024nmol) both significantly increased IBAT temperature (T(IBAT)) and pretreatment with the MC4R antagonist, HS024 (0.072nmol) blocked the MC4-R agonist-induced increased T(IBAT) in conscious, freely-moving Siberian hamsters. Injection of the MC4-R antagonist alone significantly decreased T(IBAT) up to 3h post injection. Collectively, these results highlight the identification of a brain area that possesses high concentrations of MC4-R mRNA and SNS outflow neurons to IBAT that has not been previously reported to be involved in the control of T(IBAT). These results add to previously identified neural nodes that are components of the central circuits controlling thermogenesis.

Fig 1

Location of the subZI using the mouse atlas (Paxinos and Franklin, 2007).

Fig 2

Photomicrographs of melanin concentrating hormone immunoreactivity in the rostral subZI (A) and caudal subZI (B). in the subZI. AHP, anterior hypothalamic nucleus, posterior part; AHC, anterior hypothalamic nucleus, central part; 3V, third ventricle. Scale bar = 200 μm.

Fig 3

Photomicrographs of oxytocin (OXY)-immunoreactivity in the rostral (A) and caudal (B) subZI. Arginine vasopressin (AVP)-immunoreactivity in the rostral (C) and caudal (D) subZI. AHP, anterior hypothalamic nucleus, posterior part; AHC, anterior hypothalamic nucleus, central part; 3V, third ventricle.

Fig 4

Photomicrograph of agouti related protein (AgRP) fibers in the hypothalamus. No AgRP is present in the rostral subZI (A) but is present in caudal subzi (B). α-MSH fibers are present in rostral (C) and caudal (D) subZI. AHP, anterior hypothalamic nucleus, posterior part; AHC, anterior hypothalamic nucleus, central part; 3V, third ventricle.

Fig 5

Percent change in IBAT temperature from baseline. MTII (A) and MC4-R agonist (cyclo ßβ-Ala-His-D-Phe-Arg-Trp-Glu]-NH2) (B) unilaterally into the subZI increases IBAT temperature. P<.05, * greater than saline, ** greater than 0.05 nmol.

Fig 6

Schematic showing all hits (circles) and misses (triangles) using the mouse atlas (A) (Paxinos and Franklin, 2007). Each circle represents more than one hamster. Example of a correct unilateral cannula placement (B). PVH, paraventricular nucleus of the hypothalamus; ZI, zona incerta.

Fig 7

Percent change in IBAT temperature from baseline. MC4-R agonist and MC4-R antagonist alone or together bilaterally into the subZI. P<.05, * different from saline

Fig 8

Schematic showing all hits (circles) and misses (triangles) using the mouse atlas (A) (Paxinos and Franklin, 2007). Each circle represents more than one hamster. Example of a correct bilateral cannula placement (B). ZI, zona incerta.