Effects of estradiol on cerebrospinal fluid levels of agouti-related protein in ovariectomized rhesus monkeys

Abstract

Hypothalamic proopiomelanocortin (POMC)-derived MSH peptides and the melanocortin receptor antagonist, agouti-related protein (AgRP), interact to regulate energy balance. Both POMC and AgRP neurons express estrogen receptors, but little is known about estrogen regulation of the melanocortin system in the primate. We have therefore examined the effects of physiological doses of estradiol (E2) on POMC and AgRP in lumbar cerebrospinal fluid (CSF) of ovariectomized monkeys. POMC prohormone was measured by ELISA. AgRP was measured by RIA (sensitive for the more biologically active C-terminal AgRP(83-132) but also detects full-length AgRP) and by ELISA (measures primarily full length AgRP). In the first experiment, 14 animals were studied before and after 3 wk of E2. CSF POMC did not change, but AgRP(RIA) decreased from 7.9 +/- 1.2 to 4.7 +/- 1.2 fmol/ml after E2 (P = 0.03) and the POMC/AgRP(RIA) ratio increased from 4.2 +/- 0.89 to 6.8 +/- 1.04 (P = 0.04). AgRP(ELISA) did not change, but the ratio of AgRP(RIA) compared with AgRP(ELISA) was reduced after E2 (P = 0.02). In the second experiment, 11 animals were studied after 6 wk of E2, and similar changes were noted. The degree of AgRP(RIA) suppression with E2 was inversely related to body mass index (r = 0.569; P = 0.03). These results show for the first time that E2 suppresses AgRP(C-terminal) in CSF, increases the POMC to AgRP ratio, and may decrease AgRP processing, thus leading to increased melanocortin signaling. Furthermore, obesity was associated with resistance to the suppressive effects of E2 on AgRP, analogous to what is seen with obesity and leptin resistance.

Figure 1

Upper panel, Mean (±sem) CSF AgRP (RIA) levels shown on the left axis in 14 OVX monkeys before (solid bars) and after 3 wk of E2 replacement (hatched bars). CSF AgRP (RIA) decreased significantly after E2 (P = 0.03). The corresponding POMC to AgRP (RIA) ratio is shown on the right axis and increased after E2 (*, P = 0.03). Lower panel, The degree of AgRP (RIA) suppression with E2 (expressed as percent of baseline OVX CSF AgRP level) was inversely related to BMI (r = 0.569; P = 0.03) and was significantly more in animals with a BMI below the mean (n = 6) than in those with a BMI above the mean (n = 8) (P = 0.017).

Figure 2

Upper panel, Mean (± sem) CSF AgRP (RIA) levels shown on the left axis in 11 OVX monkeys before (solid bars) and after 6 wk of E2 replacement (hatched bars). CSF AgRP (RIA) decreased significantly after E2 (*, P = 0.016). The corresponding POMC to AgRP (RIA) ratio is shown on the right axis and increased after E2 (*, P = 0.01). Lower panel, Mean (±sem) CSF AgRP (RIA) levels in four OVX monkeys studied before (solid bar) and after 1 and 6 wk of E2 replacement (hatched bars). CSF AgRP (RIA) levels were significantly lower after 6 wk of E2 (*, P < 0.05) but not after 1 wk of E2.

Figure 3

Characterization of AgRP immunoactivity in the hypothalamic homogenate of an OVX monkey by gel filtration (Sephadex-G-75) chromatography (lA and 1B) and HPLC (2A and 2B). AgRP immunoactivity in the eluted fractions was assayed by RIA (1A and 2A) and ELISA (1B and 2B). Arrows indicate elution positions of synthetic AgRP (83-132)-NH2 and of FL AgRP. The majority of the AgRP immunoactivity eluted in the same position as AgRP (83-132) when characterized by gel filtration and HPLC using either the RIA [more sensitive in detecting AgRP (83-132)] or the ELISA (more sensitive in detecting FL AgRP).

Figure 4

Characterization of AgRP immunoactivity by HPLC in CSF pools from OVX monkeys before (left) and after (right) E2 replacement. AgRP immunoactivity in the eluted fractions was assayed by RIA. Arrows indicate elution positions of synthetic AgRP (83-132)-NH2 and of FL AgRP. Two prominent peaks of immunoactivity were found in CSF, eluting in the positions of both AgRP (83-132) and FL AgRP. The relative amount of AgRP (83-132) compared with FL AgRP was less in the CSF of E2-treated animals.