Testosterone Rapidly Augments Retrograde Endocannabinoid Signaling in Proopiomelanocortin Neurons to Suppress Glutamatergic Input from Steroidogenic Factor 1 Neurons via Upregulation of Diacylglycerol Lipase-α

Abstract

Testosterone exerts profound effects on reproduction and energy homeostasis. Like other orexigenic hormones, it increases endocannabinoid tone within the hypothalamic feeding circuitry. Therefore, we tested the hypothesis that testosterone upregulates the expression of diacylglycerol lipase (DAGL)α in the hypothalamic arcuate nucleus (ARC) to increase energy intake via enhanced endocannabinoid-mediated retrograde inhibition of anorexigenic proopiomelanocortin (POMC) neurons. Energy intake, meal patterns, and energy expenditure were evaluated in orchidectomized, male guinea pigs treated subcutaneously with testosterone propionate (TP; 400 μg) or its sesame oil vehicle (0.1 mL). TP rapidly increased energy intake, meal size, O₂ consumption, CO₂ production, and metabolic heat production, all of which were antagonized by prior administration of the DAGL inhibitor orlistat (3 μg) into the third ventricle. These orlistat-sensitive, TP-induced increases in energy intake and expenditure were temporally associated with a significant elevation in ARC DAGLα expression. Electrophysiological recordings in hypothalamic slices revealed that TP potentiated depolarization-induced suppression of excitatory glutamatergic input onto identified ARC POMC neurons, which was also abolished by orlistat (3 μ<smlcap>M</smlcap>), the CB₁ receptor antagonist AM251 (1 μ<smlcap>M</smlcap>), and the AMP-activated protein kinase inhibitor compound C (30 μ<smlcap>M</smlcap>) and simulated by transient bath application of the dihydrotestosterone mimetic Cl-4AS-1 (100 n<smlcap>M</smlcap>) and testosterone-conjugated bovine serum albumin (100 n<smlcap>M</smlcap>). Thus, testosterone boosts DAGLα expression to augment retrograde, presynaptic inhibition of glutamate release onto ARC POMC neurons that, in turn, increases energy intake and expenditure. These studies advance our understanding of how androgens work within the hypothalamic feeding circuitry to affect changes in energy balance.

Endocannabinoid Signaling at Hypothalamic Steroidogenic Factor-1/Proopiomelanocortin Synapses Is Sex- and Diet-Sensitive

“…However, in animals fed a HFD this effect is completely abrogated (Figure 5D ). The composite line graph seen in Figure 5E further illustrates the fact that retrograde, EC-mediated decreases in eEPSC amplitude (Conde et al, 2017 ) were significantly greater in HFD-fed animals compared to chow-fed controls at each time bin, an effect that was completely abolished in the presence of SC79. These data indicate that the HFD-induced enhancement of retrograde, EC-mediated signaling at VMN/ARC POMC synapses is facilitated by the reduction in Akt activation within the mediobasal hypothalamus.…”

“…The feeding and metabolic studies were performed using a four-station Comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH, USA) from which we monitored cumulative food intake, meal size, and several measures of energy expenditure (O 2 consumption, CO 2 production and respiratory exchange ratio (RER)) as previously described and validated (Farhang et al, 2010 ; Borgquist et al, 2015a ; Conde et al, 2017 ). These studies were conducted under conditions in which food (standard chow or HFD) and water were available ad libitum .…”

Opposite-sex effects of the Tac2 pathway blockade in fear memory consolidation: implications for fear-related disorders

“…All mice received osanetant 30 min after FA and were tested for memory recall 24 hours later to assess for differences in the effect of the drug between dominant and submissive mice. 50 uL of blood was collected from each animal by tail-nick two days before the rst habituation to the tube, 2 hours before the rst habituation session, 2 hours before the rst test and 2 hours before the rst habituation to the fear conditioning chamber, thus, with every two days since the rst extraction. Blood was centrifuged (8000 g, 15 min, 4ºC) and serum was stored at -20ºC.…”

“…The dose of intra-Central Amygdala (CeA) Androgen Receptor (AR) agonist CI-4AS-1 (Tocris, UK) was 100 nM based on previous research showing decreased depolarization-induced suppression of excitation in POMC neurons (50), dissolved in 1% DMSO (v/v). We used the AR agonist instead of testosterone in order to avoid its possible conversion to 17-b-Estradiol by aromatase.…”

Opposite-sex effects of the Tac2 pathway blockade in fear memory consolidation

“…For Fear Acquisition (FA) and Fear Expression (FE) test, a computerized Startle system was employed (Panlab-Harvard, Barcelona, Spain) as previously described (49). Delivery of tones and shocks was simultaneously controlled by Freezing v1.3.04 software (Panlab-Harvard, Barcelona, Spain).…”