Sympathetic innervation of interscapular brown adipose tissue is not a predominant mediator of oxytocin-elicited reductions of body weight and adiposity in male diet-induced obese mice

Abstract

Previous studies indicate that CNS administration of oxytocin (OT) reduces body weight in high fat diet-induced obese (DIO) rodents by reducing food intake and increasing energy expenditure (EE). We recently demonstrated that hindbrain (fourth ventricular [4V]) administration of OT elicits weight loss and elevates interscapular brown adipose tissue temperature (T_IBAT, a surrogate measure of increased EE) in DIO mice. What remains unclear is whether OT-elicited weight loss requires increased sympathetic nervous system (SNS) outflow to IBAT. We hypothesized that OT-induced stimulation of SNS outflow to IBAT contributes to its ability to activate BAT and elicit weight loss in DIO mice. To test this hypothesis, we determined the effect of disrupting SNS activation of IBAT on the ability of 4V OT administration to increase T_IBAT and elicit weight loss in DIO mice. We first determined whether bilateral surgical SNS denervation to IBAT was successful as noted by ≥ 60% reduction in IBAT norepinephrine (NE) content in DIO mice. NE content was selectively reduced in IBAT at 1-, 6- and 7-weeks post-denervation by 95.9 ± 2.0, 77.4 ± 12.7 and 93.6 ± 4.6% (P<0.05), respectively and was unchanged in inguinal white adipose tissue, pancreas or liver. We subsequently measured the effects of acute 4V OT (1, 5 µg ≈ 0.99, 4.96 nmol) on T_IBAT in DIO mice following sham or bilateral surgical SNS denervation to IBAT. We found that the high dose of 4V OT (5 µg ≈ 4.96 nmol) elevated T_IBAT similarly in sham mice as in denervated mice. We subsequently measured the effects of chronic 4V OT (16 nmol/day over 29 days) or vehicle infusions on body weight, adiposity and food intake in DIO mice following sham or bilateral surgical denervation of IBAT. Chronic 4V OT reduced body weight by 5.7 ± 2.23% and 6.6 ± 1.4% in sham and denervated mice (P<0.05), respectively, and this effect was similar between groups (P=NS). OT produced corresponding reductions in whole body fat mass (P<0.05). Together, these findings support the hypothesis that sympathetic innervation of IBAT is not necessary for OT-elicited increases in BAT thermogenesis and reductions of body weight and adiposity in male DIO mice.

Keywords: brown adipose tissue (BAT); food intake; obesity; oxytocin; white adipose tissue (WAT).

Figure 1

Effect of IBAT surgical denervation procedure on IBAT NE content at 1-week post-sham or IBAT denervation in lean mice. Mice were maintained on chow for approximately 2.5 months prior to undergoing a sham (N=5/group) or bilateral surgical IBAT denervation (N=5/group) procedures and were euthanized at 1-week post-sham or denervation procedure. NE content was measured from IBAT, IWAT and liver. Data are expressed as mean ± SEM. *P<0.05 denervation vs sham.

Figure 2

Effect of IBAT surgical denervation procedure on IBAT NE content at 1-, 6- and 7-weeks post-sham or IBAT denervation in DIO mice. Mice were maintained on a HFD (60% kcal from fat) for approximately 4.25 months prior to undergoing sham (N=5/group) or bilateral surgical IBAT denervation (N=5/group) procedures and were euthanized at (A) 1-, (B) 6- and (C) 7-weeks post-sham (N=15/group) or denervation procedures (N=15/group). NE content was measured from IBAT, IWAT, liver and pancreas. Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 denervation vs sham.

Figure 3

Effect of diet-induced obesity (DIO) on SNS Innervation (NE content) of IBAT, IWAT, EWAT, liver and pancreas at 1-week post-sham in age-matched lean and DIO mice. Age-matched mice were maintained on either chow (16% kcal from fat) or HFD (60% kcal from fat) for approximately 4.5 months prior to undergoing a sham denervation (N=5/group) procedure and were euthanized at 1-week post-sham (N=5/group). NE content was measured from IBAT, IWAT, EWAT, liver and pancreas. Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 lean vs DIO.

Figure 4

(A–F) Effect of systemic beta-3 receptor agonist (CL 316243) administration (0.1 and 1 mg/kg) on IBAT temperature (T_IBAT), energy intake and body weight post-sham or IBAT denervation in DIO mice. Mice were maintained on HFD (60% kcal from fat; N=9-10/group) for approximately 4.5 months prior to undergoing a sham or bilateral surgical IBAT denervation and implantation of temperature transponders underneath IBAT. Animals were subsequently adapted to a 4-h fast prior to receiving IP injections of (CL 316243) (0.1 or 1 mg/kg, IP) or vehicle (sterile water) where each animal received each treatment at approximately 7-day intervals. (A, C), Effect of CL 316243 on T_IBAT in (A) sham operated or (C) IBAT denervated DIO mice; (B, D), Effect of CL 316243 on change in T_IBAT relative to baseline T_IBAT (delta T_IBAT) in (B) sham operated or (D) IBAT denervated DIO mice; (E), Effect of CL 316243 on change in energy intake in sham or IBAT denervated DIO mice; (F), Effect of CL 316243 on change in body weight in sham or IBAT denervated DIO mice. Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 CL 316243 vs. vehicle.

Figure 5

(A–D) Effect of acute 4V OT administration (1 and 5 μg) on T_IBAT post-sham or IBAT denervation in male DIO mice. Mice were maintained on HFD (60% kcal from fat; N=11-13/group) for approximately 4.25 months prior to undergoing a sham or bilateral surgical IBAT denervation and implantation of temperature transponders underneath IBAT. Mice were subsequently implanted with 4V cannulas and allowed to recover for 2 weeks prior to receiving acute 4V injections of OT or vehicle. Animals were subsequently adapted to a 4-h fast prior to receiving acute 4V injections of OT or vehicle (A, C), Effect of acute 4V OT on T_IBAT in (A) sham operated or (C) IBAT denervated DIO mice; (B, D), Effect of acute 4V OT on change in T_IBAT relative to baseline T_IBAT (delta T_IBAT) in (B) sham operated or (D) IBAT denervated DIO mice; Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 OT vs. vehicle. The T_IBAT data from sham-operated DIO mice has been previously published (22).

Figure 6

(A–D) Effect of chronic 4V OT infusions (16 nmol/day) on body weight, adiposity and energy intake post-sham or IBAT denervation in male DIO mice. (A), Mice were maintained on HFD (60% kcal from fat; N=7-9/group) for approximately 4.25-4.5 months prior to undergoing a sham or bilateral surgical IBAT denervation. Mice were subsequently implanted with 4V cannulas and allowed to recover for 2 weeks prior to being implanted with subcutaneous minipumps that were subsequently attached to the 4V cannula. A, Effect of chronic 4V OT or vehicle on body weight in sham operated or IBAT denervated DIO mice; (B), Effect of chronic 4V OT or vehicle on body weight change in sham operated or IBAT denervated DIO mice; (C), Effect of chronic 4V OT or vehicle on adiposity in sham operated or IBAT denervated DIO mice; (D), Effect of chronic 4V OT or vehicle on adiposity in sham operated or IBAT denervated DIO mice. Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 OT vs. vehicle.

Figure 7

(A, B) Effect of chronic 4V OT infusions (16 nmol/day) on adipocyte size post-sham or IBAT denervation in male DIO mice. (A), Adipocyte size (pixel²) was measured in IWAT from mice that received chronic 4V infusion of OT (16 nmol/day) or vehicle (VEH) in sham or IBAT denervated DIO mice (N=9-10/group). (B), Adipocyte size was measured in EWAT from mice that received chronic 4V infusion of OT (16 nmol/day) or vehicle in sham operated or IBAT denervated mice (N=9-10/group). Data are expressed as mean ± SEM. *P<0.05 OT vs. vehicle.

Figure 8

(A–D) Effect of chronic subcutaneous OT infusions (16 and 50 nmol/day) on body weight, adiposity and energy intake in male DIO mice. (A), Mice were maintained on HFD (60% kcal from fat; N=11-14/group) for approximately 4-4.25 months prior to being implanted with temperature transponders and allowed to recover for 1-2 weeks prior to being implanted with subcutaneous minipumps. (A), Effect of chronic subcutaneous OT or vehicle on body weight in DIO mice; (B), Effect of chronic subcutaneous OT or vehicle on body weight change in DIO mice; (C), Effect of chronic subcutaneous OT or vehicle on adiposity in DIO mice; (D), Effect of chronic subcutaneous OT or vehicle on adiposity in DIO mice. Data are expressed as mean ± SEM. *P<0.05, †0.05<P<0.1 OT vs. vehicle.