Orexin activation counteracts decreases in nonexercise activity thermogenesis (NEAT) caused by high-fat diet

Abstract

Overweight and obesity result from an imbalance between caloric intake and energy expenditure, including expenditure from spontaneous physical activity (SPA). Changes in SPA and resulting changes in non-exercise activity thermogenesis (NEAT) likely interact with diet to influence risk for obesity. However, previous research on the relationship between diet, physical activity, and energy expenditure has been mixed. The neuropeptide orexin is a driver of SPA, and orexin neuron activity can be manipulated using DREADDs (Designer Receptors Exclusively Activated by Designer Drugs). We hypothesized that HFD decreases SPA and NEAT, and that DREADD-mediated activation of orexin neuron signaling would abolish this decrease and produce an increase in NEAT instead. To test these ideas, we characterized behaviors to determine the extent to which access to a high-fat diet (HFD) influences the proportion and probability of engaging in food intake and activity. We then measured NEAT following access to HFD and following a DREADD intervention targeting orexin neurons. Two cohorts of orexin-cre male mice were injected with an excitatory DREADD virus into the caudal hypothalamus, where orexin neurons are concentrated. Mice were then housed in continuous metabolic phenotyping cages (Sable Promethion). Food intake, indirect calorimetry, and SPA were automatically measured every second. For cohort 1 (n=8), animals were given access to chow, then switched to HFD. For cohort 2 (n=4/group), half of the animals were given access to HFD, the other access to chow. Then, among animals on HFD, orexin neurons were activated following injections of clozapine n-oxide (CNO). Mice on HFD spent significantly less time eating (p<0.01) and more time inactive compared to mice on chow (p<0.01). Following a meal, mice on HFD were significantly more likely to engage in periods of inactivity compared to those on chow (p<0.05). NEAT was decreased in animals on HFD, and was increased to the NEAT level of control animals following activation of orexin neurons with DREADDs. Food intake (kilocalories) was not significantly different between mice on chow and HFD, yet mice on chow expended more energy per unit of SPA, relative to that in mice consuming HFD. These results suggest that HFD consumption reduces SPA and NEAT, and increases inactivity following a meal. Together, the data suggest a change in the efficiency of energy expenditure based upon diet, such that SPA during HFD burns fewer calories compared to SPA on a standard chow diet.

Keywords: Designer receptors exclusively activated by designer drugs (DREADDs); High-fat diet; Non-exercise activity thermogenesis (NEAT); Orexin; Spontaneous physical activity (SPA).

Copyright © 2017 Elsevier Inc. All rights reserved.

Figures

Figure 1

Cre-dependent genetic targeting of orexin neurons. (A) Schematic diagram of AAV vector encoding DREADD-mCherry driven by Elongation factor alpha (EFa1) promoter sequence and flanked by dual flox sites for recombination in the presence of Cre-recombinase. Cre is driven by the prepro-orexin-promoter of Orexin:Cre mice. (B) Schematic of anatomical targeting with stereotactic viral infusion. Photomicrographs of coronal sections containing immunofluorescent orexin neurons (green) and hM3Dq-mCherry (red; C). Percent of orexin neurons expressing mCherry (orange arrow head) in Cre::hM3Dq (D) and Wt::hM3Dq (E) mice.

Figure 2

Experimental timeline. Cohort 1 was injected with the DREADD virus and then allowed 2 weeks to recover. All animals (n = 8) were then placed on chow for 10 days, and then switched to high-fat diet (HFD) for another 10 days. Cohort 2 was injected with the DREADD virus and then allowed 2 weeks to recover. Half of the animals (n = 4) were fed chow and the other half fed HFD (n = 8) within the Sable cages for 10 days. The animals on HFD (n = 4) were then injected with CNO daily, for 10 days.

Figure 3

Mean +/− time spent engaging in food intake (panel A), short bouts of inactivity (panel B), and long bouts of inactivity (panel C) expressed as a percentage of total time in a 24 h period, averaged across all 10 days of access to chow and all 10 days of access to high-fat diet (HFD; n = 8). **p

Figure 4

Mean +/− SEM probability, expressed as a percentage of total time in a 24 h period, that a mouse would engage in long (panel A) and short (panel B) bouts of inactivity following a bout of food intake, averaged across all 10 days of access to chow and all 10 days of access to high=fat diet (HFD). **p

Figure 5

Mean +/− SEM time spent engaging in food intake (panel A) or inactivity (panel B) in animals with access to high-fat diet (HFD; n=4) or chow (n=4),, expressed as a percentage of total time in a 24 h period, for 10 total days. Also shown is the time spent engaging in food intake or inactivity for animals on high-fat diet (n=4) during the subsequent 10 days of CNO injections. **p

Figure 6

Mean +/− SEM energy expenditure of SPA every 30 minutes averaged across 10 days (panel A) and the total cumulative energy expenditure of SPA over 23 hours following injections of CNO (injections took place at time ‘0’) averaged across 10 days (panel B). Energy expenditure of SPA was defined as total energy expenditure every 30 minutes minus the energy expenditure during the 30 minutes of least activity for each animal, divided by that animal’s lean mass, for animals (n =8) on chow, high-fat diet (HFD), and HFD plus CNO. @@p

Figure 7

Mean +/− SEM ratio of NEAT to SPA, presented as the mean across all 10 days of testing, for animals on high-fat diet (HFD; n = 4), chow (n = 4), and HFD+CNO (n = 4). The ratio was calculated as the mean total NEAT (total energy expenditure of SPA / lean mass) divided by the mean total SPA (meters traveled) for each animal across 10 days of testing. ##p

Figure 8

Mean +/− SEM meters traveled per second for each animal, averaged across all 10 days of access to chow or HFD, and across all 10 days of CNO injections. **p