Adipocyte REVERBα dictates adipose tissue expansion during obesity

The circadian clock component REVERBα is considered a dominant regulator of lipid metabolism, with global Reverbα deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (ReverbαFlox2-6AdipoCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of REVERBα regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, ReverbαFlox2-6AdipoCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT REVERBα cistrome with differential gene expression reveals broad control of metabolic processes by REVERBα which is unmasked in the obese state. Adipocyte REVERBα does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, REVERBα action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.


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The mammalian circadian clock directs rhythms in behaviour and physiology to coordinate our 41 biology with predictable changes in food availability and daily alternations between fasted and 42 fed states. In this way, profound cycles in nutrient availability and internal energy state can be

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Extensive work over the past 20 years has demonstrated that circadian clock function and its REVERBα as a regulator responsive to the metabolic state of the tissue, rather than one which 94 delivers an anticipatory daily oscillation to the WAT metabolic programme.    A-E. Under light:dark conditions, Reverbα -/mice maintain robust diurnal rhythms in physiology and behaviour. Day/night food intake (A) (n=12-14/group), and energy expenditure (B) (n=3-4/group). Diurnal activity profile, mean activity (C) and body temperature (D) of adult male Reverbα -/mice (activity is reported as the % daily activity, n=9-13/group). Diurnal profiles in oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) (E) (n=9-13/group). F. Western blot showing REVERBα expression in adipose tissue over 24 hours. G. Molar percentages of fatty acid species in WT and Reverbα -/-gWAT. n=6/group. a P<0.05. H. Daytime (ZT6) fasted blood glucose levels (n=11/group). I. Reverbα -/mice are highly susceptible to diet-induced obesity, showing significantly higher body weights and fat mass than control mice after 10 weeks of HFD feeding (n=6/group). J. In a separate study, food intake was tracked for individual mice over 3 weeks of HFD feeding (n = 13/group). Mean daily food intake in Reverbα -/mice showed a significant positive correlation with body weight.
Data presented as individual data points with mean (A-D, H, I), as mean +/-SEM (F), or as individual data points with line of best fit (J). **P<0.01, two-way ANOVA with Tukey's multiple comparisons tests (A-D), unpaired ttests with correction for multiple testing (G), unpaired two-tailed t-tests (H,I), linear regression (J).

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In marked contrast to global Reverbα -/mice, adult Reverbα Flox2-6 Adipo Cre mice did not show an 149 increase in adiposity when maintained on a standard chow diet ( Figure 2D; n=7/group), with 150 no differences in mean body weight, fat and lean mass observed. In parallel with this, we saw (Cre -ve ) and Reverbα Flox2-6 Adipo Cre (Cre +ve ) mice (n=4-5/group). C. REVERBα protein expression (arrowhead) in Cre -ve and targeted Cre +ve mice. Lower blot shows Ponceau S protein staining. D. Body weight, fat mass and lean mass in 13-week old Cre -ve and Cre +ve male mice (n=7/group). E-G. Both Reverbα Flox2-6 Adipo Cre Cre +ve and Cre -ve mice demonstrate diurnal rhythms in behaviour and physiology, with no genotype differences observed in food intake (E), energy expenditure and daily activity (F) or body temperature (G) in 13-week old males (n=4-7/group).
Data presented as mean +/-SEM (B,) or as mean with individual data points (D-G). *P<0.05, **P<0.01, unpaired t-tests corrected for multiple comparisons (B), unpaired t-tests (D), two-way ANOVA with Tukey's multiple comparisons tests (E-G). Cre +ve mice, when compared to control Cre -ve littermate controls (A; n=5-6/group), despite showing increased UCP1 expression (B; n=3/group). C. No intergenotype genotype differences were observed in body temperature profiles recorded from mice housed under thermoneutral conditions (28-30 ∘ C) for 3 weeks (n=5-6/group). D-E. Brown adipose tissue (BAT) gene expression studies (qPCR) demonstrated expected de-repression of Bmal1 expression in Cre +ve mice, and expected reduction in Ucp1 expression at thermoneutral conditions in both genotypes (compared to room temperature) (n=5-6/group). F. Reverbα Flox2-6 Adipo Cre Cre +ve mice and control littermates were exposed to an acute cold challenge (4 ∘ C for 6 h) with body temperature recording throughout (n=5-6/group). No genotype difference in thermogenic response was observed. G-H. As previously reported, Reverbα expression was reduced by cold exposure; however, no genotype differences were observed cold-induced increases in Ucp1 or Dio2 gene expression (n=5-6/group).

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As brown adipose tissue (BAT) makes an important contribution to whole body energy 155 metabolism, we studied the thermoregulation of Reverbα Flox2-6 Adipo Cre mice in greater detail.

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It has previously been proposed that REVERBα is key in conferring circadian control over

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We saw no differences in adipocyte size between the two genotypes, indicating that our     (Figure 6A(iii,iv)). We 318 observed a highly significant proximity enrichment of these commonly up-regulated genes (863) to sites of REVERBα chromatin binding (Figure 6A(iii)), but again, saw no enrichment 320 of the commonly down-regulated genes (Figure 6A(iv)). This integration of transcriptome and

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We set out to define the role of REVERBα in the regulation of white adipose tissue metabolism,

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The reduced inflammation seen in obese Reverbα Flox2-6 Adipo Cre mice is likely multifactorial, but 363 may be secondary to a reduction in the pro-inflammatory free fatty acid pool, resulting from

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FAMEs were identified by their retention times compared to a standard containing 31 known 515 fatty acids and quantified in micromolar from the peak area based on their molecular weight.

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The micromolar quantities were then totalled and each fatty acid was expressed as a 517 percentage of this value (molar percentage; mol%).

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Proteomics. Mice were culled by cervical dislocation and the gWAT was immediately removed        indicating +/-SEM. In these tests, significance is defined as *P<0.05 or **P<0.01 (P values 670 below 0.01 were not categorised separately, i.e. no more than two stars were used, as we 671 deemed this to be a meaningful significance cut-off). Statistical analyses of proteomics, RNA-672 seq and ChIP-seq data were carried out as described above in Methods, using the significance 673 cut-offs mentioned. Plots were produced using GraphPad Prism or R package ggplot2.