Time-restricted feeding mitigates obesity through adipocyte thermogenesis

Abstract

Misalignment of feeding rhythms with the light-dark cycle leads to disrupted peripheral circadian clocks and obesity. Conversely, restricting feeding to the active period mitigates metabolic syndrome through mechanisms that remain unknown. We found that genetic enhancement of adipocyte thermogenesis through ablation of the zinc finger protein 423 (ZFP423) attenuated obesity caused by consumption of a high-fat diet during the inactive (light) period by increasing futile creatine cycling in mice. Circadian control of adipocyte creatine metabolism underlies the timing of diet-induced thermogenesis, and enhancement of adipocyte circadian rhythms through overexpression of the clock activator brain and muscle Arnt-like protein-1 (BMAL1) ameliorated metabolic complications during diet-induced obesity. These findings uncover rhythmic creatine-mediated thermogenesis as an essential mechanism that drives metabolic benefits during time-restricted feeding.

Figure 1.. Circadian mistiming of feeding promotes obesity through reducing energy expenditure.

(A) Experimental design depicting the timing of high fat diet (HFD) access as Ad libitum, restricted to the light period (ZT0–12; “Light-restricted”) or restricted to the dark period (ZT12–24; “Dark-restricted”) for one week in wildtype male mice at thermoneutrality (30°C). (B) Body weight of mice fed ad libitum, light-restricted, or dark-restricted HFD at day 0 and 7 of HFD (n=5). (C) Average daily food intake of mice, average feeding distribution for ad libitum HFD fed mice from days 5–7 of HFD, and digestive efficiency at days 6–7 of HFD (n=5). (D-F) RER rhythms (D), activity rhythms and average total activity over 12 hours (E), and VO₂ rhythms and average VO₂ levels over 12 hours (F) during days 5–7 of HFD (n=5). (G) Model of adipocyte-specific deletion of ZFP423 to drive thermogenic programming in white adipocytes. (H) Body weight during isocaloric light-restricted or dark-restricted HFD for 10 weeks in control (Adiponectm-rtTA;Zfp423^flox//lox) and Zfp423-KO (Adiponectin-rtTA;TRE-Cre;Zfp423^flox//lox) male mice (n=5–6). (I) Glucose tolerance test (GTT), area under curve (AUC) during the GTT, and insulin during the GTT at ZT2 at 10 weeks of HFD (n=5–6). Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2.. Genetic disinhibition of adipocyte thermogenesis enhances glycolytic flux and creatine metabolism.

(A) Oxygen consumption rate of adipocytes differentiated from inguinal WAT stromal vascular cells from control and adipocyte Zfp423-KO mice. After 3 basal recordings, 100 nM norepinephrine was added onto the cells (n=5). (B) Labeling schematic of U-¹³C-glucose tracing into glycolysis and the TCA cycle. Isotopic labeling profile of m+6 glucose, m+6 glucose-6-phosphate (G6P), m+3 pyruvate, and m+3 lactate following addition of U-¹³C-glucose in the presence or absence of 100 nM norepinephrine for 5 hours in differentiated adipocytes from control and adipocyte Zfp423-KO mice (n=6). (C) Heatmap of differentially abundant metabolites (p<0.05) in differentiated adipocytes (n=6). (D) Relative abundance of ATP, ADP, creatine, and phosphocreatine in differentiated adipocytes (n=6). (E) Phosphocreatine to creatine (PCr/Cr) ratio in differentiated adipocytes (left) from control and Zfp423-KO mice and iWAT (right) from 3-month-old male control and adipocyte Zfp423-KO mice after 4 weeks of dox-chow harvested at ZT2 (light period) and ZT14 (dark period) (n=4). Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3.. Rhythmic chromatin profiling in adipocytes reveals distinct phases of accessibility in BAT and iWAT.

(A) Following Cre expression, Adiponectin-Cre;NuTRAP^fc/+ mice express nuclear membrane labeled with mCherry and biotin and labeling of the translating mRNA polysome complex with EGFP-fused ribosomal protein L10a. Adipocyte nuclei from BAT and iWAT of male Adiponectin-Cre;NuTRAP^fc/+ mice housed at thermoneutrality were isolated every 4 hours throughout the 24-hour day (ZT1, 5, 9, 13, 17, 21) by FACS for ATAC-seq analysis. (B) Heatmaps showing rhythmic activation of adipocyte chromatin in BAT and WAT (n=3 per timepoint) and radial histograms showing the phases of maximal accessibility for rhythmic peaks within each 2-hr window. (C) Overlap of genes near oscillating peaks in BAT and WAT. (D) Rhythmic opening of chromatin at the circadian Cryl and thermogenic Ucpl enhancer with location of BMAL1 motifs. (E) Motif enrichment at oscillating peaks separated by phase of maximal accessibility in BAT and WAT throughout the day.

Figure 4.. Ribosomal RNA profiling reveals diurnal control of adipocyte metabolism.

(A) Adipocyte ribosomal RNA for RNA-sequencing was isolated from BAT and iWAT harvested every 4 hours throughout the 24-hour day (ZT1, 5, 9, 13, 17, 21) from male Adiponectin-Cre;NuTRAP^fx/+ mice housed at thermoneutrality. (B) Heatmaps showing rhythmic adipocyte RNA expression in BAT and iWAT (n=3 per timepoint) and radial histograms showing the number of genes whose oscillations peak within each 2-hr window in BAT and iWAT. (C) KEGG pathway analysis of oscillating genes in BAT from ZT7–19 and ZT19–7 and in WAT from ZT3–15 and ZT15–3. (D) Overlap of rhythmic genes identified through RNA-seq and genes near oscillating ATAC-seq peaks. (E) Examples of rhythmic gene expression (shown in transcripts per million “TPM”) identified through RNA-seq in BAT and iWAT (n=3 per timepoint).

Figure 5.. The adipocyte clock regulates metabolic health through creatine metabolism.

(A) Body weight of control (Bmal^flox/flox) and Bmal1-KO (Adiponectin-Cre;Bmal1^flox/flox) male mice fed ad libitum HFD supplemented with 2% creatine for 6 weeks at thermoneutrality (n=5) with average daily food intake and % of feeding during the light period from weeks 5–6 of HFD. (B-C) Average daily activity (B) detected by infrared sensors during week 5–6 of HFD feeding and relative metabolite abundance (C) in iWAT after 6 weeks of HFD (n=5). (D) Experimental design depicting mice with doxycycline-inducible transgenic expression of the clock activator Bmal1 in adipocytes have enhanced amplitude of core clock expression. (E) Body weight of control (Adiponectin-rtTA) and Bmal1-Tg (Adiponectin-rtTA;TRE-Bmal1) mice during 6 weeks of ad libitum HFD feeding at thermoneutrality (n=8). (F) Body composition and adipose tissue weights after 6 weeks of HFD (n=8). (G) VO₂ rhythms and average VO₂ levels from weeks 5–6 of HFD (n=8). (H) Glucose tolerance test, AUC, and insulin during the GTT at ZT2 at 6 weeks of HFD (n=8). (I- J) Expression of circadian and creatine metabolism genes (I) and the PCr/Cr ratio (J) in iWAT after 6 weeks of HFD (n=8). Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).