Obesity in mice with adipocyte-specific deletion of clock component Arntl

Journal name:
Nature Medicine
Volume:
18,
Pages:
1768–1777
Year published:
DOI:
doi:10.1038/nm.2979
Received
Accepted
Published online

Abstract

Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

At a glance

Figures

  1. Adipocyte-specific deletion of Arntl disrupts molecular rhythms in clock and clock-output gene expression.
    Figure 1: Adipocyte-specific deletion of Arntl disrupts molecular rhythms in clock and clock-output gene expression.

    (a) Schematic diagram illustrating the region surrounding the basic helix-loop-helix (bHLH, purple) domain of the mouse Arntl locus, the targeted ArntlfxNeo allele, the conditional Arntlfx allele and the Arntlfx-excised allele. (b,c) PCR products amplified from adipocyte genomic DNA isolated from epididymal WAT (b) and interscapular BAT (c). 1, ubiquitous Ella-Cre+/− Arntlfx/+; 2, aP2-Cre+/− Arntlfx/fx; 3, Arntlfx/fx, no Cre; 4, Arntl+/+, no Cre. (d,e) mRNA levels of Arntl quantified in primary adipocytes isolated from epididymal WAT (d) or interscapular BAT (e) from WT, aP2-Cre+/− Arntlfx/fx (Ad-Arntl−/−), Arntlfx/fx, no Cre (control) or ubiquitous Ella-Cre+/− Arntlfx/fx (Ella-Cre) mice. Error bars, s.e.m. (f,g) Western blot analysis of Arntl in white (f) and brown (g) adipocytes. 1, aP2-Cre Arntl+/+; 2, Arntlfx/fx, no Cre; 3, aP2-Cre+/− Arntlfx/fx; 4, Arntl+/+, no Cre; 5, Arntl−/−; 6, negative control; 7, positive control. (h) PCR products amplified from genomic DNA isolated from different tissues of aP2-Cre+/− Arntlfx/fx mice. 1, WAT; 2, BAT; 3, liver; 4, skeletal muscle; 5, hypothalamus; 6, adrenals; 7, peritoneal macrophages. (i,j) Clock and clock-controlled gene expression in epididymal WAT (i) and subscapular BAT (j) from adipocyte-specific Arntl knockout mice (Ad-Arntl−/−) with ad libitum access to food (20-week-old mice on a regular diet; n = 4 for each time point and group). Relative expressions are normalized to Gapdh and plotted in arbitrary linear units (mean ± s.e.m.).

  2. Obesity in adipocyte-specific Arntl knockout (Ad-Arntl-/-) mice.
    Figure 2: Obesity in adipocyte-specific Arntl knockout (Ad-Arntl−/−) mice.

    (a) Body weights of adipocyte-specific Arntl knockout mice (aP2-Cre Arntlfx), littermate Arntlfx controls (No Cre Arntlfx), aP2-Cre controls (aP2-Cre), WT littermates of aP2-Cre controls (WT) and WT mice (WT C57BL/6J) (n = 12 per group). (b) Body weights of adipocyte-specific Arntl knockout mice (adiponectin-Cre Arntlfx), littermate Arntlfx controls (No Cre Arntlfx), adiponectin-Cre controls (adiponectin-Cre) and WT littermates of adiponectin-Cre controls (WT Aghouti) fed either regular chow (open symbols) (n = 6) or HFD starting at 6 weeks of age (n = 9). (c) Body composition of 32-week-old Ad-Arntl−/− and control mice fed a regular diet, and 16-week-old Ad-Arntl−/− and control mice fed HFD (n = 5 per group). *P < 0.05 by two-sample parametric t test. (d) Representative H&E stain images of epididymal WAT tissue isolated from 20-week-old weight-matched Ad-Arntl−/− and control mice on regular diet. Scale bars, 100 μm. (e) Adipocyte area size quantification for white adipocytes from epididymal WAT from weight-matched Ad-Arntl−/− and control mice (20-week-old mice fed a regular diet). *P < 0.05 by two-sample parametric t test. (f) Plasma concentrations of leptin, triglycerides and glucose at different time points of circadian time in 20-week-old mice kept under constant darkness with ad libitum access to a regular chow diet (n = 4 for each time point and group). *P < 0.05 by two-sample Mann-Whitney test. All data in the figure are the mean ± s.e.m.

  3. Attenuation of the diurnal rhythm in feeding activity and lower energy expenditure in adipocyte-specific Arntl knockout mice (Ad-Arntl-/-).
    Figure 3: Attenuation of the diurnal rhythm in feeding activity and lower energy expenditure in adipocyte-specific Arntl knockout mice (Ad-Arntl−/−).

    (af) Locomotor activity (a,b), O2 consumption rate (vO2) (c,d) and food intake (e,f) monitored over two consecutive 24-h cycles for 20-week-old mice kept in 12-h light, 12-h dark conditions and fed HFD for 1 week. Results are represented as the totals for the light and dark periods in b, d and f (n = 8 per group). *P < 0.05, **P < 0.01 by two-sample Mann-Whitney test. vO2 values are corrected for body weight, and correction for lean body mass produced similar results. All data in the figure are the mean ± s.e.m.

  4. Higher food intake during the light period leads to obesity.
    Figure 4: Higher food intake during the light period leads to obesity.

    (a) Body weight in adipocyte-specific 20-week-old Arntl knockout mice (Ad-Arntl−/−) fed the same amount of daily calories with access to food (i) only during the light period (L), (ii) only during the dark period (D) or (iii) ad libitum (AD) (n = 6 per group). Mice required an adjustment period to the feeding regimen, after which they were introduced to HFD (day 17). Body weight was monitored twice daily at the beginning of the light and dark phases. Results are the average of the two measurements (mean ± s.e.m.). (bf) Time-specific alterations in hypothalamic expression of appetite-regulating neuropeptides. mRNA levels are quantified in hypothalamic sections isolated from regular diet–fed 20-week-old mice kept in constant darkness and fasted for 24 h before tissue collection (n = 4 for each time point and group). Relative expressions are normalized to Gapdh and plotted in arbitrary linear units (mean ± s.e.m.). *P < 0.05 by two-sample Mann-Whitney test. Results are representative of three independent experiments. (g) Food intake in response to a fasting and re-feeding challenge monitored over 24 h for mice kept in 12-h light, 12-h dark conditions. Refeeding started at time 0 after a 24-h fast (n = 8 20-week-old mice fed regular diet per group). *P < 0.05 by two-sample Mann-Whitney test.

  5. Lower concentrations of polyunsaturated fatty acids in adipose tissue, plasma and hypothalamus of adipocyte-specific Arntl knockout mice (Ad-Arntl-/-).
    Figure 5: Lower concentrations of polyunsaturated fatty acids in adipose tissue, plasma and hypothalamus of adipocyte-specific Arntl knockout mice (Ad-Arntl−/−).

    (a,b) Relative concentration of triglycerides in WAT from regular diet–fed mice. (c) mRNA levels in WAT isolated from regular diet–fed mice. (d) Binding of Arntl to the promoters of Elovl6 and Scd1 as determined in epididymal adipose tissue by ChIP at zeitgeber time 10 (ZT10). Quantitative PCR–determined binding of DNA fragments of the Elovl6 and Scd1 promoters to Arntl expressed as a percentage of the detection of the same fragments in the input DNA used for ChIP. Regions close to the transcriptional starting site of the insulin and Arbp genes served as negative controls. (eg) Amounts of fatty acids in plasma from regular diet–fed mice. (h) mRNA levels in WAT isolated from regular diet–fed mice. (i) Hypothalamic amounts of arachidonic acid (AA), EPA and DHA in regular diet–fed mice. In all experiments, data are expressed as the mean ± s.e.m. Relative expression mRNA levels are normalized to Gapdh and plotted in arbitrary linear units. For ac and ei, experiments were performed on regular diet–fed 20-week-old mice kept in constant darkness and fasted for 24 h before tissue collection (n = 6 for each time point and group).

  6. Polyunsaturated fatty acid-rich diets restore hypothalamic polyunsaturated fatty acid content and correct body weight, feeding behavior, energy homeostasis and hypothalamic neuropeptide expression in adipocyte-specific Arntl knockout mice (Ad-Arntl-/-).
    Figure 6: Polyunsaturated fatty acid–rich diets restore hypothalamic polyunsaturated fatty acid content and correct body weight, feeding behavior, energy homeostasis and hypothalamic neuropeptide expression in adipocyte-specific Arntl knockout mice (Ad-Arntl−/−).

    (a) Hypothalamic amounts of nonesterified arachidonic acid (AA), EPA and DHA after 8 weeks on the indicated diet at CT4 and CT16 of 30-week-old Ad-Arntl−/− and control mice kept in constant darkness and fasted for 24 h before tissue collection. HFD, high-fat diet providing 43% of the total energy from fat; 2% PUFA HFD, diet providing 2% of the total energy from EPA (1.2%) and DHA (0.8%) and 43% from fat; 10% PUFA HFD, diet providing 10% of the total energy from EPA (6%) and DHA (4%) and 43% from fat; PUFA, polyunsaturated fatty acid. n = 6 for each group and diet throughout. (b) Body weights of Ad-Arntl−/− and control mice during the 8 weeks on the three diets. (c,d) Food intake (c) and vO2 (d) monitored over two consecutive 24-h cycles of mice on the three diets kept in 12-h light, 12-h dark conditions. (e) Hypothalamic expression of appetite-regulating neuropeptides after 8 weeks on the diet at CT4 and CT16. mRNA levels were quantified in hypothalamic sections isolated from mice kept in constant darkness and fasted for 24 h before tissue collection. Relative expressions are normalized to Gapdh and plotted in arbitrary linear units. All data in the figure are the mean ± s.e.m. NS, not significant, *P < 0.05, **P < 0.01 by two-sample Mann-Whitney test.

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Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Perelman School of Medicine, Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Georgios K Paschos,
    • Salam Ibrahim,
    • Wen-Liang Song,
    • Takeshige Kunieda,
    • Gregory Grant,
    • Teresa M Reyes,
    • John A Lawson &
    • Garret A FitzGerald
  2. McArdle Laboratory, Department of Oncology, University of Wisconsin, School of Medicine and Public Health, Madison, Wisconsin, USA.

    • Christopher A Bradfield
  3. Department of Biology, Center for Obesity Reversal, Georgia State University, Atlanta, Georgia, USA.

    • Cheryl H Vaughan
  4. Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Cambridge, UK.

    • Michael Eiden,
    • Mojgan Masoodi &
    • Julian L Griffin
  5. Perelman School of Medicine, Institute for Diabetes, Obesity and Metabolism, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Fenfen Wang

Contributions

G.K.P., S.I., W.-L.S., T.K., C.H.V. and F.W. contributed to the acquisition, analysis and interpretation of the data. G.K.P. and G.A.F. initiated and designed the study. G.G. performed statistical analyses and analysis of the microarray data. T.M.R. performed indirect calorimetric analyses. C.A.B. provided the mice with the conditional Arntl allele. M.E., M.M., J.L.G. and J.A.L. performed the liquid chromatography mass spectrometry (LC-MS) analysis. G.K.P. and G.A.F. wrote the paper.

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The authors declare no competing financial interests.

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