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. 2016 Jun 1;310(11):E900-11.
doi: 10.1152/ajpendo.00328.2015. Epub 2016 Apr 5.

Effect of Peripheral Circadian Dysfunction on Metabolic Disease in Response to a Diabetogenic Diet

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Free PMC article

Effect of Peripheral Circadian Dysfunction on Metabolic Disease in Response to a Diabetogenic Diet

Sonja S Pijut et al. Am J Physiol Endocrinol Metab. .
Free PMC article

Abstract

BMAL1 is a core component of the transcription/translation machinery that regulates central and peripheral circadian rhythms that coordinate behavior and metabolism, respectively. Our objective was to determine the impact of BMAL1 in adipose alone or in combination with liver on metabolic phenotypes. Control, adipose-Bmal1 knockout (ABKO), and liver- and adipose-Bmal1 knockout (LABKO) female mice were placed in TSE System metabolic chambers for metabolic phenotyping. A second cohort of male mice was fed a control or diabetogenic diet, and body weight and composition, glucose tolerance, insulin sensitivity, and serum and hepatic lipids were measured. Both female ABKO and LABKO mice exhibited increased food consumption compared with control mice. ABKO mice also exhibited increased overall activity predominantly during the light phase compared with both control and LABKO mice and were protected from increased weight gain. When the male cohort was challenged with a diabetogenic diet, LABKO mice had increased body weight due to increased fat mass compared with control and ABKO mice. However, these mice did not present further impairments in glycemic control, adipose inflammation, or liver injury. LABKO mice had increased hepatic cholesterol and elevated expression of cholesterol synthesis and uptake genes. Our data indicate that deletion of this allele in adipose or in combination with liver alters feeding behavior and locomotor activity. However, obesity is exacerbated only with the combination of liver and adipose deletion.

Keywords: brain and muscle arnt-like protein-1; cholesterol; circadian; insulin resistance; obesity.

Figures

Fig. 1.
Fig. 1.
Deletion of Bmal1 in white adipose tissue (WAT) and liver of 13- to 24-wk-old female mice on standard rodent chow. Hour 0 represents the beginning of lights-on; n = 3–9/group. A: Bmal1 expression in WAT, genotype F(2, 49) = 18.23, P < 0.0001; time F(5, 49) = 29.76, P < 0.0001; interaction F(10, 49) = 6.604, P < 0.0001, and in liver, genotype F(2, 55) = 8.709, P = 0.0005; time F(5, 55) = 22.75, P < 0.0001; interaction F(10, 55) = 1.860, P = 0.0714. B: Western blot and densitometry analyses of BMAL1 in WAT of mice negative or positive for the aP2-Cre transgene; T = 4.944, DF = 6, and P = 0.0026, and in liver of mice injected with Ad-empty or Ad-Cre virus T = 7.732, DF = 6, and P = 0.0002 at hour 0. C: Dbp expression in WAT, genotype F(2, 49) = 2.481, P = 0.0941; time F(5, 49) = 6.358, P = 0.0001; interaction F(10, 49) = 2.223, P = 0.0317, and in liver, genotype F(2, 55) = 0.9127, P = 0.4075; time F(5, 55) = 11.35, P < 0.0001; interaction F(10, 55) = 1.874, P = 0.0695. *P < 0.05, comparison between control and adipose-Bmal1 knockout (ABKO); #P < 0.05, comparison between control and liver- and adipose-Bmal1 knockout (LABKO); +P < 0.05, comparison between ABKO and LABKO for A and C; ** and ##P < 0.01; *** and ###P < 0.001, **** and ####P < 0.0001.
Fig. 2.
Fig. 2.
Food intake, physical activity, and energy expenditure changes in 12-wk-old female mice on standard rodent chow. Hour 0 represents the beginning of lights-on; n = 7–9/group. A: food intake over 24 h. Genotype: F(2, 3,842) = 10.28, P < 0.0001; time: F(47, 3,842) = 3.594, P < 0.0001; interaction: F(94, 3,842) = 1.361, P = 0.0124. B: food intake during the light phase, F(2, 20) = 9.791, P = 0.001; dark phase, F(2, 20) = 5.956, P = 0.0093; both phases, F(2, 20) = 16.65, P < 0.0001. C: locomotor activity defined as the sum of ambulatory (break of 2 adjacent beams in succession) and fine (break of the same beam twice) activity over 24 h. Genotype: F(2, 3,888) = 54.14, P < 0.0001; time: F(47, 3,888) = 16.03, P < 0.0001; interaction: F(94, 3,888) = 1.741, P < 0.0001. D: locomotor activity during the light phase, F(2, 20) = 7.541, P = 0.0036; dark phase, F(2, 20) = 3.555, P = 0.0477; both phases, F(2, 20) = 5.853, P = 0.01. E: resting energy expenditure relative to lean mass; slope P = 0.458, intercept P = 0.171. F: respiratory exchange ratio, F(2, 20) = 25.67, P < 0.0001. Effects of genotype are indicated by horizontal lines terminating in vertical lines. *P < 0.05, comparison between control and ABKO; #P < 0.05, comparison between control and LABKO; +P < 0.05, comparison between ABKO and LABKO in A and C; **, ##, and ++P < 0.01; ***, ###, and +++P < 0.001; **** and ++++P < 0.0001.
Fig. 3.
Fig. 3.
Body weight, body composition, and blood glucose of 13- to 24-wk-old female mice fed a standard rodent chow; n = 7–9/group. A: body weight, F(2, 20) = 37.99, P < 0.0001. B: lean mass, F(2, 20) = 10.63, P = 0.0007. C: fat mass, F(2, 20) = 124.9, P < 0.0001. D: %adiposity, F(2, 20) = 106.3, P < 0.0001. E: blood glucose measured beginning at circadian time 0. Genotype: F(2, 74) = 10.72, P < 0.0001; time: F(5, 370) = 65.51, P < 0.0001; interaction: F(10, 370) = 1.058, P = 0.3938 (n = 17–31/group). Effects of genotype are indicated by horizontal lines terminating in vertical lines. *P < 0.05, comparison between control and ABKO; +P < 0.05, comparison between ABKO and LABKO in E; ***P < 0.001; ****P < 0.0001.
Fig. 4.
Fig. 4.
Deletion of Bmal1 in WAT and liver of 24-wk-old male mice on control diet (CD) or diabetogenic diet (DD) for 16 wk. A: Bmal1 expression at circadian time 0 in WAT. Genotype: F(2, 39) = 8.433, P = 0.0009; diet: F(1, 39) = 9.94, P = 0.0031; interaction: F(2, 39) = 4.676, P = 0.0151 (n = 4–11/group). B: Western blot analysis of BMAL1 in WAT of control mice on CD or DD. C: Bmal1 expression at circadian time 0 in liver. Genotype: F(2, 37) = 8.64, P = 0.0008; diet: F(1, 37) = 2.305, P = 0.1374; interaction: F(2, 37) = 2.611, P = 0.0869 (n = 5–9/group). Effects of genotype are indicated by horizontal lines terminating in vertical lines. Effects of diet are indicated by brackets terminating in horizontal lines. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 5.
Fig. 5.
Body weight and composition of male mice fed a control diet (CD) or a diabetogenic diet (DD) for 16 wk; n = 5–18/group. A: body weight prior to initiation of diets at week 8; F(2, 59) = 8.981, P = 0.0004. B: body weight at the end of study at week 24. Genotype: F(2, 56) = 15.12, P < 0.0001; diet: F(1, 56) = 204.1, P < 0.0001; interaction: F(2, 56) = 2.003, P = 0.1444. C: change in body weight from week 8 to week 24. Genotype: F(2, 56) = 9.023, P = 0.0004; diet: F(1, 56) = 228.7, P < 0.0001; interaction: F(2, 56) = 2.032, P = 0.1407. D: %adiposity at week 24. Genotype: F(2, 56) = 8.202, P = 0.0008; diet: F(1, 56) = 95.17, P < 0.0001; interaction: F(2, 56) = 3.521, P = 0.0363. Effects of genotype are indicated by horizontal lines terminating in vertical lines. Effects of diet are indicated by brackets terminating in horizontal lines. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 6.
Fig. 6.
Adipose histology, quantification, and measurement of adipogenesis and lipolysis in WAT at circadian time 0 in 24-week-old male mice fed a control diet (CD) or a diabetogenic diet (DD) for 16 wk. A: representative images of hematoxylin and eosin (H & E) stain from epididymal adipose. B: adipocyte area. Genotype: F(2, 14) = 1.392, P = 0.281; diet: F(1, 14) = 6.985, P = 0.0193; interaction: F(2, 14) = 1.599, P = 0.2369 (n = 2–4/group). C: histogram of adipocyte count distribution of specified adipocyte area. D: gene expression of adipogenic and lipolytic genes. For C/ebpβ, F(2, 20) = 5.893, P = 0.0097; n = 5–9/group. Effects of genotype are indicated by horizontal lines terminating in vertical lines. Effects of diet are indicated by brackets terminating in horizontal lines. *P < 0.05; **P < 0.01.
Fig. 7.
Fig. 7.
Glycemic control in 24-wk-old male mice fed a control diet (CD) or a diabetogenic diet (DD) for 16 wk; n = 5–18/group. A: fasting blood glucose concentrations following a 4-h fast. Genotype: F(2, 56) = 1.956, P = 0.1509; diet: F(1, 56) = 8.497, P = 0.0051; interaction: F(2, 56) = 0.05681, P = 0.9448. B: glucose tolerance following a 4-h fast in mice fed CD, genotype F(2, 28) = 0.8944, P = 0.4202, time F(4, 112) = 75.99, P < 0.0001, interaction F(8, 112) = 1.436, P = 0.1894; and in mice fed DD, genotype F(2, 28) = 1.29, P = 0.291, time F(4, 112) = 80.27, P < 0.0001, interaction F(8, 112) = 1.909, P = 0.0654. C: area under the curve (AUC) calculated from glucose tolerance test. Genotype: F(2, 56) = 0.3693, P = 0.6929; diet: F(1, 56) = 18.25, P < 0.0001; interaction: F(2, 56) = 1.989, P = 0.1464. D: insulin sensitivity following a 1-h fast in mice fed DD. Genotype: F(2, 28) = 7.759, P = 0.0021; time: F(4, 112) = 33.28, P < 0.0001; interaction: F(8, 112) = 3.42, P = 0.0015. E: AUC calculated from insulin sensitivity test. F(2, 28) = 7.768, P = 0.0021. F: Western blot and densitometry analyses of total and phosphorlated Akt (p-Akt) in WAT of mice on DD injected with insulin, F(2, 6) = 3.495, P = 0.0985. Effects of genotype are indicated by horizontal lines terminating in vertical lines. Effects of diet are indicated by brackets terminating in horizontal lines. *P < 0.05, comparison between control and ABKO; #P < 0.05, comparison between control and LABKO; +P < 0.05, comparison between ABKO and LABKO in B and D; **P < 0.01; ****P < 0.0001.
Fig. 8.
Fig. 8.
Liver histology, hepatic lipids, and gene expression from liver isolated at circadian time 0 in 24-wk-old male mice fed a control diet (CD) or a diabetogenic diet (DD) for 16 wk. A: representative images of H & E stain. B: total cholesterol. Genotype: F(2, 26) = 5.095, P = 0.0136; diet: F(1, 26) = 25.42, P < 0.0001; interaction: F(2, 26) = 0.9054, P = 0.4167. C: triglycerides. Genotype: F(2, 26) = 2.519, P = 0.1000; diet: F(1, 26) = 44.3, P < 0.0001; interaction: F(2, 26) = 0.3547, P = 0.7047 (n = 4–7/group). D: gene expression of Hmgcr. Genotype: F(2, 39) = 6.118, P = 0.0049; diet: F(1, 39) = 1.268, P = 0.267; interaction: F(2, 39) = 3.374, P = 0.0445. E: gene expression of Hmgcs. Genotype: F(2, 39) = 3.582, P = 0.0373; diet F(1, 39) = 12.54, P = 0.001; interaction: F(2, 39) = 2.471, P = 0.0976. F: gene expression of Abcg5. Genotype F(2, 39) = 0.8746, P = 0.4251; diet: F(1, 39) = 19.89, P < 0.0001; interaction: F(2, 39) = 1.024, P = 0.3685. G: gene expression of Abcg8. Genotype: F(2, 39) = 2.004, P = 0.1484; diet: F(1, 39) = 18.92, P < 0.0001; interaction: F(2, 39) = 1.747, P = 0.1876 (n = 5–9/group). Effects of genotype are indicated by horizontal lines terminating in vertical lines. Effects of diet are indicated by brackets terminating in horizontal lines. *P < 0.05; **P < 0.01; ****P < 0.0001.

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