Metabolic Profile and Lipid Metabolism Phenotype in Mice with Conditional Deletion of Hepatic BMAL1

In our previously study, we found that exposure to PM_2.5 disturbed Bmal1 circadian oscillations at ZT0 and disrupted hepatic lipid metabolism at ZT12 [12]. To build upon our previous findings, we generated Bmal1^hep−/− mice by crossing Bmal1^f/f mice with Alb-Cre mice (Figure 1A). In Bmal1^hep−/− mice, Bmal1 expression was markedly reduced and abolished in liver tissue (Figure 1B,C).

We examined the expression of core CCGs in the liver of mice at ZT0 and ZT12. At ZT0, Bmal1^hep−/− mice displayed a significant upregulation of Cry1, Cry2, and Per2, with a significant downregulation of Rev-erbα. At ZT12, Bmal1^hep−/− mice exhibited a significant upregulation of Clock, Cry1, and Rev-erbα, while Per1, Per2, and Rorα were significantly downregulated (Figure 1D–J). These findings collectively indicated substantial alterations in hepatic CCGs resulting from hepatocyte-specific Bmal1 knockout.

Liver-clock mutant mice exhibit impaired lipid homeostasis, which is partially regulated by BMAL1/LIPIN/DGAT signaling [16,17]. Combined with our previous research findings, we investigated hepatic lipid metabolism in Bmal1^hep−/− mice, focusing on changes in lipolysis, lipid uptake, and synthesis. At ZT0, the level of TG was significantly lower in Bmal1^hep−/− mice (Figure 2A), and this finding was confirmed by Oil Red O staining (Figure 2B). The expression of enzymes for lipolysis (Lpl but not Atgl or Hsl) significantly increased (Figure 2C), while the expression of fatty acid uptake receptors (Fabp1, Fabp2, and Fabp5) (Figure 2D) and lipogenic enzymes (Fas, Scd1, and Dgat) (Figure 2E) significantly decreased. At ZT12, the expression of Lpl still exhibited a significant increase in Bmal1^hep−/− mice (Figure 2C) with no change in the level of TG (Figure 2A,B), while other genes that previously exhibited changes at ZT0 did not show significant differences (Figure 2D,E). No significant difference was observed in lipid export genes (Mttp and Apob) between Bmal1^f/f and Bmal1^hep−/− mice (Supplementary Figure S1). Based on the findings, the decrease in hepatic TG levels at ZT12 in the 24th week could be attributed to heightened lipolysis, diminished lipogenesis, and decreased expression of lipid receptors resulting from the deletion of Bmal1.

The rhythmic metabolism of liver lipids is closely related to mitochondrial beta-oxidation involving molecules such as PPAR-α, CPT1, DBP1, and PGC1 [18,19]. Considering the enhanced lipolysis in Bmal1^hep−/− mice, we conducted assays on mitochondrial function at ZT12 (end of light phase) using mitochondria isolated from mouse livers. At the Leak state (using glutamate and malate for Leak state respiration), respiration in both groups was unaffected by Bmal1 deletion. In the CI-linked OXPHOS and CI&II-linked OXPHOS states, Bmal1^hep−/− mice showed a decrease of 43.3% and 32.5%, respectively, compared to Bmal1^f/f mice, indicating a reduction in ATP production through oxidative phosphorylation. To assess the maximal capacity of the electron transfer system (ETS), uncoupler CCCP was added to reach the maximum capacity. Uncoupled respiration demonstrated that Bmal1^hep−/− caused a reduction of CII-linked ETS capacity by 34.6%. CIV-driven respiration was decreased by 12.1% in Bmal1^hep−/− mice compared to Bmal1^f/f mice (Figure 3A). However, further analysis of mitochondrial function throughout the entire process from CI-Leak to CIV state revealed that deletion of Bmal1 led to a decline in mitochondrial function, but no statistically significant difference was observed between groups (Figure 3B). Accordingly, Bmal1^hep−/− mice exhibited a significant downregulation of Acox1, Ppara, and Pgc1β at ZT12 (Figure 3C), whereas Cpt1α, Ppara, Dbp1, Pgc1α, Pgc1β, and Pparγ were significantly downregulated at ZT0 (Figure 3D).

Considering the impact of BMAL1 on mitochondrial respiratory substrates, we utilized a Promethion Core Metabolic System to assess the respiratory exchange ratio during the 14th week of the experiment (at 22 weeks of age in mice). The Bmal1^f/f mice had a respiratory exchange ratio of 0.89643, while the Bmal1^hep−/− mice had a respiratory exchange ratio of 0.902038 (Supplementary Figure S2), indicating no significant difference between the two groups. These results suggested that glucose was the primary substrate for aerobic respiration and that liver-specific BMAL1 knockout does not affect the overall aerobic respiratory substrates in the body.

Taken together, these results indicated decreased hepatic mitochondrial function due to Bmal1 deletion at both ZT0 and ZT12.

At the 4th week, Bmal1^hep−/− mice showed a higher fat mass percentage and a lower lean mass percentage compared to the Bmal1^f/f mice (Figure 4A,B). Additionally, at the 4th week, GTT revealed that Bmal1^hep−/− mice demonstrated enhanced glucose tolerance compared to Bmal1^f/f mice at the 30-min mark (Figure 4D). This observation was confirmed by the area under the curve (AUC) of GTT at the 4th week (Figure 4E). Throughout the experimental period, no discernible differences were observed in body weight, food intake, and free body fluid between Bmal1^f/f and Bmal1^hep−/− mice (Figure 4C and Supplementary Figure S3A,B). No other differences were noted in GTT and ITT examination between Bmal1^f/f and Bmal1^hep−/− mice throughout the experimental period (Supplementary Figure S3C–P).

Considering the impact of BMAL1 on feeding rhythms, we utilized a Promethion Core Metabolic System to assess the impact of hepatocyte-specific Bmal1 deletion on whole-body metabolism during the 14th week of the experiment (mice at 22 weeks of age). During the light phase (ZT0–ZT12), Bmal1^hep−/− mice consumed more food compared to Bmal1^f/f mice (Figure 4G). No significant differences were noted in cumulative water consumption, pedestrian distance, or intra-cage distance (Supplementary Figure S4).

The chemicals used in this study included EGTA (103777-10G), lactobionic acid (153516), taurine (T0625-25G), HEPES (H7523-50G), D-sucrose (84097), BSA (SRE0098-10G), pyruvate (P2256-5G), glutamate (49621-250G), malate (M1125-5G), ADP (A2754-1G), cytochrome c (C7752-50MG), succinate (S9637-100G), CCCP (C2759-250MG), rotenone (R8875-1G), antimycin A (A8674-25MG), ascorbate (PHR1279-1G), and TMPD (T3134-5G), all of which were procured from MilliporeSigma Canada Ltd. (Oakville, ON, Canada). MgCl₂·6H₂O (S24121-500G) was obtained from Shanghai YuanYe Biotechnology Co., Ltd (Shanghai, China). KH₂PO₄ (A501211-0500) was sourced from Sangon Biotech Co., Ltd (Shanghai, China). Humulin (human regular insulin) was acquired from Lilly (Indianapolis, IN, USA). Rabbit anti-mouse antibodies against BMAL1 (ab231793) were obtained from abcam Inc. (Cambridge, UK). Mouse anti-mouse antibodies against β-ACTIN (Catalog #66009-1) were obtained from Proteintech Group, Inc. (Wuhan, China).

All mice of the C57BL/6J background were used in this study. Bmal1 floxed (Bmal1^f/f) mice were bred by Gem Pharmatech (Gem Pharmatech Co., Ltd., NanJing, China). Hepatocyte-specific Bmal1 knockout (Bmal1^hep−/−) mice were generated by crossing Bmal1^f/f mice with Alb-Cre transgenic mice (Figure 1A). Seven-week-old female Bmal1^f/f and Bmal1^hep−/− mice were separately housed in cages with ad libitum access to a normal chow diet and water under a 12/12-h light/dark cycle at a temperature of 22 ± 2 °C. After a one-week acclimation, all mice received the same feeding regimen. ZT0 was set as 6 a.m., when the lights were turned on, and ZT12 was set as 6 p.m., when the lights were turned off. To build upon our previous findings that exposure to PM_2.5 increases Bmal1 expression at ZT0/24 and significantly affects the liver lipid metabolism enzymes ACL and FAS at ZT12 [12], we focused our comprehensive metabolic analysis on Bmal1^hep−/− mice at the critical time points of ZT0 and ZT12. At the end of the 24th week, mice were anesthetized with pentobarbital sodium (20 mg/kg, intraperitoneal injection) and liver tissues were obtained at ZT0 or ZT12 time points. The animal experiment protocol was reviewed and approved by the Animal Care and Use Committee of Zhejiang Chinese Medical University (animal use grant NO. 202107-0403).

At the end of the 24-week experiment (32 weeks of age in mice), an appropriate amount of liver tissue was added to the radioimmunoprecipitation assay (RIPA) lysis and centrifuged to extract the supernatant. Protein samples were prepared by quantifying the protein concentration with the bicinchoninic acid (BCA) method. Equal amounts of protein were loaded onto 8% or 10% homemade gels and then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk for 90 min at room temperature, membranes were washed three times with 1 × tris-buffered saline-tween (TBST) for 10 min each time and then incubated with the corresponding primary antibodies overnight at 4 °C. After washing three times for 10 min, membranes were incubated for 1 h with the corresponding secondary antibody at ambient temperature. Immunoreactive protein levels were analyzed using a chemiluminescence imaging system (Bio-Rad, USA), and β-actin was used as an internal reference correction. Grayscale values of the protein were analyzed quantitatively with the ImageJ software (Version 1.54f, NIH, Bethesda, MD, USA).

At the end of the 24-week experiment (32 weeks of age in mice), RT-PCR analysis was conducted using RNA isolated from liver tissues. Total RNA extraction was performed using the Trizol reagent (TaKaRa, Shiga, Japan). Subsequently, cDNA was synthesized from the RNA using the PrimeScript RT Master Mix (TaKaRa, Shiga, Japan) following the manufacturer’s protocol. Gene expression levels were assessed using the QuantStudio Q7 system (Applied Biosystems, Carlsbad, CA, USA). The relative gene expression was calculated using the 2^−△△Ct method normalized to the expression of the β-actin gene. Details of the primer sequences are provided in Table 1.

At the end of the 24-week experiment (32 weeks of age in mice), the experimental setup followed the guidelines provided by the TG test kits (DiaSys Diagnostics, Frankfurt, Germany) and was conducted in a 96-well plate. Approximately 100 mg of liver tissue was homogenized in the reaction buffer. Following the incubation period, absorbance readings were obtained using a Varioscan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The obtained data were subsequently normalized to the corresponding body weight.

At the end of the 24-week experiment (32 weeks of age in mice), the liver tissues of mice were fixed and embedded in an optimal cutting temperature (OCT) compound (Servicebio, Wuhan, China). The embedded specimens were sectioned into 5 μm thick slices for subsequent staining with Oil Red O. A 5 μm thick slice was immersed in a freshly prepared Oil Red O working solution and kept in the dark for 8–10 min. Afterward, the slices were removed, allowed to stand for 3 s, and sequentially immersed in two containers of 60% isopropanol for differentiation for either 3 or 5 s. Subsequently, the slices were immersed in two containers of pure water for 10 s each. Hematoxylin staining was performed followed by microscopic examination of the staining effect and final sealing. The histopathological changes in the liver were observed using a Nano Zoomer S60 imaging system (Hamamatsu, Hamamatsu City, Japan). The percentage of positive areas in the liver Oil Red O staining was quantified using ImageJ software (Version 1.54f, NIH, Bethesda, MD, USA).

At ZT12 at the end of the 24-week experiment (32 weeks of age in mice), the mice were euthanized, and 2 mg of liver tissue was mechanically homogenized in a respiration medium, namely MIR05 buffer. The composition of the MIR05 buffer included 0.5 mM EGTA, 3 mM MgCl₂·6H₂O, 60 mM lactobionic acid, 20 mM taurine, 10 mM KH₂PO₄, 20 mM HEPES, 110 mM D-sucrose, and 1 g/L essentially fatty-acid-free BSA, with the pH adjusted to 7.1 at 37 °C. The liver tissue suspension was loaded into the chamber of the Oroboros 2K oxygraph system (Oroboros Instruments, Innsbruck, Austria), which was filled with the MIR05 buffer. The measurement of oxygen consumption rates proceeded with the sequential addition of substrates and specific inhibitors:

At weeks 4, 8, 12, 16, 20, and 24 of the experiment (12, 16, 20, 24, 28, and 32 weeks of age in mice), the whole-body composition analysis was conducted. During the analysis procedure, the mice remained awake and were placed in an acrylic cylinder. The Bruker TD-NMR system (minispec LF50, Bruker, Billerica, MA, USA) was used to measure the whole-body composition. The magnetic field was set at 0.17 T and 7.5 MHz frequency pulse. The measurements of fat, lean mass, and fluid were recorded, and mice were returned to their home cage in 1 min. Data were normalized to body weight [32].

At weeks 4, 8, 12, and 20 of the experiment (12, 16, 20, and 28 weeks of age in mice), the glucose homeostasis was evaluated. Mice were fasted for 12 h for intraperitoneal glucose tolerance testing (IPGTT) and were subsequently intraperitoneally injected with dextrose (2 mg/g body weight). Blood was drawn from the tail vein at 0, 30, 60, 90, and 120 min, and glucose concentration was determined using a FreeStyle glucometer (Abbott Diabetes Care Inc., Alameda, CA, USA). For insulin tolerance testing (ITT), mice were fasted for 4.5 h and then injected intraperitoneally with insulin (0.5 U/kg body weight), and blood glucose was measured at 0, 30, 60, 90, and 120 min [33]. IPGTT and ITT were performed at the 4th, 8th, 12th, and 20th weeks.

At the 14th week of the experiment (22 weeks of age in mice), the Promethion Core Metabolic System (Sable Systems International, North Las Vegas, NV, USA) was used to measure a series of metabolic parameters, including food consumption, water consumption, locomotor activity, oxygen consumption (VO₂), carbon dioxide exhalation (VCO₂), respiratory exchange ratio (RER), and energy expenditure (heat production). Mice were housed in individual monitoring cages of the Promethion Core Metabolic System and acclimated for 48 h before recording. Light and feeding conditions were kept the same as in the home cages. The concentrations of O₂ and CO₂ were measured by calculating the air entering and leaving the chambers. The respiratory exchange ratio is the ratio of CO₂ production to O₂ consumption. Data were normalized to body weight [34].

All data were expressed as the mean ± standard error of the mean (S.E.M) unless otherwise indicated. The analyses were performed using Graphpad Prism software (Version 10.0.0, GraphPad Software, Boston, MA, USA). For the analysis, the t-test was used when comparing the Bmal1^f/f group with the Bmal1^hep−/− group. A p-value of <0.05 was considered statistically significant.

The data that support the findings of this study are available in the methods and/orof this article. Supplementary Material