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. 2021 Oct 1;321(4):G389-G399.
doi: 10.1152/ajpgi.00204.2021. Epub 2021 Aug 25.

Circadian clock core component Bmal1 dictates cell cycle rhythm of proliferating hepatocytes during liver regeneration

Huaizhou Jiang  1   2 , Veronica Garcia  2 , Jennifer Abla Yanum  2 , Joonyong Lee  2 , Guoli Dai  2
Affiliations

Circadian clock core component Bmal1 dictates cell cycle rhythm of proliferating hepatocytes during liver regeneration

Huaizhou Jiang et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

After partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/- hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/- hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators and dysregulated activation patterns of mitogenic signaling molecules c-Met and epidermal growth factor receptor. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/- hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, as well as eventually determines the redox state of regenerated livers.NEW & NOTEWORTHY We demonstrated that Bmal1 centrally controls the synchronicity and rhythmicity of the cell cycle and lipid accumulation in replicating hepatocytes during liver regeneration. Bmal1 plays these roles, at least in part, by ensuring formation of the expression peaks of cell cycle components and regulators, as well as the timing and levels of activation of mitogenic signaling molecules.

Keywords: Bmal1; cell cycle; fat accumulation; hepatocyte proliferation; liver regeneration.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Numbers of cycling hepatocytes after partial hepatectomy (PH). Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to PH and euthanized at the indicated time points. Ki-67 immunostaining was performed with liver sections. A: representative liver sections showing Ki67-positive hepatocytes. B: Ki67-positive hepatocytes were counted at ×200 magnification in five randomly chosen fields per section. The results are shown as mean numbers per field ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01).
Figure 2.
Figure 2.
Numbers of hepatocytes undergoing DNA synthesis after partial hepatectomy (PH). Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to PH and euthanized at the indicated time points. One hour before mice were euthanized, BrdU was injected (100 mg/kg ip). Liver sections were subjected to BrdU immunostaining. A: representative liver sections showing BrdU-positive hepatocytes. B: BrdU-positive hepatocytes were counted at ×200 magnification in 5 randomly chosen fields per section. The data are shown as the mean numbers per field ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01). BrdU, bromodeoxyuridine.
Figure 3.
Figure 3.
Numbers of hepatocytes undergoing mitosis after partial hepatectomy (PH). Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to PH and euthanized at the indicated time points. Liver sections were immunostained with anti-p-Histone H3 antibody. A: representative liver sections show p-Histone H3-positive hepatocytes. B: p-Histone H3-positive hepatocytes were counted at ×200 magnification in five randomly chosen fields per section. The data are shown as means per field ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01).
Figure 4.
Figure 4.
Fat accumulation in regenerating livers. Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3-4-mo-old male mice were subjected to partial hepatectomy (PH) and euthanized at the indicated time points. Frozen liver sections were prepared and used for Oil red O staining. A: representative liver sections show Oil Red O staining. B: the stained areas were quantified in five randomly chosen microscopic fields per liver section (×200 magnification) with Image-Pro software. Data are presented as the means of percent stained areas ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01).
Figure 5.
Figure 5.
mRNA expression of a subset of circadian clock components and cell cycle components and regulators in regenerating liver. Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to partial hepatectomy (PH) and euthanized at the indicated time points. Total liver RNA samples were prepared and used for quantifying mRNA levels of the genes indicated by RT-qPCR. A: mRNA expression of a subset of circadian clock components. B: mRNA expression of a subset of cell cycle components and regulators. Data are expressed as the mean fold change relative to mRNA levels pre-PH in WT mice ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 6.
Figure 6.
Protein expression of a subset of cell cycle components and mitogenic signaling molecules in regenerating liver. Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to partial hepatectomy (PH) and euthanized at the indicated time points. Liver lysates prepared from 5 mice per time point per genotype were pooled with equal amount of proteins from each preparation. Western blotting was performed using antibodies against the proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used as a loading control. Relative densitometry was normalized against GAPDH. NL, normal liver.
Figure 7.
Figure 7.
mRNA expression of a group of genes involved in lipid metabolism in regenerating liver. Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to partial hepatectomy (PH) and euthanized at the indicated time points. Total liver RNA samples were prepared and used for quantifying mRNA levels of the genes indicated by RT-qPCR. Data are expressed as the mean fold change relative to mRNA levels pre-PH in WT mice ± SD (n = 5 mice/time point/genotype; *P < 0.05; **P < 0.01; ***P < 0.001).
Figure 8.
Figure 8.
A: liver-to-body weight ratios before partial hepatectomy (PH). B: liver regrowth patterns after PH. Bmal1+/+ (WT) and hepatocyte-specific Bmal1 knockout (KO) 3- to 4-mo-old male mice were subjected to PH and euthanized at the indicated time points. Liver and body weights were recorded. Liver-to-body weight ratio was used as a liver regrowth index. The results are presented as mean liver-to-body weight ratio ± SD (n = 5 mice/time point/genotype; *P < 0.05). C: Nrf2 immunofluorescence analysis was performed on liver sections prepared from formalin-fixed and paraffin-embedded liver tissues collected from mice euthanized at day 7 post-PH. Representative liver sections show Nrf2 (red, cytosol or nucleus) and DAPI (blue, nucleus).
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