Real-time monitoring in three-dimensional hepatocytes reveals that insulin acts as a synchronizer for liver clock.
Resetting the peripheral clock and understanding the integration between the circadian rhythm and metabolic pathways are fundamental questions. To test whether insulin acts as a synchronizer for the hepatic clock by cell-autonomous mechanisms, the phase-resetting capabilities of insulin were investigated in cultured hepatic cells. We provide evidence that three-dimensional (3D) cell culture conditions that preserve the differentiated state of primary hepatocytes sustained the robustness of the molecular clock, while this robustness rapidly dampened under classical monolayer cell culture conditions. Herein, we established a 3D cell culture system coupled with a real-time luciferase reporter, and demonstrated that insulin directly regulates the phase entrainment of hepatocyte circadian oscillators. We found that insulin-deficient diabetic rats had a pronounced phase advance in their hepatic clock. Subsequently, a single administration of insulin induced phase-dependent bi-directional phase shifts in diabetic rat livers. Our results clearly demonstrate that insulin is a liver clock synchronizer.
Molecular Medicine Research Laboratories, Drug Discovery Research, Astellas Pharma, Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan.
Daisuke
D
Yamajuku
Takahiko
T
Inagaki
Tomonori
T
Haruma
Shingo
S
Okubo
Yutaro
Y
Kataoka
Satoru
S
Kobayashi
Keisuke
K
Ikegami
Thomas
T
Laurent
Tomoko
T
Kojima
Keiji
K
Noutomi
Seiichi
S
Hashimoto
Hiroaki
H
Oda
D000818:Animals; D001683:Biological Clocks / Q000235:genetics / Q000502:physiology*; D015152:Blotting, Northern; D018929:Cell Culture Techniques; D002460:Cell Line; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000235:genetics / Q000502:physiology; D003921:Diabetes Mellitus, Experimental / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000378:metabolism / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism* / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D056950:Period Circadian Proteins / Q000235:genetics; D051381:Rats; D055647:Rats, Transgenic; D017208:Rats, Wistar
D007004:Hypoglycemic Agents; D007328:Insulin; D056950:Period Circadian Proteins; D008156:Luciferases