AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Abstract

Background: AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/principal finding: THE CATALYTIC SUBUNIT OF AMPK HAS TWO ISOFORMS: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1-/- and AMPKα2-/- mice. We found that both α1-/- and α2-/- mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1-/- mice have a shorter circadian period whereas α2-/- mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1-/- mice, but not in α2-/- mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1-/- mice, but it was severely disrupted in the heart and skeletal muscle of α2-/- mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1-/- and α2-/- mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD+, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/significance: This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.

Figure 1. Circadian oscillation of AMPK activity in hypothalamus across the 24 hr light-dark cycle.

Left, A representative Western blot showing the phosphorylation level of AMPK (T172) in extracts from mouse hypothalamus which were harvested at 4 hr intervals in a 12h light:12h dark (LD) cycle. The entire hypothalamus from 3 months old male C57BL/6J mice was used for this experiment. Right, Quantification of phosphorylated AMPKα (T172) is shown in arbitrary units (n = 3–4 per time point). Average AMPK activity (arbitrary unit) was calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Lights-on (6am; light) is indicated by a white bar and lights-off (6pm; dark) is indicated by a black bar. Results are means ± S.E. * P<0.05 between 10 am and 10 pm.

Figure 2. Rhythmic expression of AMPK activity is cell autonomous.

(A) WT mefs were synchronized by forskolin and harvested at the indicated time point. Phosphorylated AMPKα (T172) and phosphorylated-ACC (S79) were assessed by Western blot. Average AMPK and ACC activity (arbitrary unit) were calculated by densitometric quantification of phosphorylated proteins normalized to total proteins. Experiments were repeated at least three times. (B) Expression level (arbitrary units) of mPer2, Bmal1 and PGC-1α in WT and AMPKα1/α2 double knockout (AMPK KO) mefs after synchronization with forskolin. Results are means ± S.E. * P<0.05, # P<0.001between WT and AMPK KO mefs.

Figure 3. Altered free-running period in AMPKα-deficient mice.

(A–B) The activity rhythm was monitored by wheel running under light:dark cycles 12 hr:12 hr (LD) or under constant darkness (DD). Activity records of representative AMPKα1−/− and AMPKα2 −/− mice and their wild-type littermates are shown in double plotted actograms. Each horizontal line represents a 48 hr period and the vertical bars represent wheel running in 10-minute bins (n = 5–6). # P<0.0001, between AMPKα1+/+ and AMPKα1−/− mice. The free-running period was determined by using the χ2 periodogram for days 1–14 in DD. The periods of AMPKα1−/−, AMPKα2−/− and their wild type littermate mice are shown in the right panel. (C,D) Total 24 hr food intake, % of food intake during the light period and body weight of AMPKα1−/− and AMPKα2−/− and their wild-type littermates (n = 10). Results are means ± S.E. * P<0.05 between AMPKα1+/+ and AMPKα1−/−mice. *** P<0.001 between AMPKα2+/+ and AMPKα2−/− mice.

Figure 4. Disruption of circadian physiology in AMPKα-deficient mice.

(A,B) Core body temperature was measured by telemetry. AMPKα1−/−, AMPKα2−/− and WT mice were monitored in LD for 7 days followed by DD for 14 days. Representative data (mean ± SE) is LD day 7. Lights on is indicated by a white bar and lights off is indicated by a black bar. The same WT data is plotted in A and B. (C) Amplitude calculated from cosinor analysis of WT, AMPKα1−/− and AMPKα2−/− mice in LD, DD day 3, 7, 10. Results are expressed as mean ± S.E. * P<0.05 between WT mice versus AMPKα1 deficient mice (n = 3–5 for each genotype). (D) Cosinor analysis of core temperature for WT, AMPKα1−/− and AMPKα2−/− mice. The cosine curve (black line) is superimposed on core temperature raw data (gray line). A representative plot is shown for each genotype (E) VO₂ (left) and RER (right) in AMPKα1−/− and WT mice. Average data is shown under LD on day 7. Light on is indicated by a white bar and light off is indicated by a black bar (n = 4 for each genotype).

Figure 5. Clock and clock related gene expression in heart, skeletal muscle and fat in (A) AMPKα1−/−, (B) AMPKα2−/− and WT mice.

mRNA levels of mPer2, Bmal1, Clock, PGC-1α and leptin were measured by real-time PCR. The relative levels of mRNA are presented in arbitrary units. Light on is indicated by a white bar and light off is indicated by a black bar. The same WT data is plotted in A and B. Results are mean ± S.E. * P<0.05, # P<0.001 between WT versus AMPKα deficient mice (n = 4–5 for each time point).

Figure 6. Circadian oscillation of NAD+ and Nampt gene expression requires AMPK.

(A) Nampt gene expression patterns of heart, skeletal muscle and fat tissue during 24 hr for AMPKα1−/−, AMPKα2 −/− and WT mice. The relative levels of mRNA are presented in arbitrary units. Results are expressed as mean ± S.E., * P<0.05, # P<0.001 between WT and AMPKα deficient mice. (n = 4–5 for each time point). The same WT data is plotted for α1 KO and α2 KO. (B) Nampt gene expression (arbitrary units) and cellular NAD+ and NADH level in WT and AMPKα1/α2 KO mefs synchronized with forskolin. Results are expressed as mean ± S.E., * P<0.05, between WT mefs and AMPKα1/α2 KO mefs (n = 3).