(416b) Effect of Circadian Disruption on Hepatic Gluconeogenesis

The circadian machinery is biological time-keeping
mechanisms which orchestrate the biological changes that follow a 24-hour
cycle. In mammals, the superchiasmatic nuclei (SCN) in the hypothalamus takes
the light/dark cycle as the entraining signal and acts as the central pace
maker that synchronizes the clock genes in the peripheral organs, such as
muscles, liver, heart, and kidneys. The rhythmicity of hormonal and metabolic
signals play an important role in immune [1]
and metabolic functions [2]
by maintaining and controlling the phase relations among the various clocks.
The robustness of circadian rhythms is associated with a homeostatic state of
the host while disruption in the circadian rhythms is observed in a diseased
state of the host [3].

Aside from the light/dark cycle, food intake is a strong zeitgeber,
a cue given by the environment on host to reset the internal body clock.
Feeding rhythms are a more potent zeitgeber compared to the light/dark
cycle eventually synchronizing the peripheral clocks away from the SCN,
especially in organs governing the energy homeostasis of the host such as the
liver. Therefore, an interconnection between the circadian clocks and
metabolism is evident, and it is supported by a multitude of studies. At a
clinical level, several studies correlate the disruption of circadian rhythms
to increased rate of metabolic syndrome. Shift-work and sleep deprivation
dampen the circadian rhythms, and are linked with obesity and high level of
triglycerides [4].
At a molecular level, Clock mutant mice showed decreased metabolic rate [5] while liver-specific Bmal1 knockout disrupted glucose homeostasis in mice [6].
These studies suggest that a bidirectional relationship exists between the
circadian clocks and metabolism, probably as parts of a network of complex
signaling cascades that intricately regulate one another.

Moreover, one can realize from these observations that a
broader environmental or behavioral cues (such as feeding patterns) can
influence specific, low-level targets (such as liver enzymes). To explore this
hypothesis, we would like to demonstrate by a mathematical model how the
feeding/fasting cycle influences a specific metabolic function in relation to
the circadian rhythms entrained to the light/dark cycle. As a first step toward
answering this question, we have previously demonstrated via mathematical
modeling how the rhythms of the peripheral clock genes (PCGs) in the liver can
be completely entrained to the feeding rhythms, synchronizing away from the
light/dark cycle. We further developed this model to explore the effects of
light-feeding phase relations on hepatic metabolism. We model the transcription
of two genes, G6pc and Pck1, as a surrogate for glucose
metabolism in the human liver.

In our model, PPARγ coactivator-1α (PGC1-α)
is a key molecule that incorporates the effects of light and feeding to the
transcription of the gluconeogenic genes. Transcription of PGC-1α is
activated by forkhead box protein O1 (FOXO1) binding upstream of Pck1
and G6pc [7]. When in the nucleus, PGC-1α is deacetylated by SIRT1,
which results in an increased activity [8].
The glucocorticoid receptor is also required along with HNF-4α for full
activation of Pck1 promoter [9]. PGC-1 has shown to be induced
synergistically in primary liver cultures by cAMP and glucocorticoids.
Additionally, Pck1 promotor requires co-activation of the glucocorticoid
receptor and the liver-enriched transcription factor HNF-4α by PGC-1 for
full transcriptional activation. HNF-4α is necessary for the action of
PGC1, as hepatocytes from mice lacking HNF-4α loses the ability of PGC-1
to activate key genes of gluconeogenesis such as Pck1 and G6Pc [10]. The role of HNF-4α and PGC-1α were lumped for simplicity in our model. FOXO1
also plays an important role in gluconeogenesis. PGC-1α binds and
co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation [11]. FOXO1 is known to be required for robust activation of gluconeogenic gene
expression in hepatic cells and in mouse liver. Since FOXO1 binds to a DNA
motif upstream of the gluconeogenic genes, we used it to model the signal
transmission from PGC-1α to transcription of gluconeogenic genes [11]. Finally, PGC-1α also activates the transcription of Bmal1 (Equation 7) [12].

Our model builds upon earlier works [13]
of a semi-mechanical model for light entrainment on the peripheral clock genes
through the HPA axis.  The main driver for oscillations in the HPA axis are the
negative feedback between glucocorticoid and corticotropin-releasing hormone
(CRH)/adrenocorticotropic hormone (ACTH) using a modified Goodwin oscillator.
The glucocorticoid released from the central compartment regulates the
peripheral clock genes (Per/Cry, Bmal1, and Clock). The
feeding/fasting cycle influences the periphery by modifying the redox reactions
between NAD⁺ and NADH [14].
The feeding-entrained NAD⁺ activates human sirtuin 1 (SIRT1), a
class II histone deacetylase. SIRT1 then acts in coordination with CLOCK/BMAL1
complex, interacting with the PCGs in several ways. It facilitates the
transcription of NAMPT, which is the rate-limiting enzyme for the NAD⁺
salvage cycle, regulated by the CLOCK/BMAL1 complex. NAD⁺, in turn,
activates SIRT1, as well as entraining glucocorticoids through a transit compartment
that represents neural input through the ventromedial arcuate nucleus [15].
SIRT1 also facilitates the degradation of PER/CRY proteins in the nucleus,
exerting more influence on the peripheral clock genes. Finally, the
transcription of G6pc/Pck1 (gluconeogenic gene) is controlled by binding
of glucocorticoid-receptor complex to the glucocorticoid response element on
the promotor region, as well as FOXO1 binding also to the promotor region.

Current model successfully predicts some observations from
experimental time-restricted feeding studies. Some basic predictions include
that restricting feeding during the active phase results in phase shift of
total gluconeogenic enzyme amount and the increase of amplitude in PEPCK total
enzyme amount [16].
Another interesting prediction is that when the system is subjected to constant
lighting, the oscillation in G6Pase activity becomes less robust [17].
Furthermore, overexpression of CRY protein results in decreased transcription
of gluconeogenic genes [18]. Disruption of circadian clocks by the means of
Clock gene knockout resulted in over-expression of G6pc and Pck1,
while the nominal expression level could be partially recovered by altering the
light-feeding phase relations. To analyze this complex network, sensitivity
analysis of the model was performed in three parts: 1) local sensitivity
analysis of all parameters; 2) selection of important elementary effects by
Morris method; and 3) global uncertainty analysis employing the high
dimensional model representations (HDMR).  Interpreting and integrating the
results from all three methods revealed that transcription and activation of
PGC-1α and FOXO1 along with Per/Cry transcription had the greatest
influence over the expression level of gluconeogenic genes, demonstrating the
close relationship between circadian rhythms and hepatic metabolic activities.

References

1.            Silver, A.C., et al., The circadian clock controls toll-like receptor 9-mediated
innate and adaptive immunity. Immunity, 2012. 36(2): p. 251-61.

2.            Feillet,
C.A., U. Albrecht, and E. Challet, "Feeding time" for the brain: a
matter of clocks. J Physiol Paris, 2006. 100(5-6): p. 252-60.

3.            Fang,
M., et al., Enhancement of NAD(+)-dependent SIRT1 deacetylase activity by
methylselenocysteine resets the circadian clock in carcinogen-treated mammary
epithelial cells. Oncotarget, 2015. 6(40): p. 42879-91.

4.            Spiegel,
K., et al., Effects of poor and short sleep on glucose metabolism and
obesity risk. Nat Rev Endocrinol, 2009. 5(5): p. 253-61.

5.            Turek,
F.W., et al., Obesity and metabolic syndrome in circadian Clock mutant mice.
Science, 2005. 308(5724): p. 1043-5.

6.            Lamia,
K.A., K.F. Storch, and C.J. Weitz, Physiological significance of a
peripheral tissue circadian clock. Proc Natl Acad Sci U S A, 2008. 105(39):
p. 15172-7.

7.            Daitoku,
H., et al., Regulation of PGC-1 promoter activity by protein kinase B and
the forkhead transcription factor FKHR. Diabetes, 2003. 52(3): p.
642-9.

8.            Schwer,
B. and E. Verdin, Conserved metabolic regulatory functions of sirtuins.
Cell Metab, 2008. 7(2): p. 104-12.

9.            Yoon,
J.C., et al., Control of hepatic gluconeogenesis through the transcriptional
coactivator PGC-1. Nature, 2001. 413(6852): p. 131-8.

10.          Rhee,
J., et al., Regulation of hepatic fasting response by PPARgamma
coactivator-1alpha (PGC-1): requirement for hepatocyte nuclear factor 4alpha in
gluconeogenesis. Proc Natl Acad Sci U S A, 2003. 100(7): p. 4012-7.

11.          Puigserver,
P., et al., Insulin-regulated hepatic gluconeogenesis through
FOXO1-PGC-1alpha interaction. Nature, 2003. 423(6939): p. 550-5.

12.          Green,
C.B., J.S. Takahashi, and J. Bass, The meter of metabolism. Cell, 2008. 134(5):
p. 728-42.

13.          Mavroudis,
P.D., et al., Mathematical modeling of light-mediated HPA axis activity and
downstream implications on the entrainment of peripheral clock genes.
Physiol Genomics, 2014. 46(20): p. 766-78.

14.          Diaz-Munoz,
M., et al., Anticipatory changes in liver metabolism and entrainment of
insulin, glucagon, and corticosterone in food-restricted rats. Am J Physiol
Regul Integr Comp Physiol, 2000. 279(6): p. R2048-56.

15.          Yi,
C.X., et al., Ventromedial arcuate nucleus communicates peripheral metabolic
information to the suprachiasmatic nucleus. Endocrinology, 2006. 147(1):
p. 283-94.

16.          Perez-Mendoza,
M., J.B. Rivera-Zavala, and M. Diaz-Munoz, Daytime restricted feeding
modifies the daily variations of liver gluconeogenesis: adaptations in
biochemical and endocrine regulators. Chronobiol Int, 2014. 31(7):
p. 815-28.

17.          Ashman,
P.U. and J.R. Seed, Biochemical studies in the vole, Microtus montanus. II.
The effects of a Trypanosoma brucei gambiense infection on the diurnal
variation of hepatic glucose-6-phosphatase and liver glycogen. Comp Biochem
Physiol B, 1973. 45(2): p. 379-92.

18.          Lamia,
K.A., et al., Cryptochromes mediate rhythmic repression of the
glucocorticoid receptor. Nature, 2011. 480(7378): p. 552-6.