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Endocrinology, doi:10.1210/en.2008-0944
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Endocrinology Vol. 150, No. 1 161-168
Copyright © 2009 by The Endocrine Society

High-Fat Diet Delays and Fasting Advances the Circadian Expression of Adiponectin Signaling Components in Mouse Liver

Maayan Barnea, Zecharia Madar and Oren Froy

Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food, and Environmental Quality, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Address all correspondence and requests for reprints to: Oren Froy, Institute of Biochemistry, Food Science, and Nutrition, Faculty of Agricultural, Food, and Environmental Quality, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel. E-mail: froy{at}agri.huji.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The circadian clock controls energy homeostasis by regulating circadian expression and/or activity of enzymes involved in metabolism. Disruption of circadian rhythms may lead to obesity and metabolic disorders. We tested whether the biological clock controls adiponectin signaling pathway in the liver and whether fasting and/or high-fat (HF) diet affects this control. Mice were fed low-fat or HF diet and fasted on the last day. The circadian expression of clock genes and components of adiponectin metabolic pathway in the liver was tested at the RNA, protein, or enzyme activity level. In addition, serum levels of glucose, adiponectin, and insulin were measured. Under low-fat diet, adiponectin signaling pathway components exhibited circadian rhythmicity. However, fasting and HF diet altered this circadian expression; fasting resulted in a phase advance, and HF diet caused a phase delay. In addition, adenosine monophosphate-activated protein kinase levels were high during fasting and low during HF diet. Changes in the phase and daily rhythm of clock genes and components of adiponectin signaling pathway as a result of HF diet may lead to obesity and may explain the disruption of other clock-controlled output systems, such as blood pressure and sleep/wake cycle, usually associated with metabolic disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mammals have developed an endogenous circadian clock located in the suprachiasmatic nuclei of the anterior hypothalamus that responds to the environmental light-dark cycle (1). Similar clock oscillators have been found in peripheral tissues, such as the liver, intestine, and adipose tissue (2, 3, 4, 5). Light is the most potent synchronizer for the suprachiasmatic nuclei, yet there is a growing body of evidence that metabolism, food consumption, timed meals, and some nutrients also feed back to entrain the clock (6, 7, 8).

The core clock mechanism is encoded by the genes Clock, brain-muscle-Arnt-like 1 (Bmal1), Period1 (Per1), Period2 (Per2), Period3 (Per3), Cryptochrome1 (Cry1), and Cryptochrome2 (Cry2) (1). The protein products CLOCK and BMAL1 dimerize to activate transcription. CLOCK:BMAL1 heterodimers mediate transcription of a large number of genes including those of the negative feedback loop Pers and Crys. When PERs and CRYs are produced in the cytoplasm, they oligomerize and translocate to the nucleus to inhibit CLOCK:BMAL1-mediated transcription. All the aforementioned clock genes exhibit a 24-h rhythm (2, 8). In addition, casein kinase I{epsilon} (CKI{epsilon}) is thought to phosphorylate the PER proteins and, thereby, enhance their instability and degradation. CKI{epsilon} also phosphorylates and partially activates the transcription factor BMAL1 (9).

The circadian clock has been reported to regulate metabolism and energy homeostasis in the liver and other peripheral tissues (10). This is achieved by mediating the expression and/or activity of certain metabolic enzymes and transport systems (11). Animals with mutations in clock genes that disrupt cellular rhythmicity have provided evidence to the relationship between the circadian clock and metabolic homeostasis. Clock mutation-attenuated obesity induced by high-fat (HF) diet in ICR mice through impaired dietary fat absorption (12), possibly through the suppression of acyl-CoA synthase long chain family member 4 (Acsl4) and fatty acid binding protein 1 (Fabp1) gene expression (13). On the other hand, disrupted circadian rhythms led to attenuated circadian feeding rhythms, hyperphagia, obesity, and metabolic syndrome in mice (14). Clock mutant mice showed impaired glucose tolerance and reduced plasma insulin and plasma glucose nocturnally suggesting impaired insulin secretion (15). Similarly, Bmal1–/– knockout mice exhibited suppressed diurnal variations in glucose and triglycerides as well as abolished gluconeogenesis (16). In addition, the core clock mechanism has been shown to be linked with lipogenic pathways. 1) REV-ERB{alpha}, the negative regulator of Bmal1, is induced during normal adipogenesis (17). 2) ROR{alpha}, the positive regulator of Bmal1 (18), has been shown to regulate lipogenesis and lipid storage in skeletal muscle (19). 3) CLOCK:BMAL1 heterodimer regulates the expression of Rev-erb{alpha} and Ror{alpha} (17, 18, 20). 4) Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}), involved in lipid and lipoprotein metabolism, binds directly to the Bmal1 promoter (21), and in turn, the CLOCK:BMAL1 heterodimer regulates PPAR{alpha} expression (21, 22, 23). 5) Fibroblasts derived from Bmal1–/– embryos are resistant to adipogenesis when exposed to dexamethasone and insulin (24). 6) In mPpar{alpha}-null mice, temporally restricted food access caused prolonged phase shifts of circadian transcription factors and PPAR{alpha}-responsive genes (25).

Adiponectin is secreted from differentiated adipocytes and is involved in glucose and lipid metabolism (26). It increases fatty acid oxidation and potentiates insulin inhibition of hepatic gluconeogenesis, thus promoting insulin sensitivity. Adiponectin binds to two different receptors, AdipoR1 and AdipoR2, and activates a protein kinase cascade (27) leading to the activation of AMP-activated protein kinase (AMPK) (28, 29), which, in turn, phosphorylates and, thus, inactivates acetyl-coenzyme A carboxylase (ACC), the key enzyme in fatty acid synthesis. AMPK activation by adiponectin results in mitochondrial fatty acid oxidation through the activation of PPAR{alpha} (30). Recently, it has been shown that HF diet disrupts circadian rhythms (31). Because little is known about the involvement of the circadian clock in the signaling pathway of adiponectin, we set out to evaluate whether the different components of this pathway exhibit daily rhythmicity in the liver and whether HF diet and fasting affect their oscillation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and experimental design
The 3- to 4-wk-old C57BL/6 male mice (Harlan Laboratories, Jerusalem, Israel) were housed in a temperature- and humidity-controlled facility (23–24 C, 60% humidity). Mice were entrained to a lighting cycle of 12 h light and 12 h darkness (LD) for 4 wk and then were divided into two groups that were fed either HF diet or low-fat (LF) diet for 7 wk. The HF diet was based on soybean oil and palm stearin (fatty-acid composition: C:12 0.3%, C:14 1.3%, C:16 55%, C:18 5.1%, C:18-1 29.5%, C:18-2 7.4%, C:18-3 0.7%) and contained 22% wt/wt fat (42% kcal from fat) vs. soybean oil diet 7% wt/wt (16% kcal from fat) of the LF group. The difference between the LF and HF diet was that the above-mentioned fat replaced some of the starch content of the diet to prevent caloric difference. Body weight was recorded weekly, and on the fourth and ninth week, fasting blood glucose levels were determined (Fig. 1AGo). After 7 wk on the diets, half of the LF group and the whole of the HF group were placed on a 24-h fast, whereas the other half of the LF group was maintained with the food. Every 3 h around the circadian cycle, animals of each group were tested for blood glucose levels (n = 4), anesthetized by ip injection of ketamine/xylazine (100/7.5 mg/kg) under dim red light in total darkness (DD), and liver was removed. Blood was collected from the inferior vena cava into EDTA-free tubes and centrifuged at 2000 x g for 15 min. Animals were humanely killed. Institutional approval was granted for these experiments. Experiments were conducted in full compliance with the strict guidelines of the Hebrew University policy on animal care and use.


Figure 1
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  		FIG. 1. Experimental design, body weight, and blood glucose levels. A, Forty-eight C57BL/6 male mice were entrained to a lighting cycle of 12 h light and 12 h darkness for 4 wk. After 4 wk, mice were tested for fasting glucose (FG) and divided into two groups: a group fed a HF diet and a group fed a LF diet. At the end of the 11th week, half of the LF group and the entire HF group were placed on a 24-h fast, whereas the other half of the LF group were kept on ad libitum feeding. B and C, Body weight (B) was recorded weekly, and fasting blood glucose levels (C) were determined at the beginning and on the fifth week of the feeding regimen (fourth and ninth weeks, respectively, since the beginning of the experiment). *, P < 0.05.

 
ELISA
Serum insulin levels were determined by a mouse ultrasensitive insulin ELISA kit (Mercodia AB, Uppsala, Sweden). Serum adiponectin levels (dilution 1:1000) were determined using a specific ELISA kit (Linco Research, St. Charles, MO). Assays were performed according to the manufacturers’ instructions.

Homeostasis model assessment of insulin resistance (HOMA-IR)
The insulin-resistance index from fasting serum insulin and plasma glucose levels was determined by the HOMA parameter: HOMA = fasting serum insulin (µU/ml) x fasting plasma glucose (mg/dl)/405. The greater the HOMA value, the higher the level of insulin resistance (32).

RNA extraction and quantitative real-time PCR
For gene expression analyses, RNA was extracted from tissue using TRI Reagent (Sigma, Jerusalem, Israel). Total RNA was deoxyribonuclease (DNase) I treated using RQ1 DNase (Promega, Madison, WI) for 2 h at 37 C, as was previously described (8). Three micrograms of DNase I-treated RNA were then reverse transcribed using Maloney murine leukemia virus reverse transcriptase (Promega) and random hexamers. Then, 1/200 of the reaction was subjected to quantitative real-time PCR using the Sybr Green Master kit (Applied Biosystems, Foster City, CA) and the ABI Prism 7300 Sequence Detection System. Genes were normalized to mouse glyceraldehyde-3-phosphate dehydrogenase (mGapdh). The fold change in target gene expression was calculated by the {Delta}Ct method (Applied Biosystems). The primers used are listed in supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Western blot analysis
Liver tissue samples (200 mg) were homogenized in 1 ml lysis buffer (20 mM Tris, 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 100 µM phenylmethylsulfonyl fluoride, 50 µmol/liter NaF, 1 mM sodium orthovanadate). Samples were run onto an SDS-polyacrylamide gel (10% for mAMPK, 7.5% for mACC), and after electrophoresis, proteins were transferred onto nitrocellulose membranes. Blots were incubated with mAMPK/mACC antibodies (Cell Signaling Technology, Beverly, MA) and, after several washes, with horseradish peroxidase-labeled secondary antibody (Pierce, Rockford, IL). The immune reaction was detected by enhanced chemiluminescence. Finally, bands were quantified by scanning and densitometry and expressed as arbitrary units.

Phosphoenolpyruvate carboxykinase (PEPCK) activity
Liver homogenates were prepared in homogenization buffer [50 mM HEPES-KOH (pH 7.4), 140 mM NaCl, 250 mM sucrose, 1 mM CaCl2, 2 mM EDTA, 2.5 mM Na3VO4, 20 mM NaF, 10% glycerol, 1 mM MgCl2, 200 µM phenylmethylsulfonyl fluoride]. The homogenates were centrifuged for 30 min at 48,000 x g, and the supernatants were recentrifuged for 30 min at 48,000 x g. The supernatant was tested for mPEPCK activity using the rate of exchange between KH14CO3 and unlabeled oxaloacetate, according to the method by Chang and Lane (33).

Statistical analyses
All results are expressed as means ± SEM. Student’s t test was used for comparison between groups. One-way ANOVA was used to analyze circadian pattern with several time points. The acrophase and P value of gene oscillation were calculated using cosinor analysis software (version 2.3). For all analyses, the significance level was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We set out to test whether the biological clock controls the expression of genes and enzymes involved in the adiponectin signaling pathway and whether fasting and/or HF diet disrupt this control. After 4 wk entrainment, mice were tested for fasting glucose and divided into two groups that were fed one of two locally produced experimental diets for 7 wk, HF diet and LF diet (Fig. 1AGo). Weekly weight gain was greater in the HF group compared with the LF group. The final mean body weight of the LF group was significantly lower than that of the HF group (Student’s t test, P < 0.0001) (Fig. 1BGo). Fasting blood glucose levels were determined on the fifth week of the feeding experiment (ninth week, including the 4-wk entrainment), and its levels were found to be significantly higher in the HF group compared with the LF group (Student’s t test, P < 0.0001) (Fig. 1CGo). Because feeding may affect the expression of genes involved in the adiponectin pathway, at the end of the seventh week (11th week, including the 4-wk entrainment), half of LF group and the entire HF group fasted for 24 h. Thus, 24 h later, there were two fasting groups (HF+F and LF+F) and one nonfasting group (LF) (Fig. 1AGo). Animals of each of the three groups were weighed and tested for blood glucose. Liver, the adiponectin target tissue, and serum were collected every 3 h around the circadian cycle under dim red light in DD. Tissue was removed under total darkness to eliminate possible light effects. At the end of the experiment, HF+F mice weighed significantly more than LF+F mice (Student’s t test, P < 0.0001) (Fig. 2AGo).


Figure 2
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  		FIG. 2. Body weight and insulin, glucose, and adiponectin levels. A–C, Final average body weight (A), glucose (B), and insulin (C) were measured on the day of experiment termination and calculated using measurements of all time points collected every 3 h around the circadian cycle. D, HOMA-IR was calculated from fasting insulin and glucose levels in LF+F or HF+F groups. E, Adiponectin was measured on the day of experiment termination and calculated using measurements of all time points collected every 3 h around the circadian cycle. F, Normalized adiponectin level was calculated from adiponectin in E and body weight in A. Values are means ± SE; n = 4 for each time point in each group. *, P < 0.05.

 
Insulin, glucose, and adiponectin levels in mice fed LF and HF diet
As expected, glucose (Fig. 2BGo) and insulin levels (Fig. 2CGo) in the nonfasting LF group were significantly higher compared with both the LF+F and HF+F groups (Student’s t test, P < 0.0001). An insulin-resistance index based on fasting insulin and glucose levels (HOMA-IR) was significantly higher in HF+F group compared with LF+F (Student’s t test, P < 0.05) (Fig. 2DGo), indicating a decline in insulin sensitivity in animals fed a HF diet. Neither blood glucose nor insulin levels exhibited a daily rhythm in any of the groups (one-way ANOVA, P > 0.05) (data not shown). Adiponectin levels were higher in the LF and HF+F groups compared with LF+F (Fig. 2EGo). However, when these levels were normalized to body weight, there was no difference among the groups (Fig. 2FGo). Adiponectin levels did not oscillate in the serum (one-way ANOVA, P > 0.05) (data not shown).

Circadian mRNA expression of clock genes in mice fed LF and HF diet
To study whether fasting and/or HF diet affect the biological clock, we tested the phase and expression amplitude of the clock genes mPer1, mPer2, mBmal1, and mClock at the RNA level in the liver. Quantitative real-time PCR analysis revealed that all clock genes exhibited circadian oscillation (one-way ANOVA, P < 0.05) (Fig. 3Go). However, compared with the LF group, fasting resulted in the attenuation of clock gene amplitude and a phase advance of mPer2, mClock, and mBmal1 (Fig. 3Go and supplemental Table 2). HF diet resulted in a 3-h phase delay of mPer1 in the liver (Fig. 3Go and supplemental Table 2). There was 3-h phase difference between LF+F and HF+F in all clock genes tested (supplemental Table 2).


Figure 3
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  		FIG. 3. The effect of diet on clock gene, mPpar{alpha} and mAdipoR1 mRNA expression. Liver was collected every 3 h around the circadian cycle in DD from the different mouse groups [LF+F (Figure 3) HF+F ({triangleup}) or LF({diamondsuit})], and total RNA was extracted and reverse transcribed. Expression levels of mPer1, mPer2, mBmal1, mClock, mPpar{alpha}, and mAdipoR1 were determined by quantitative real-time PCR. Values are mean ± SE; n = 4 for each time point in each group. The gray and black bars designate the subjective day (formerly the light period) and dark cycles, respectively. CT0 and CT12 represent the circadian times at which the lights would have been turned on and off, respectively, had the animals remained in light-dark.

 
Circadian mRNA expression of mPpar{alpha} in mice fed LF and HF diet
Because PPAR{alpha} is a known component affecting the circadian clock (21) and affected by adiponectin and the biological clock (21, 22, 23), its daily rhythm and the impact of fasting and HF diet on its expression was evaluated. When analyzed by quantitative real-time PCR, mPpar{alpha} mRNA in the liver oscillated robustly and peaked during the light period in the liver of the LF group. Fasting (LF+F group) resulted in the attenuation of mPpar{alpha} mRNA amplitude. Fasting had even a stronger attenuation effect when combined with HF diet (HF+F group) (Fig. 3Go) leading to almost a complete disruption of mPpar{alpha} mRNA oscillation. In addition to the dampened rhythm, HF caused a phase delay in mPpar{alpha} similarly to the effect on clock gene expression (Fig. 3Go and supplemental Table 2).

Circadian mRNA expression of mAdipoR1 and mAdipoR2 in mice fed LF and HF diet
The expression level and oscillation of the genes encoding adiponectin receptors, mAdipoR1 and mAdipoR2, were then tested by quantitative real-time PCR. The mRNA of mAdipoR1 exhibited a daily rhythm in the liver with approximately a 2-fold oscillation between peak and trough (one-way ANOVA, P < 0.05) in the LF group (Fig. 3Go). Fasting caused a phase advance and HF diet resulted in a phase delay in mAdipoR1 expression (one-way ANOVA, P < 0.05) (Fig. 3Go and supplemental Table 2). mAdipoR2 mRNA did not exhibit a clear circadian oscillation, but fasting increased its abundance in the liver (data not shown).

Circadian mRNA and protein expression of mAmpk/mAMPK in mice fed LF and HF diet
The effect of HF diet and fasting on mAMPK, a key enzyme in the adiponectin signaling pathway, was then studied at the mRNA and protein levels. The expression of mAmpk mRNA in the liver showed a 2.2-fold increase between peak in the middle of the subjective day and trough in the subjective night in the nonfasting LF group (one-way ANOVA, P < 0.0001). Similarly, mAMPK protein levels of the LF group peaked in the middle of the subjective day, as determined by Western blot (one-way ANOVA, P < 0.05). Although fasting did not affect mAmpk daily rhythm, HF dramatically dampened its mRNA daily rhythm with a phase delay in the liver (Fig. 4Go and supplemental Table 2). In the LF+F group, the oscillation of the protein was maintained (one-way ANOVA, P < 0.05), the amplitude decreased, and the expression peak shifted to the beginning of the subjective night (Fig. 4Go). Similarly to the results achieved at the RNA level, HF diet led to the loss of mAMPK protein rhythmicity in the liver (Fig. 4Go). Total AMPK levels were higher in the LF+F compared with HF+F in the liver (Fig. 4Go). Phosphorylated mAMPK did not oscillate, and the average levels did not differ among the groups (data not shown).


Figure 4
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  		FIG. 4. Daily rhythm of mAmpk and mAcc mRNA and protein levels. mRNA expression levels of mAmpk and mAcc were determined by quantitative real-time PCR and protein levels of mAMPK and mACC by Western blotting in the liver collected every 3 h around the circadian cycle from mice fed LF+F (Figure 4), HF+F ({triangleup}), or LF({diamondsuit}). Average mAMPK levels were calculated using all the time-point measurements. Values are mean ± SE; n = 4 for each time point in each group. *, P < 0.05. The gray and black bars designate the subjective day (formerly the light period) and dark cycles, respectively. CTO and CT12 represent the circadian times at which the lights would have been turned on and off, respectively, had the animals remained in light-dark.

 
Circadian mRNA and protein expression of mAcc/mACC in mice fed LF and HF diet
Because mAMPK phosphorylates, and, hence, inactivates mACC, we evaluated the circadian rhythm of mACC. In the LF and LF+F, mAcc mRNA exhibited a daily rhythm with a peak in the middle of the subjective day in the liver (one-way ANOVA, P < 0.01) (Fig. 4Go). HF diet abolished mAcc mRNA oscillation in the liver (Fig. 4Go). mACC protein did not oscillate in any of the groups (one-way ANOVA, P > 0.05) (Fig. 4Go). Similarly, phospho-mACC did not oscillate, and the average levels did not differ among the groups (data not shown).

Circadian expression and enzyme activity of mPepck/mPEPCK in mice fed LF and HF diet
The effect of fasting and HF diet on mPEPCK, a key enzyme in gluconeogenesis that is affected by adiponectin, was then evaluated. mPepck mRNA oscillated robustly in the LF and LF+F animals, with a peak in the middle of the subjective day (Fig. 5AGo). HF diet caused a marked decrease in the amplitude and a 3-h phase delay (Fig. 5AGo and supplemental Table 2). Evaluation of mPEPCK average activity according to the method by Chang and Lane (33), at circadian time 6 and circadian time 18, as representatives of activity levels during the mid-light period and mid-dark period, respectively, revealed an expected increase (40%) in the fasting groups compared with the LF group (Fig. 5BGo).


Figure 5
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  		FIG. 5. Daily rhythm of mPepck mRNA and mPEPCK protein activity levels. A, Expression levels of mPepck were determined by quantitative real-time PCR in the liver collected every 3 h around the circadian cycle from mice fed either LF+F (Figure 5), HF+F ({triangleup}), or LF({diamondsuit}). B, Average activity of mPEPCK was calculated according to Chang and Lane (33 ) using circadian time 6 and circadian time 18, as representative levels in the middle of the light period and dark period, respectively. Values are mean ± SE; n = 4 for each time point in each group. *, P < 0.05. The gray and black bars designate the subjective day (formerly the light period) and dark cycles, respectively. CT0 and CT12 represent the circadian times at which the lights would have been turned on and off, respectively, had the animals remained in light-dark.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that the components of the adiponectin metabolic pathway in the liver exhibit circadian rhythmicity under LF conditions. In addition, the effect of fasting and HF diet was different on the various clock genes and components of adiponectin signaling.

Effect of fasting and HF diet on clock gene expression
Our results show quite clearly that fasting results in the attenuation of clock gene amplitude and a phase advance in mPer2, mClock, and mBmal1 (Fig. 3Go and supplemental Table 2). Previously, it has been reported that fasting resulted in an increase in mPer1 and a decrease in mPer2 and mBmal1 mRNA expression in the liver (34, 35). However, Kobayashi et al. (34) and Kawamoto et al. (35) conducted their experiments under light-dark conditions, whereas our tissue collection was conducted in total darkness. Because light is a strong synchronizer for the biological clock, it is possible that it masked the effect of the food signal. In contrast with the effect of fasting, HF diet resulted in a 3-h phase delay of mPer1 in the liver (Fig. 3Go and supplemental Table 2). Yanagihara et al. (36) found only minimal effects of HF diet on the rhythmic expression of clock genes. However, our study examined eight time points under constant darkness, whereas Yanagihara et al. (36) assessed only four time points under light-dark conditions.

Oscillation of components of the adiponectin signaling pathway
We found that mAdipoR1, mPpar{alpha}, mAmpk, mAcc, and mPepck oscillated at the mRNA level and mAMPK at the protein level. The increased expression of adiponectin receptors as a result of fasting is in concert with previous results in the liver (37) and in brown and epigonadal adipose tissue (38). Because each of these receptors has a distinct role in mediating adiponectin effects, it seems that they are regulated differently. This diversity may enable the tissues to adapt to changes in the metabolic state. mPpar{alpha} was found to oscillate similarly to what was previously reported (39). Interestingly, we found dampening of mAMPK levels under HF diet (Fig. 4Go), which could result in further accumulation of lipids in the liver in obesity, as was previously reported (40). Our findings that phosphorylated mAMPK did not oscillate and that the average levels did not differ among the groups are in concert with previous studies in which HF diet did not affect phospho-AMPK levels in the liver (41). It seems that liver regulation of AMPK phosphorylation is complex and influenced by a combination of several factors including serum leptin levels and AMP/ATP ratio (42). Although mAcc mRNA exhibited a daily rhythm with a peak in the middle of the subjective day in the liver, as was previously reported (43), we could not detect any oscillation at the protein level (Fig. 4Go). Although ACC protein levels have never been evaluated, ACC activity has been analyzed. Although some described diurnal variation reaching maximal activity at night and minimal during the light period (44, 45), others did not observe any circadian variation (43). mPepck mRNA oscillated robustly in the LF and LF+F animals, with a peak in the middle of the subjective day (Fig. 5AGo), as was previously reported (15). These findings are expected, because mPEPCK is a key enzyme in gluconeogenesis, a pathway that is up-regulated during the inactive period. mPepck transcription was also found to be regulated by albumin D-site binding protein (46), a well-characterized clock-controlled gene. In addition, mClock mutation resulted in the loss of mPepck rhythmicity (15). All these findings support the notion that mPepck is a circadian-controlled gene.

Expression phase of clock genes and components of the adiponectin signaling pathway
In addition to the oscillation of mAdipoR1, mPpar{alpha}, mAmpk, mAcc, and mPepck at the mRNA level and mAMPK at the protein level, fasting results in a phase advance and dampening of the mRNA amplitude of components of the adiponectin signaling pathway. HF diet had the most dramatic effect causing disruption and a 3-h phase delay of components of the adiponectin signaling pathway (supplemental Table 2). These results are consistent with the effects seen on clock genes, as discussed above. Kohsaka et al. (31) tested the effect of HF diet, and their results matched previous reports in which obesity had been shown to disrupt circadian rhythms (47). However, in Kohsaka et al. (31), the animals were not fasted before tissue analyses. The fact that a phase shift (either delay or advance) was not observed in their experiments emphasizes the importance of fasting in comparing between LF and HF diet. This is further emphasized in light of the known effects of feeding on circadian rhythms (7). Thus, our results demonstrate the importance of fasting when analyzing the effect of diet on the circadian clock. In addition, we show for the first time that HF diet leads not only to disrupted rhythms but also to a phase delay. Fasting by itself leads to a phase advance. Recently, mAMPK has been found to phosphorylate Ser-389 of CKI{epsilon}, resulting in increased CKI{epsilon} activity and degradation of mPer2. mPer2 degradation leads to a phase advance in the circadian expression pattern of clock genes in wild-type mice (48). As the levels of mAMPK decline under HF diet (Fig. 4Go), it is plausible that the changes seen in the expression phase of genes under HF diet vs. fasting are mediated by changes in AMPK levels (supplemental Fig. 1). Thus, HF diet disrupts clock gene expression (31, 47), which in turn affects the daily rhythm of important components of glucose and lipid metabolism (14). In addition, the levels of mAMPK, which are controlled by the diet, can alter the phase of gene expression. Changes in the phase and daily rhythm of components of glucose and lipid metabolism may lead to obesity, steatosis (14), and disruption of clock-controlled output systems, such as blood pressure, sleep/wake cycle, etc. (49).

In summary, the constant levels of adiponectin in the serum under HF diet, similarly to what was previously shown (50), alongside changes in the expression of the adiponectin receptor, mAMPK, mACC, and mPpar{alpha} reflect a defect in downstream adiponectin signaling that might lead to adiponectin resistance similarly to insulin resistance (Fig. 2Go). Moreover, disruption in the phase and circadian expression of the clock mechanism and adiponectin components, such as mPpar{alpha} and mAmpk, which promote β-oxidation, may lead to the impairment of lipid metabolism in the liver. These findings may represent a vicious circle, in which obesity-inducing diet results in disrupted circadian rhythms, which, in turn, lead to obesity and steatosis (8, 14).


   Footnotes
 
This work was supported by Nutricia Research Foundation (Grant 2008-16).

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 18, 2008

Abbreviations: ACC, Acetyl-coenzyme A carboxylase; AMPK, AMP-activated protein kinase; BMAL1, brain-muscle-Arnt-like 1; CKI{epsilon}, casein kinase I{epsilon}; CRY, cryptochrome; DD, total darkness; DNase, deoxyribonuclease; HF, high-fat; HOMA-IR, homeostasis model assessment of insulin resistance; LF, low-fat; m, mouse; PEPCK, phosphoenolpyruvate carboxykinase; PER, period; PPAR{alpha}, peroxisome proliferator-activated receptor-{alpha}.

Received June 25, 2008.

Accepted for publication September 5, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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