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Endocrinology, doi:10.1210/en.2007-0816
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Endocrinology Vol. 149, No. 4 1914-1925
Copyright © 2008 by The Endocrine Society

Daily Rhythms in Metabolic Liver Enzymes and Plasma Glucose Require a Balance in the Autonomic Output to the Liver

Cathy Cailotto, Caroline van Heijningen, Jan van der Vliet, Geoffrey van der Plasse, Caroline Habold, Andries Kalsbeek, Paul Pévet and Ruud M. Buijs

Netherlands Institute for Neuroscience (C.C., C.v.H., J.v.d.V., G.v.d.P., A.K., R.M.B.), 1105 BA Amsterdam, The Netherlands; Department de Neurobiologie des Rythmes (C.C., P.P.), Institut des Neurosciences Cellulaires et Intégratives Unité Mixté de Recherche/LC2 7168, 67000 Strasbourg, France; Département d’Ecologie, Physiologie et Ethologie (C.H.), Institut Pluridisciplinaire Hubert Curien, Unité Mixté de Recherche 7178 Centre National de la Recherche Scientifique-ULP, 67087 Strasbourg Cedex 2, France; and Instituto Investigaciones Biomédicas (R.M.B.), Universidad Nacional Autónoma México, México

Address all correspondence and requests for reprints to: Cathy Cailotto, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. E-mail: c.cailotto{at}nin.knaw.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Daily variations in plasma glucose concentrations are controlled by the biological clock, located in the suprachiasmatic nucleus. Our previous studies indicated an important role for the sympathetic innervation of the liver in the generation of the daily glucose rhythm. In the present study, we investigated further the role of the autonomic nervous system (ANS) in the genesis of the plasma glucose rhythm. First, we showed that complete removal of the autonomic inputs to the liver did not impair the plasma glucose rhythm or the daily expression of the glucoregulatory enzymes in the liver. Consequently, we studied whether the daily glucose rhythm is driven by the daily feeding activity in denervated animals. Surprisingly, complete denervation combined with a noncircadian feeding schedule also did not abolish the 24-h profile in plasma glucose or all daily rhythms in the gene expression of liver enzymes. These results demonstrate that the mechanisms used by the suprachiasmatic nucleus to control the rhythmic expression of glucose-metabolizing enzymes and the 24-h rhythm in plasma glucose concentrations are highly versatile and the glucose rhythm can be maintained in absence of hepatic ANS input and/or a day/night rhythm in feeding activity. Interestingly, a hepatic sympathectomy or parasympathectomy did abolish the plasma glucose rhythm, demonstrating that a unilateral denervation of the liver is more deleterious to maintaining the rhythmic liver metabolism than a complete removal of both branches. This observation supports the notion that an unbalanced ANS in obesity and diabetes accounts for the disturbed glucose balance in these disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BRAIN IS intimately involved in regulating blood glucose levels not only within an optimal physiological range (1) but also according to the time of the day (2, 3). In the latter process, the biological clock, located in the suprachiasmatic nucleus (SCN), is of the utmost importance (4, 5). In fact in rodents, the daily variation in plasma glucose concentrations, characterized by a peak before the onset of the activity period, is driven by the SCN, independent of the daily rhythm in feeding activity (6). A physiologically quite meaningful fact is that the highest plasma glucose concentration and glucose tolerance coincide just before awakening, suggesting that an increase of glucose output by hepatic glucose production is required to compensate for the increased glucose uptake (7, 8). In agreement with this hypothesis, human studies also indicated hepatic glucose output as a major factor in the morning rise of glucose (3). Recently we demonstrated that the sympathetic input to the liver plays an essential role in the daily rise in plasma glucose concentrations (8, 9). Interestingly, within the liver, key enzymes involved in glucose metabolism display an oscillatory pattern in their gene expression along the 12-h light, 12-h dark (L/D) cycle, suggesting a role of the circadian pattern of hepatic gene expression in the genesis of the 24-h rhythm in plasma glucose concentrations (10, 11, 12). Here we show that, in addition to a loss of the daily rhythm in blood glucose concentrations (9), hepatic sympathectomy (HSx) combined with a six-meal-a-day feeding schedule also disrupts the daily rhythm of the glucoregulatory liver enzymes. Taking these observations together, we questioned whether any autonomic nervous system (ANS) input to the liver is essential to drive the daily rhythm in plasma glucose concentrations and to what extent a daily rhythm in the expression of glucose-metabolizing liver enzymes can be responsible for the daily glucose rhythm. We investigated these questions by removing both branches of ANS input to the liver and determining the daily gene expression profile of a number of glucoregulatory liver enzymes. Because plasma glucose rhythms persisted after a complete hepatic denervation, we decided to investigate whether the day/night rhythm in feeding activity could be involved in the daily variation of plasma glucose concentrations. To achieve this goal, we combined complete hepatic denervation with a noncircadian feeding regimen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Adult male Wistar rats (Wu, Harlan Nederland, Horst, The Netherlands) were kept in a L/D cycle [lights on at 0700 h, Zeitgeber time (ZT) 0]. All experiments were conducted with the approval of the Animal Care Committee of the Royal Netherlands Academy of Arts and Sciences.

Hepatic denervation
The parasympathetic (HPx) and sympathetic (HSx) denervations of the liver were performed according to our previously published methods (8, 9). An intraatrial silicone cannula was implanted through the jugular vein according to the method of Steffens (13) in all groups in which glucose, insulin, and corticosterone concentrations had to be measured.

Histology
For the physiological groups, HPx completeness [in complete denervated rats (CD)] was checked by injection of cholera toxin subunit B (CTB)-alexa fluor 488 (1%, no. C22841; Molecular Probes, Leiden, The Netherlands), as previously described (14). Ten days after tracer injection, the animals were perfused with saline and then with 4% paraformaldehyde (pH 7.4). At the time the animals were killed, pieces of liver were quickly taken from all animals (before perfusion) and directly frozen in nitrogen for the measurement of noradrenalin (NA) content. The immunocytochemical staining for CTB of the brainstem sections (Fig. 1Go) was performed according to our published method (14). The parasympathetic denervation was successful in 26 of the 26 animals, i.e. a 100% success rate.


Figure 1
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  		FIG. 1. Transversal brain sections at the level of the brain stem stained for CTB. The liver-intact animals (i.e. parasympathetic branch intact) show CTB staining in the dorsal motor nucleus of the vagus (DMV) (A), whereas in the HPx animals, no CTB labeling in the DMV is observed (B). Black bar, 250 µm. AP, Area postrema; C, central canal. In A, the level of the transversal section is not identical between the left and right sides; therefore, CTB staining is observed only on the left side of the section in the liver-intact animal.

 
The HSx completeness was systematically checked in all experimental groups (including CD animals) by measuring NA content in the liver (liver tissue was collected immediately after the killing of the animals and put directly in liquid nitrogen) with HPLC according to our published method (9). The HSx was considered successful when the NA content measured in the liver was less than 15% of the NA content measured in intact liver (9). The HPLC detection limit (ratio signal to noise = 2) was 1 pg of NA injected on column. ANOVA indicated no significant difference in NA content (P = 0.49) between nonoperated control livers (n = 9) and CTB-injected livers (n = 9), indicating that the CTB injection did not affect the hepatic NA content.

Experimental set-up
Experiment 1: effect of HSx on glucoregulatory gene expression in animals subjected to scheduled feeding
In the first part of this study, we checked whether the loss of the glucose rhythm, observed previously in HSx rats under scheduled feeding (9), could be correlated with an abnormal daily expression profile of glucoregulatory enzymes in the liver. To this end, we analyzed the daily expression of glucose-6-phosphatase (G6Pase), glucokinase (GK), pyruvate kinase (PY), glucose transporter (GLUT)-2, phosphoenolpyruvate kinase (PEPCK) and the glycogen content in the liver along the L/D cycle in a group of sham-operated animals placed under ad libitum conditions (n = 5 per time point) and a group of complete HSx rats (n = 5 per point) subjected to a six-meal schedule (9).

Experiment 2: complete hepatic denervation and ad libitum conditions
Effect on the daily hormonal profile.
Rats were divided into two groups: a sham-operated group (n = 10) and a liver CD group (n = 14). Two weeks after surgery, 0.2 ml of blood was collected for the measurement of plasma glucose, insulin, and corticosterone concentrations, once every hour for 12 consecutive hours on two different occasions within a period of 2 wk. The two runs started at ZT6.5 and ZT18.5. Blood samples were kept in –80 C until analysis.

Effect on gene expression of glucoregulatory enzyme/glycogen content.
Rats were divided into two groups: sham-operated (n = 30) and CD (n = 42). After 2 postoperative weeks, both groups were killed in a schedule of every 4 h along the L/D cycle (ZT2, 6, 10, 14, 18, and 22).

Experiment 3: complete hepatic denervation combined with scheduled feeding regimen
For this experiment, rats were entrained to the scheduled feeding regimen according to our published method (9). Briefly, rats were entrained to six meals spread equally over the L/D cycle (ZT2, 6, 10, 14, 18, and 22). The access to food was 11 and 9 min for the daytime and nighttime meals, respectively. The rats were given 3 wk to adapt to the feeding schedule. This regular feeding schedule was maintained until the end of the experiments.

Effect on the daily hormonal profile.
Two weeks after surgery, blood sampling was performed, as in experiment 2, on sham-operated (n = 8) and CD (n = 12) animals.

Effect on gene expression of glucoregulatory enzyme/glycogen content.
The CD group contained 36 rats, i.e. six animals per time point. After 2 postoperative weeks, animals were killed every 4 h along the L/D cycle.

Real-time PCR (RT-PCR)
Quantitative analysis of gene expression was done using RT-PCR. RNA extraction and single-stranded cDNA was synthesized using a kit provided by QIAGEN (Courtaboeuf, France) and Invitrogen (Leiden, The Netherlands), respectively (for more details, see (9)]. PCRs and the primers for G6Pase (forward, 5'-CCCATCTGGTTCCACATTCAA-3'; reverse, 3'-GGCGCTGTCCAAAAAGAATC-5'), GK (forward, 5'-TCCTCCTCAATTGGACCAAGG-3'; reverse, 3'-TGCCACCACATCCATCTCAA-5'), GLUT2 (forward, 5'-GAAGGATCA-AAGCCATGTTGG-3'; reverse, 3'-CCTGATACGCTTCTTCCAGCA-5'), PY (forward, 5'-GAGAGTTTTGCAACCTCCCCA-3'; reverse, 3'-CCTTCACAATTTCCACCTCCG-5'), Pepck (forward, 5'-TGCCCTCTCCCCTTAAAAAAG-3'; reverse, 3'-CGCTTCCGAAGGA-GATGATCT-5') and the two reference genes (ubiquitin conjugate enzyme, Ubc2e, and TATA box binding protein, Tbp) were done as previously described (9). The mRNA levels of the metabolic enzymes were quantified in each sample collected throughout the L/D-cycle. The amount of each transcript was first normalized independently, with the average calculated from the transcript levels along the L/D cycle. The value obtained for each transcript was then divided by the average of the normalized values of the two reference genes.

Measurement of the hepatic glycogen content
Frozen liver tissue was crushed on dry ice to obtain powder. Liver powder was homogenized into 0.1 M potassium hydroxide (pH 13). Homogenization tubes were placed in a water bath (80 C) for 30 min and then placed on ice and left to reach room temperature. Formic acid was added to the homogenized solution to adjust the pH to 4 and then centrifuged for 1 min at 14,000 rpm. The aliquots were collected in new tubes. For each sample, 100 µl were incubated with 10 µl of amylo-{alpha}-1.4-{alpha}1,6-glucosidase (from stock solution diluted half, catalog no. 102857; Roche, The Netherlands) at 40 C for 2 h (15, 16, 17). By way of control, i.e. free glucose concentrations in the liver, 100 µl of the sample were incubated without the amyloglucosidase for 2 h. As a standard, replicate rabbit liver standard (G-8876; Sigma, The Netherlands) was used and treated in the same way as the samples. Glucose concentrations were determined in the supernatant, using the glucose/GOD-Perid method (Boehringer Mannheim GmGH, Mannheim, Germany). The glycogen content in the liver tissue was calculated by (glucose concentration measured in the sample, the control) and converted to glycogen (picograms per milligram) of tissue by using the replicate rabbit liver glycogen standard.

Measurement of the hepatic PEPCK activity
PEPCK activity was measured by the decarboxylation assay previously described (18, 19). Briefly, 100 mg of liver tissue were homogenized in 900 µl buffer solution [i.e. HEPES 10 mM + sucrose 250 mM (pH 7.3)] and centrifuged at 100,000 x g for 1 h at 4 C. Fifty microliters of the cytosolic supernatant were then transferred in 850 µl of reaction buffer [i.e. Tris-base 50 mM (pH 8), MnCl2 0.75 mM, NAD+ 1 mM, malate dehydrogenase 6 U/ml, GTP 1 mM]. Reaction was started by adding 100 µl of 100 mM malate (end concentration 10 mM). Oxaloacetate formation from malate by malate dehydrogenase was then determined spectrophotometrically at 37 C by measuring nicotinamide adenine dinucleotide hydroxide (NADH) formation. The measurement was repeated every 10 sec during 10 min. A reaction blank was obtained without malate. The samples were performed in duplicate. The calculations were made as follow: PEPCK activity (moles per liter per minute) = ({delta}OD/{delta}t) x (1/{epsilon}), where {epsilon} is the extinction coefficient for nicotinamide adenine dinucleotide hydroxide = 6.22 mM/cm at 340 nm, and 1 is the curve thickness (in centimeters). The results were then expressed as mean of micromoles per minute per gram (unit per gram) of liver tissue.

Plasma measurements
Plasma glucose concentrations were determined using the glucose/GOD-Perid method, whereas plasma insulin and corticosterone concentration were measured using a RIA kit (Linco Research, St. Charles, MO, and ICN Biomedicals, Costa Mesa, CA, respectively). For more details see (9).

Statistical analysis
Glucose, corticosterone and insulin profiles.
The plasma concentrations of glucose, corticosterone, and insulin are expressed as mean ± SEM. Repeated-measures ANOVA was used to test for an effect of time. If ANOVA detected a significant effect of time, cosinor analysis was performed using constrained nonlinear regression analysis (with the SPSS Advanced Statistic 11.0), as reported in our previous article (9). For the ANOVA, (paired) t and the cosinor analysis, P < 0.05 was considered to be a significant difference. In all cases, statistics and cosine analysis were done on absolute values.

RT-PCR and glycogen data analysis
Data from all experiments are presented as mean ± SEM. To demonstrate a statistically significant effect of time within each experimental group, the P value of one-way ANOVA was calculated on the basis of the normalized data (SPSS package, version 12.0; SPSS, Chicago, IL). RT-PCR data were also analyzed by a mixed one-way ANOVA (group (2 levels), i.e. sham vs. denervated or ad libitum vs. six-meal schedule; time (six levels), i.e. ZT 2, 6, 10, 14, 18, and 22). If significant effects were detected, it was followed by a post hoc least significant differences test. The differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The daily mRNA profile of liver metabolic enzymes
Under ad libitum conditions, glucokinase, Pepck mRNA expression, and glycogen content (Fig. 2Go) in sham liver-denervated animals show rhythmic profiles along the L/D cycle (P < 0.05) in accordance with data previously described for nonoperated animals (11, 12, 20, 21, 22). GK displayed a higher mRNA level during the night; Pepck shows an elevated expression at the end of the day (ZT10). Despite the fact that G6Pase gene expression did not show a significant effect of time (P = 0.19), we did observe a clear difference between the day and night mRNA levels as previously described (23). The diurnal gene expression of G6Pase also fits with the daily profile of its enzymatic activity (24, 25). No daily pattern in gene expression was observed for PY and GluT2 (Table 1Go).


Figure 2
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  		FIG. 2. Daily profile of G6Pase, GK, GluT2, PY, and PEPCK mRNA levels and glycogen content in the liver of sham-operated rats (data are represented as the mean ± SEM). Animals were housed in L/D cycle with lights on at 0700 h under ad libitum conditions and were killed (n = 5 for ZT10/18 and 22; n = 4 for ZT2/6 and 14) at the indicated times of the daily cycle. The peak of mRNA expression for each liver enzyme was compared with that of other time points and was considered significantly different (*) for P < 0.05 (see Table 1Go). The black bars indicate the dark period.

 

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  		TABLE 1. Statistical analysis of the daily variations of hepatic enzyme mRNA expression and glycogen content in groups of rats placed under ad libitum condition (sham vs. CD) and groups of CD rats (ad libitum vs. six-meal schedule)

 
Effect of HSx on glucoregulatory gene expression in animals subjected to scheduled feeding
Two HSx animals did not recover from the surgery. Thirteen of the 28 animals left displayed no detectable levels of NA in the liver, and four animals showed NA content less than 15% of the control levels. In the remaining 17 animals, the rhythmic profiles of the mRNA of all the liver enzymes examined and of the liver glycogen content were affected in HSx rats subjected to scheduled feeding (Fig. 3Go and Table 2Go). In fact, in contrast to the sham-operated animals, one-way ANOVA did not reveal a significant effect of time for most of the liver enzymes. Only PY displayed a significant changes over the 24 h (P = 0.02) (Table 2Go). These results indicate that HSx combined with a six-meal schedule profoundly disturbed the daily pattern of all the studied genes and glycogen content.


Figure 3
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  		FIG. 3. Daily profile in metabolic enzyme mRNA levels and glycogen content (data are represented as the mean ± SEM) in the liver of HSx animals subjected to a six-meal schedule (n = 3 except for ZT14, n = 2). The peak of mRNA expression for each liver enzyme was compared with that of other time points and was considered significantly different (*) for P < 0.05. The daily expression of these enzymes in sham-operated animals fed ad libitum (Fig. 2Go) is represented as a dashed line.

 

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  		TABLE 2. Statistical analysis of the daily variations of hepatic enzyme mRNA expression and glycogen content in a group of HSx rats subjected to the six-meal feeding schedule regimen

 
To be able to separate the (disturbing) effects of the six-meal schedule and the HSx on the daily profile of the enzyme expression, we measured G6Pase, Gluk, and Pepck expression (the three enzymes that exhibited clear daily changes in nondenervated animals) in two groups of intact animals either fed ad libitum or subjected to a six-meal feeding schedule. No significant effect of feeding pattern on the daily changes in the liver enzymes was detected (see supplemental data Fig. S1 and Table S1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://endo.endojournals.org), indicating that arrhythmic feeding on its own does not cause an enzyme expression.

Taken all these observations together, we concluded that the abnormal daily patterns in liver enzyme expression and hepatic glycogen content after HSx are indicative for an impairment of the circadian glucose metabolism within the liver and may therefore contribute to the loss of the rhythm in plasma glucose concentrations as previously demonstrated [Fig. 4AGo; (9)].


Figure 4
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  		FIG. 4. Plasma glucose (A and C) and corticosterone (B and D) concentrations across the L/D cycle under scheduled feeding conditions in HSx (A and B), HPx (C and D), and sham-operated rats. The HSx data are derived from our prior study (9 ). The HPx data are extracted from the thesis of La Fleur (42 ). The gray areas indicate the mean ± SEM plasma glucose concentrations in sham-operated animals. The black bar indicates the dark period. The vertical dotted lines represent the times of food availability (10 min).

 
Complete hepatic denervation in ad libitum-fed animals
In experiments 2 and 3, we aimed to examine the role of the ANS and the daily feeding rhythm in the 24-h rhythm of plasma glucose concentrations and to investigate further the relation between the circadian profile of liver enzymes and rhythmic plasma glucose concentrations.

As a first step, we investigated the effect of a complete hepatic denervation on the daily rhythms of glucoregulatory liver enzymes and plasma glucose concentrations. In this experiment, the amount of food consumed during day and night periods did not significantly differ between sham-operated (n = 14) and CD animals (n = 18): 3.1 ± 0.3 vs. 3.4 ± 0.2 g during the light period (P = 0.34) and 20.2 ± 0.6 vs. 18.4 ± 0.7 g during the dark period (P = 0.10). Furthermore, 34 of 56 rats were completely denervated, i.e. no CTB staining and no detectable NA content.

Effect on the daily hormonal profile
Plasma glucose concentrations.
The 24-h profile of plasma glucose concentrations (Fig. 5AGo) measured in the sham-operated rats (n = 9) showed a significant variation along the L/D cycle (P < 0.001) with a peak occurring at ZT 11.3 ± 1.21 [goodness of fit (R2) = 0.37 ± 0.12]. Plasma glucose concentrations of the eight CD animals also showed a significant variation along the L/D cycle (P < 0.001). The data from the eight CD animals could be fitted to a cosinor curve, showing that the denervation did not affect the rhythmic profile of the plasma glucose concentrations. However, the significant Group x Time interaction revealed that CD animals displayed higher plasma glucose concentrations during the daytime, compared with those in sham-operated rats. Indeed the Student’s t test indicated a significant difference in the mean plasma glucose concentrations during the daytime between sham-operated and CD rats (6.1 ± 0.24 vs. 6.9 ± 0.20; P = 0.02). On the other hand, no significant difference was indicated between the means of their nighttime plasma glucose concentrations (5.98 ± 0.21 vs. 6.13 ± 0.13 mmol/liter).


Figure 5
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  		FIG. 5. Plasma glucose (A and D), corticosterone (B and E), and insulin (C and F) concentrations across the L/D cycle in CD (black circles; n = 8) and sham-operated rats (white circles; n = 9) under ad libitum conditions (A–C) and in animals subjected to a scheduled feeding regimen (D–F). The data are represented as the mean ± SEM. The black bars indicate the dark period. The vertical dotted lines represent the times of food availability (10 min).

 
Plasma corticosterone concentrations.
The 24-h profiles of plasma corticosterone concentrations (Fig. 5BGo) measured in the sham-operated and CD rats showed a significant variation along the L/D cycle (P < 0.001). The cosinor analysis revealed a peak at ZT 12.12 ± 1 (R2 of 0.56 ± 0.14) and ZT 12.19 ± 0.5 (R2 of 0.53 ± 0.15) for sham and CD rats, respectively. These data indicate a similar 24-h rhythm in plasma corticosterone concentrations in both groups. The 24-h mean plasma corticosterone concentrations in CD rats did not differ significantly (P = 0.141) from those of sham-operated rats (50.23 ± 5.22 vs. 62.32 ± 5.68 mmol/liter).

Plasma insulin concentrations.
Despite the higher daytime glucose concentrations, the basal insulin concentrations in sham-operated and CD animals (Fig. 5CGo) did not show a variation over the L/D cycle (P = 0.07 and P = 0.21, respectively). The absence of group and Group x Time effects indicates that the denervation does not affect daily plasma insulin concentrations over the L/D cycle. Furthermore, the 24-h mean plasma insulin concentrations in CD rats did not differ significantly from those of sham-operated rats (2.74 ± 0.25 vs. 2.81 ± 0.31 mmol/liter).

Effect on gene expression levels of liver enzymes/glycogen content
The absence of a group effect, for the studied enzymes and glycogen content, shows that the daily profile of the glucoregulatory enzymes in the liver persisted after a complete hepatic denervation in animals fed ad libitum (Fig. 6Go). Only Pepck mRNA expression did not show a rhythmic pattern in the CD group (P = 0.9), in contrast to the sham-operated group (P = 0.02). The absence of a Group or Group x Time effect may be explained by the intermediate levels of Pepck mRNA throughout the L/D cycle in the CD animals. The circadian profile in GK mRNA expression in CD animals exhibited a somewhat higher levels during daytime, compared with the sham-operated animals (grouptime effect). Glycogen content in CD rats remained rhythmic (P < 0.001), but a significant effect of group and grouptime was detected, showing that denervation does modify the 24-h profile of the hepatic glycogen content, i.e. lower levels at the beginning of the light period (Table 1Go).


Figure 6
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  		FIG. 6. Daily profile in glucose-metabolizing enzyme and hepatic glycogen content in CD (black circles; n = 5 for ZT2/6, n = 4 for ZT10, n = 3 for ZT14/22, and n = 6 for ZT18) and sham-operated animals (white circles; same as in Fig. 2Go) fed ad libitum. The data are represented as the mean ± SEM. The black bars indicate the dark period. For statistics see Table 1Go. *, P < 0.05, compared with the control group.

 
To examine whether Pepck mRNA expression can be translated into Pepck enzyme activity, we also examined in this experiment next to the daily rhythm in Pepck mRNA expression enzyme activity. PEPCK activity displayed a significant effect of time (P ≤ 0.001) with a peak at ZT14 in nondenervated animals in contrast to CD animals (P = 0.803) (Fig. 7Go). These results are in line with previous findings in which a tight correlation has been found between Pepck gene expression and its enzymatic activity (1, 38, 39). In addition, these data demonstrated that the loss of daily changes in Pepck gene expression in the CD animals is also reflected in a loss of rhythmicity in enzyme activity.


Figure 7
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  		FIG. 7. PEPCK activity in sham-operated (gray bars; n = 4 for ZT6/14 and n = 5 for ZT10/18) and CD rats (black bars; n = 5 for ZT18/6, n = 3 for ZT10 and n = 2 for ZT14) fed ad libitum. The data are represented as the mean ± SEM. The peak of PEPCK activity at ZT14 in the sham-operated animals was compared with that of the other time points and was considered significantly different (*) for P < 0.001 A bracket with an asterisk indicates a time point in which the two experimental groups differ significantly from each other (unpaired t test, *, P < 0.001).

 
In summary, the integrity of the ANS is necessary to maintain a rhythmic gene expression of Pepck, whereas the sustained oscillatory mRNA expression of GK, G6Pase, and glycogen in CD animals seems to rely on another rhythmic signal, e.g. rhythmic feeding activity. Therefore, we decided to study blood glucose levels and liver metabolic enzyme expression after CD, independent of a daily rhythm in food intake.

Complete hepatic denervation combined with a scheduled feeding regimen
The amount of food consumed before denervation (n = 23) did not significantly differ from the amount consumed after the animals had undergone a complete denervation of the liver (10.79 ± 0.07 vs. 10.50 ± 0.12 g for the light period (i.e. three meals, P = 0.235); 10.12 ± 0.05 vs. 9.91 ± 0.11 g for the dark period (i.e. three meals, P = 0.428). Furthermore, 35 of the 48 animals operated were completely denervated.

Effect on the daily profile of plasma glucose, corticosterone, and insulin concentrations
Plasma glucose concentrations.
The 24-h profiles of plasma glucose concentrations (Fig. 5DGo) measured in the sham-operated rats subjected to the six-meal schedule showed a significant variation along the L/D cycle (P < 0.001) as described previously (6, 26). The data points of five of the six sham-operated rats could be fitted to a cosinor curve, indicating that the plasma glucose concentrations displayed a 24-h rhythm [peak at ZT 9.8 ± 0.6; goodness of fit (R2) = 0.34 ± 0.14]. Plasma glucose concentrations of the seven CD animals also displayed a significant variation along the L/D cycle (Fig. 4AGo) (P < 0.001) with a peak occurring at ZT 10.6 ± 1.0 [goodness of fit (R2) = 0.35 ± 0.13]. No significant effects of Group and Group x Time were detected, showing that the plasma glucose concentrations in CD animals did not differ from those of the sham-operated ones.

Plasma corticosterone concentrations.
The 24-h profiles of plasma corticosterone concentrations (Fig. 5EGo) measured in the sham-operated and CD rats showed a significant variation along the L/D cycle (P < 0.001) with a peak at ZT 12.7 ± 0.7 (R2 = 0.48 ± 0.16) and ZT 11.4 ± 0.4 (R2 of 0.34 ± 0.11), respectively. An effect of Group x Time indicated a slight shift in the daily rise in plasma corticosterone concentrations in CD groups. The 24-h mean plasma corticosterone concentrations in CD rats were not significantly different from those of sham-operated rats (36.73 ± 4.97 vs. 40.67 ± 18.19 mmol/liter).

Plasma insulin concentrations.
The basal insulin concentrations in sham-operated and CD animals (Fig. 5FGo) showed a variation over the L/D cycle (P < 0.01), with a clear increase at each meal time. The absence of Group and Group x Time effects indicates that the denervation does not affect the insulin pattern over the L/D cycle. Moreover, the 24-h mean plasma insulin concentration in CD rats was similar to those of sham-operated rats (2.35 ± 0.19 vs. 2.91 ± 0.25 mmol/liter).

Effect on gene expression of glucoregulatory enzymes and glycogen content
To investigate the influence of food intake without interference of the ANS, the 24-h profiles of liver enzymes were measured in 28 CD animals subjected to a scheduled feeding regimen and compared with those obtained in CD animals fed ad libitum (Fig. 8Go). The daily pattern in G6Pase mRNA expression is maintained in both feeding conditions, but the peak values occur at different times of the day, i.e. at ZT6 and ZT22 for ad libitum and regular feeding animals groups, respectively (Fig. 6Go). A similar phase advance was observed in the daily rhythm of GK mRNA in the CD animals with arrhythmic feeding, compared with the ad libitum-fed animals (Group x Time effect, P < 0.001). No significant effect of time for Pepck was found in either group of CD animals (Table 1Go), confirming the essential role of the ANS in the circadian control of Pepck gene expression. Interestingly, the diurnal variation in glycogen content persisted under complete denervation combined with a-rhythmic food intake, indicating that yet another rhythmic signal (i.e. not the ANS or the daily feeding rhythm) is responsible for the glycogen breakdown/storage rhythm. However, the weaker oscillation observed in the regular feeding group (P = 0.028 and P < 0.001 in six meals and ad libitum groups, respectively) indicates that food intake does contribute to the daily variation in the hepatic glycogen content.


Figure 8
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  		FIG. 8. Daily profile in glucose-metabolizing enzyme and hepatic glycogen content in CD rats subjected to a scheduled feeding regimen (black circles; n = 5 for ZT10/18 and 22, n = 4 for ZT14, n = 3 for ZT2, and n = 6 for ZT6) and under ad libitum conditions (white circles; see also Fig. 5Go). The data are represented as the mean ± SEM. The black bars indicate the dark period. For statistics see Table 1Go. *, P < 0.05, compared with the control group.

 
In summary, the daily rhythm in food intake is not the most important contributor to the rhythmic expression of glucoregulatory liver enzymes, although it does seem able to entrain the GK/G6ase rhythms.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we show that both rhythmic food intake and the autonomic inputs to the liver participate in, but are not indispensable components for, the genesis of a daily rhythm in plasma glucose concentrations. The results, observed in CD animals with a day/night rhythm in feeding behavior (i.e. ad libitum) or subjected to a noncircadian feeding schedule (i.e. six meals/day) indicate the existence of additional pathway(s) by which the SCN conveys its timing signal to drive blood glucose rhythm, i.e. via the oscillatory variation of plasma corticosterone concentrations. On the other hand, we demonstrated that a dysbalance of the ANS input [provided by the denervation of one branch of the ANS (Fig. 4Go)] does result in an arrhythmic expression of glucose metabolizing liver enzymes and the loss of rhythmic plasma glucose concentrations, despite the presence of an intact daily plasma corticosterone rhythm.

The SCN uses multiple mechanisms to control liver metabolism in a circadian manner
The analysis of experiments 2 and 3 allows us to understand better how the liver enzyme expression levels are controlled in a circadian manner and to what extend the daily expressions of these enzymes are essential to maintain the 24-h rhythm in plasma glucose concentrations. First, the observation of a loss of rhythmic expression of Pepck in CD animals fed ad libitum or subjected to six meals schedule indicates that the ANS participates in the circadian regulation of liver metabolism. Because the daily rhythm in plasma glucose levels was maintained under these two experimental conditions, we can conclude that the circadian expression of Pepck is not essential for the genesis of the glucose rhythm.

In CD animals fed ad libitum, the oscillatory pattern of G6Pase and GK mRNA expression and hepatic glycogen content are still in phase with the daily profile of the food intake, but combined with a six-meals-a-day feeding schedule, both enzyme rhythms are disturbed, suggesting that these liver enzymes are especially sensitive to the daily changes in food intake (27). Surprisingly, in both experiments the glucose rhythm was maintained despite the temporal changes in the rhythms of G6Pase, Pepck, and GK expression, indicating that the daily rhythms in the mRNA expression and activity levels of these glucoregulatory enzymes are not essential for the glucose rhythm.

Interestingly, the hepatic glycogen content exhibits a circadian profile in both experimental conditions (i.e. CD animals under the two feeding conditions) as well, showing that the enzymes involved in the storage/breakdown of glycogen are controlled in a circadian manner, independently of GK, G6Pase, and Pepck. Because glycogen depletion and the daily rise in plasma glucose concentrations occur at the same time of the day, it is tempting to suggest that the daily variation in hepatic glycogen participates in the genesis of the glucose rhythm. So, in absence of both autonomic connections and a daily rhythm in feeding behavior, the SCN is still able to provide a signal to generate this daily rhythm in hepatic glycogen content. Because glucagon has been shown to be strongly influenced by food intake (22), corticosterone appears to be a more appropriate candidate. Glucocorticoids have indeed been shown to be a powerful circadian signal for the liver (28), and the corticosterone rhythm persisted under both regular feeding and ANS denervation conditions.

Autonomic regulation of blood glucose rhythm
The loss of plasma glucose rhythmicity could be the consequence of a direct effect of hepatic denervation on hepatic glucose metabolism or an indirect effect of a change in hepatic activity on other peripheral organs such as muscle (29) or adipose tissue (30). Here we show that, in contrast to a complete disruption of the hepatic ANS input, an autonomic dysbalance induced by HSx in animals subjected to a six-meal schedule severely affects liver metabolism. Indeed the expression pattern of the enzymes GK, G6Pase, Pepck, and the glycogen content displayed abnormal circadian profiles, compared with the sham-operated animals under ad libitum feedings. Consequently, we propose that the loss of the plasma glucose rhythm is, at least partially, due to the loss of hepatic enzyme rhythms. Because animals subjected to six meals schedule (sham-operated or intact) do exhibit a clear 24-h rhythm in plasma glucose concentrations (6, 9), the dysbalance of the ANS (induced by the HSx) seems the most likely cause of the disruption of the plasma glucose rhythm. In line with this, when we investigated the plasma glucose concentrations of the five CD animals from experiment 3 that were rejected because of an incomplete sympathetic denervation (i.e. NA content > 15%), their data could not be fitted to a cosinor curve (Fig. 9Go). Therefore, these animals provide an unexpected reinforcement of our hypothesis that a dysbalance of the autonomic input (i.e. no parasympathetic innervation but partial sympathetic innervation) is more deleterious to the circadian rhythm in plasma glucose concentrations than a complete absence of the autonomic hepatic innervation.


Figure 9
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  		FIG. 9. Plasma glucose concentrations across the L/D cycle under scheduled feeding conditions in no complete CD (n = 5) rats. The black bar indicates the dark period. The vertical dotted lines represent the times of food availability (10 min). Closed symbols represent the group of animals with an incomplete sympathetic liver denervations (i.e. > 15% left); open symbols represent the group of complete CD animals.

 
In addition to sympathetic denervation, a denervation of the parasympathetic branch of the liver induces also a loss in glucose rhythm (Fig. 4CGo), reinforcing our dysbalance hypothesis. The results, obtained after complete hepatic denervation, are also in agreement with studies performed after liver transplantation in which no abnormal liver metabolism has been reported, confirming that the ANS is not essential for hepatic glucose metabolism (31, 32, 33). The absence of a glucose rhythm in HSx or HPx animals subjected to a noncircadian feeding regimen, despite an intact corticosterone rhythm (Fig. 4Go), indicates that in face of a dysbalanced autonomic output even the corticosterone rhythm is not sufficient to maintain a day/night rhythm in plasma glucose concentrations.

All these observations together lead us to propose that it is not the absence but rather the dysbalance of the ANS that induces an abnormal glucose metabolism in the liver, which in turn contributes to the loss of the daily plasma glucose rhythm. As a consequence of the autonomic dysbalance hepatic glucose metabolism is also disturbed, which is the most likely reason for the loss of the daily plasma glucose rhythm. Interestingly, a sympathovagal dysbalance has been proposed as an important mechanism underlying metabolic diseases such as obesity, type 2 diabetes, and metabolic syndrome (34, 35, 36). Our study therefore supports the notion that an unbalanced autonomic nervous system in obesity and diabetes may account for the disturbed glucose balance in these disorders.

In the present paper, we have not measured the activity of all the liver enzymes (24, 37, 38, 39). In theory their activity may compensate for the lack of rhythm in mRNA expression of the same enzymes. However, recent studies reported a clear correlation between the changes in the G6Pase and Pepck gene expression and the hepatic glucose output, resulting in changes in plasma glucose concentrations (1, 40, 41). In the present paper, we confirmed this clear correlation for the PEPCK enzyme and showed that the enzyme expression follows the changes in enzyme activity. Therefore the loss in circadian pattern of the genes for enzymes as observed in HSx animals under scheduled feeding is likely to contribute to the loss of the daily rhythm in plasma glucose concentrations. Furthermore, the results obtained with the CD groups (complete and incomplete) reinforce the idea that a loss of mRNA rhythmicity coincides with a loss of glucose rhythm. Therefore, it is clear that eventual compensatory changes in enzyme activity are not sufficient to compensate for the changes in enzyme expression and will not influence the conclusions of the present study.

In summary, the biological clock uses multiple pathways to drive liver metabolism in a circadian manner. Our data show the extreme capacity of the liver (and SCN) to use other signals to drive hepatic enzymes in a rhythmic fashion. The most striking observation is that a dysbalanced autonomic input to the liver is more harmful for a rhythmic glucose metabolism than its complete absence and is even able to block the strong synchronizing signal of the daily peak in plasma corticosterone.


   Acknowledgments
 
The authors acknowledge W. Verweij for correction of the manuscript, and E. J. W van Someren for help with the cosinor analysis.


   Footnotes
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online December 27, 2007

Abbreviations: ANS, Autonomic nervous system; CD, complete denervated rats; CTB, cholera toxin subunit B; GK, glucokinase; GLUT, glucose transporter; G6Pase, glucose-6-phosphatase; HPx, parasympathetic; HSx, hepatic sympathectomy; L/D, 12-h light, 12-h dark cycle; NA, noradrenalin; PEPCK, phosphoenolpyruvate kinase; PY, pyruvate kinase; RT-PCR, real-time PCR; SCN, suprachiasmatic nucleus; ZT, Zeitgeber time.

Received June 18, 2007.

Accepted for publication December 17, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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