What this is
- This research investigates how dietary (PAs) affect the molecular clock in the liver of rats.
- Specifically, it examines the timing of grape seed proanthocyanidin extract (GSPE) administration and its impact on gene expression and metabolic processes.
- Findings indicate that the effects of GSPE vary significantly depending on whether it is administered during the day or night.
Essence
- Dietary modulate liver clock genes and metabolic processes based on the timing of administration. GSPE affects Nampt expression and levels differently when given at ZT0 vs. ZT12.
Key takeaways
- GSPE administration at ZT12 (night) increased Nampt expression and levels, while administration at ZT0 (day) decreased them. This indicates a time-dependent effect of PAs on liver metabolism.
- The study found that GSPE administration at ZT12 led to increased acetylation of BMAL1, which is crucial for regulating Nampt expression and levels, suggesting a mechanism by which PAs influence circadian rhythms.
Caveats
- The findings are based on animal models, which may not fully translate to human physiology. Further research is needed to confirm these effects in humans.
- The study used a pharmacological dose of GSPE, which may not reflect typical dietary intake levels of .
Definitions
- Proanthocyanidins: A class of flavonoids found in many fruits and plants, known for their antioxidant properties and potential health benefits.
- NAD: Nicotinamide adenine dinucleotide, a coenzyme involved in redox reactions and essential for energy metabolism.
AI simplified
Results
Acute administration of GSPE only modulated the expression pattern of clock-core and clock-controlled genes in the liver when was administered at ZT12
The capacity of GSPE to modify the molecular clock in the liver was evaluated by measuring the 24 h mRNA oscillation ofand(clock core genes),(a component of the negative loop of the circadian clock),and(nuclear receptors whose expression is regulated by CLOCK:BMAL1 and which act as an activator or repressor, respectively, of Bmal1 gene expression),(a metabolic gene whose expression is directly regulated by CLOCK:BMAL1) and(a metabolic gene that has circadian rhythm expression but that is not directly controlled by CLOCK:BMAL1). Clock Bmal1 Per2 Rorα Rev-erbα Nampt HmgCoAR
Rats were administered GSPE at ZT0 and sacrificed at ZT0, ZT0.5, ZT1, ZT3, ZT6, ZT12 or ZT24. Nonetheless, to achieve a better visualization of the changes induced by GSPE administered at ZT0, we drew the figures () with a 24 h curve for the control group by assembling the expression values of the control group administered at ZT0 and ZT12. Overall, the ANOVA test indicated that GSPE administered at ZT0 (light turned on) did not significantly affect the mRNA rhythm of any gene studied in the liver (). Fig. 1 Fig. 1A–G
Rats are nocturnal animals and eat mainly at night. Therefore, we next studied whether PAs can modulate the molecular clock in the liver when GSPE is administered at night, when the liver actively manages the ingested nutrients. GSPE was administered when the light was turned off (ZT12), and rats were sacrificed at ZT12, ZT13, ZT15 or ZT18. Thus, the expression of clock-core and clock-controlled genes in the liver was determined at these four time points. Nonetheless, to achieve a better visualization of the changes induced by GSPE administered at ZT12, we drew the figures () with a 24 h curve for the control group by assembling the expression values of the control group from both this experiment and the former experiment. Fig. 2
GSPE, administered at ZT12, induced slight effects on the mRNA levels of clock-core genes() and() as well as on the mRNA levels of the clock-controlled genes() and(). Nonetheless, the mRNA levels of() and(), two clock-controlled genes, were significantly affected by GSPE. Moreover, the mRNA levels of(), a gene with circadian rhythm but not directly controlled by the clock-core genes, were also significantly increased by GSPE treatment. Bmal1 Clock Rorα Rev-erbα Per2 Nampt HmgCoAR Fig. 2A Fig. 2B Fig. 2C Fig. 2D Fig. 2E Fig. 2F Fig. 2G
Comparing the effects of GSPE administered at ZT12 (beginning of the night) or at ZT0 (beginning of the day), it is evident that the capacity of GSPE to modulated the clock system in the liver was dependent on the time of GSPE administration, being only effective at ZT12.
Acute administration of GSPE modulated the peripheral clock in the liver of jet-lagged rats
The capacity of GSPE to modulate the peripheral clock was also evaluated in a situation where circadian rhythm was disrupted using rats subjected to a 6 hour jet-lag. Rats at ZT6 (middle of the light period) were administered GSPE and moved to ZT12 (light turned off) and they were sacrificed at ZT12, ZT13, ZT15 or ZT18. Thus, the expression of clock-core and clock-controlled genes in the liver was determined at these four time points.
In control animals, the jet lag induced a clear shift in the mRNA rhythmicity for all of the genes that were studied () when the rhythms were compared with the 24 h control waves (built by assembling the expression values of the control groups, such as in the former experiments). Fig. 3
GSPE, administered at the beginning of jet lag, did not modulate() or(), whereas it significantly altered the expression rhythm of(),(),() and() when compared with the jet lag control group. Remarkably,andwere again two of the genes most sensitive to GSPE, as in when GSPE was administered at ZT12. Thus, next we focused in these two genes. Clock Per2 Rev-erbα Bmal1 Nampt HmgCoAR Nampt HmgCoAR Fig. 3B Fig. 3E Fig. 3D Fig. 3A Fig. 3F Fig. 3G
Acute administration of GSPE had opposite effects on both Nampt expression and NAD levels in the liver at different treatment times
Given thatgene was susceptible to GSPE in the liver and that this protein is the rate-limiting enzyme of the NAD salvage pathway, we next focused specifically on the oscillations ofRNAm and protein levels as well as NAD concentration during the first 6 hours after GSPE administration at ZT0 and ZT12. These analyses were performed using the livers of rats from the previous experiments. Nampt Nampt
Remarkably, GSPE induced opposite effects when it was administered at ZT0 or ZT12 ().protein and mRNA levels were decreased three hours after GSPE administration at ZT0 (), whilemRNA and protein levels were significantly elevated at three and six hours, respectively, after GSPE administration at ZT12 (). These modifications inexpression agreed with the alterations in NAD levels that were induced by GSPE in each situation: NAD levels were significantly decreased six hours after GSPE administration at ZT0, whereas they were significantly increased six hours after GSPE administration at ZT12. Fig. 4 Fig. 4A Fig. 4B Nampt Nampt Nampt
Altogether, these results implicateand NAD modulation as key factors in GSPE activity in the liver and confirmas a target of GSPE in the liver Nampt Nampt
Acute administration of GSPE at ZT12 increased the ratio of acetylated Bmal1 in rat liver
is a direct target gene of CLOCK:BMAL1. However, GSPE did not induce a strong modification in the mRNA rhythms oforthat could explain the observed alteration inexpression. However, the transcriptional activity of CLOCK:BMAL1 is dependent onacetylation. Thus, we focused further on the ratio ofacetylation by measuringmRNA, protein and acetylated protein during the first six hours after GSPE administration at ZT0 or ZT12.mRNA, protein and acetylated protein were analyzed in the livers of rats from the previous experiments. Nampt Clock Bmal1 Nampt Bmal1 Bmal1 Bmal1 Bmal1
GSPE administered at either time increased mRNA and protein levels ofafter 1 hour of treatment (; ZT1 and ZT13, respectively). Nonetheless, the ratio of acetylatedwas only increased when GSPE was administered at night (ZT12); these are the same circumstances under whichwas overexpressed. Bmal1 Bmal1 Nampt Fig. 5A,B
Acute administration of GSPE repressed the relative expression of HmgcoAR in rat liver
Finally, the mRNA and protein levels ofa metabolic gene not controlled by the clock molecular machinery, were determined according to the three experimental designs (day, night and jet lag) during only the first six hours after GSPE treatment (ZT1, ZT3 and ZT6 or ZT13, ZT15 and ZT18).mRNA and protein were analyzed in the livers of rats from the previous experiments. HmgcoAR, HmgcoAR
expression was significantly repressed at ZT1 (mRNA) and ZT3 (protein) after GSPE treatment was administered at ZT0 (). While mRNA levels were increased at ZT15 after GSPE treatment was administered at ZT12, the protein levels did not reflect that fact (). Finally, in the jet-lagged rats,protein and mRNA levels were decreased at ZT18 (). HmgcoAR HmgcoAR Fig. 6A Fig. 6B Fig. 6C
Discussion
While light is the major synchronizer of the central clock in the suprachiasmatic nucleus (SCN) in the hypothalamus, many other external cues such as temperature, social events or meal timingcan entrain circadian rhythms in other cerebral regions or peripheral tissues. This phenomenon is especially the case in the liver, which is the most important metabolic organ due to its involvement in glucoseand lipidmetabolism, among other crucial physiological functions. Even specific components in foods could also be important synchronizers, such as dietary fator phenolic compounds like resveratrol. Therefore, the aim of this work was to determine the capacity of an acute dose of GSPE to act as a signal to modulate the molecular clock in the liver. 10 6 5 23 24 25 26 27 28
To accomplish this outcome, three different experimental approaches were performed to determine whether PAs can modulate the liver clock: the administration of GSPE at ZT0, at the beginning of the light phase; at ZT12, at the beginning of the dusk phase; and to rats with 6 h of jet lag. The data clearly show that the power of PAs to modulate the circadian rhythm of clock-core and clock-controlled genes in the liver depends on the time of their administration.
Interestingly,and NAD emerge as molecular targets of PAs in the liver.is the rate-limiting enzyme in NAD biosynthesis through its salvage pathway. NAD plays a major role as a coenzyme in numerous oxidation-reduction reactionsand is required in a number of important signaling pathways in mammalian cells, including poly-ADP-ribosylation in DNA repair, mono-ADP-ribosylation in both the immune response and G protein-coupled signaling, and the synthesis of cyclic ADP-ribose and nicotinate adenine dinucleotide phosphate (NAADP) in intracellular calcium signaling. Furthermore, NAD activates several NAD(+)-dependent deacetylases (SIRT), such as SIRT1 and SIRT3, thus controlling the activity of many cellular proteins by cycling them between their acetylated and deacetylated forms. Specifically, the circadian oscillations of NAD levels have been shown to modulate mitochondrial respiration by controlling the activity of SIRT3, thus generating rhythms in the acetylation and activity of oxidative enzymes that synchronize mitochondrial oxidative functions across the daily cycles of fasting and feeding. Therefore, NAD is a key molecule in the synchronization of liver metabolism, and the modulation of its levels in the liver by PAs is an attractive candidate for the explanation of some of the metabolic effects of PAs. GSPE modulatedand NAD levels in opposite ways 6 h after its administration during the day versus at night, reducing or increasing their levels, respectively, suggesting thatexpression and NAD levels peaked at night. Therefore, in this sense, PAs could act as an element of adaptation in the liver, improving the energetic profile of rats and increasing mitochondrial function and oxidation at night, when rats are active. In keeping with the idea of an adaptation mechanism, as these animals are resting during the light phase, PA activity could be acting as an energy saver through the decreased levels of NAD after PA administration at ZT0. Nampt Nampt Nampt Nampt 29 30 31 32 33 34
NAD concentration oscillates in a circadian manner due to the circadian expression of, which in turn is mediated by the CLOCK:BMAL1 heterodimer. The rhythm ofandexpression was not altered by GSPE administration, either during the day or at night (and). However, whenexpression was studied during the first six hours of GSPE administration (), both mRNA and protein levels were always increased one hour after PA consumption, regardless of whether GSPE was administered diurnally, at night, or even under jet lag conditions, suggesting a robust relationship betweenand PAs. However, to be active, BMAL1 should be acetylated by CLOCK, which is its own partner. Therefore, the ratio of acetylated to total BMAL1 protein provides direct information about the transactivation activity of BMAL1:CLOCK. We found that GSPE significantly increased the ratio of BMAL1 that was acetylated at ZT13, whereas this effect was not observed at ZT1. This differential pattern of BMAL1 acetylation, which depends on the time of GSPE administration, could explain the overexpression of NAMPT and therefore the peak in NAD levels in the liver only when GSPE was administered at night. Therefore, it is globally supposed in this work that NAD levels peaked 6 hours after GSPE consumption at ZT12 (thereby at ZT18) as a consequence of an increased BMAL1 acetylation ratio at ZT13 that, in turn, increasedmRNA and protein levels at ZT15-ZT18. Nampt Clock Bmal1 Bmal1 Bmal1 Nampt 35 Figs. 1 2 Fig. 5 35
PAs modulate lipid metabolism in the liver. Therefore, we also analyzed the expression of, the key enzyme in the cholesterol biosynthetic pathway, which has circadian rhythm but is not directly controlled by the clock-core genes. GSPE had a dual effect onexpression depending on the time of its administration. GSPE repressed bothmRNA and protein levels at ZT0, whereas protein levels at ZT12 were not affected. Therefore, as in the case of BMAL1 acetylation, Nampt expression and NAD levels, the time of PA administration conditions the circadian regulation outputs. 20 36 HmgcoAR HmgcoAR HmgcoAR
Further studies are needed in order to find out the actual compounds that can modulate the molecular clock in the liver. Studies on the bioavailability of proanthocyanidins are controversial. Some studies indicate no absorption of oligomeric proanthocyanidins, thus attributing the biological effects of PA to their colonic metabolites such as phenolic acids and valerolactones. However, others studies have detected oligomeric PA in plasmaand in several tissuesof rats. Moreover, GSPE can be metabolized by intestinal and hepatic enzymes, producing a large array of metabolites. 37 37 38 39 39
Overall, these findings agree with the Xenohormesis Hypothesis, which proposes that heterotrophs are able to sense chemical cues, such as polyphenols, that are synthesized by plants in response to stress. In fact, circadian rhythms allow the anticipation of environmental changes and adaptation to the time of day and food availability, which has been shown in this work through NAD, NAMPT and BMAL1 acetylation levels. Thus, PAs can advise animals about environmental conditions by modulating biological rhythms to obtain a better ability to adapt to changing conditions over the course of their lives. Despite this study has been performed with a pharmacological dose of PAs, animals could consume such a huge amount of PAs in a wildlife situation. 40
In conclusion, PAs modulate the molecular clock in the liver even though their effectiveness depends largely on the time of administration. Specifically,andas well NAD, emerge as targets of GSPE in the liver. Bmal1 Nampt,
Materials and Methods
Grape seed proanthocyanidin extract composition
Grape seed proanthocyanidin extract (GSPE) was kindly provided by Les Dérives Résiniques et Terpéniques (Dax, France). Specifically, GSPE contains: catechin (58 μmol/g), epicatechin (52 μmol/g), epigallocatechin (5.50 μmol/g), epicatechingallate (89 μmol/g), epigallocatechingallate (1.40 μmol/g), dimericprocyanidins (250 μmol/g), trimericprocyanidins (1568 μmol/g), tetramericprocyanidins (8.8 μmol/g), pentamericprocyanidins (0.73 μmol/g) and hexamericprocyanidins (0.38 μmol/g). 41
Animals
All procedures involving the use and care of animals were reviewed and approved by The Animal Ethics Committee of the Universitat Rovira i Virgili (Permit number 4249 by Generalitat de Catalunya). All experiments were performed in accordance with relevant guidelines and regulations.
Eighty-four eight-week-old male Wistar rats (Crl: WI (Han)) were purchased from Charles River (Barcelona, Spain) and fed ad libitum with a standard chow diet (STD, Panlab 04, Barcelona, Spain) and tap water. Rats were divided into three groups according to the Zeitgeber time (ZT) when GSPE was administered.
Administration of GSPE at ZT0
Forty rats were singly caged in animal quarters at 22 °C with a 12 h light/dark cycle (light from 9:00 to 21:00 pm). After three weeks of adaptation, the rats were orally gavaged with tap water (control group) or 250 mg of GSPE/kg body weight dissolved in tap water at ZT0 (9:00 am, light turned on). Rats were sacrificed by beheading at ZT0, ZT0.5, ZT1, ZT3, ZT6, ZT12 and ZT24 (n = 3 for control and n = 3 for GSPE-treated groups).
Administration of GSPE at ZT12
Twenty-two rats were singly caged in animal quarters at 22 °C with a 12 h light/dark cycle (light from 21:00 pm to 9:00 am). After three weeks of adaptation, the rats were orally gavaged with tap water (control group) or 250 mg of GSPE/kg body weight dissolved in tap water at ZT12 (9:00 am, light off). Rats were sacrificed by beheading at ZT12, ZT13, ZT15, and ZT18 (n = 3 for control and n = 3 for GSPE-treated groups).
Administration of GSPE to jet-lagged rats
Twenty-two rats were singly caged in animal quarters at 22 °C with a 12 h light/dark cycle (light from 15:00 pm to 03:00 am). After three weeks of adaptation, rats were orally gavaged with tap water (control group) or 250 mg of GSPE /kg body weight dissolved in tap water at ZT6 (9:00 am, middle of the light day) and immediately moved to a dusk room (ZT12), thus giving rats a jet lag of 6 hours. Rats were sacrificed by beheading at ZT12, ZT13, ZT15, and ZT18 (n = 3 for control and n = 3 for GSPE-treated groups).
For the three experiments, the liver was excised, frozen immediately in liquid nitrogen and stored at −80 °C until RNA and protein extraction.
RNA extraction and cDNA synthesis
Total RNA from liver was extracted using TRIzol reagent and an RNeasy Mini Kit (Qiagen, 74106, Barcelona, Spain) according to manufacturer protocols. RNA was quantified by spectrophotometry (Nanodrop 1000 Spectrophotometer, Thermo Scientific, Madrid, Spain) at= 260 nm and tested for purity (by A260/280 ratio) and integrity (by denaturing gel electrophoresis). Complementary DNA was generated using the High-Capacity complementary DNA Reverse Transcription Kit from Applied Biosystems (4368814, Madrid, Spain). λ
mRNA quantification by real-time qRT-PCR
A total of 10 ng of cDNA was subjected to quantitative RT-PCR amplification using SYBR Green PCR Master Mix from Bio-Rad (172-5200, Barcelona, Spain). The forward and reverse primers of the genes analyzed are shown in. Reactions were run on a quantitative real-time PCR system (CFX96 touch of Bio-Rad, Barcelona, Spain); the thermal profile settings were 50 °C for 2 min, 95 °C for 2 min, and then 40 cycles at 95 °C for 15 s and 60 °C for 2 min. Finally, statistical data were converted and normalized to the linear form by the 2CT (∆∆C) calculation. The relative expression of the clock genes was normalized to cyclophilin mRNA levels. Table 1 42 − T
Western blot analyses
Protein was extracted from liver using RIPA (radio-immunoprecipitation assay) lysis buffer (15 mM Tris–HCl, 165 mM NaCl, 0.5% Na-deoxycholate, 1% Triton X-100 and 0.1% SDS) containing a protease inhibitor cocktail (1:1000; Sigma-Aldrich P8340-1 mL, Madrid, Spain) and 1 mM PMSF (phenylmethanesulfonyl fluoride solution, Sigma-Aldrich 93482, Madrid, Spain). The total protein levels of the lysates were determined using the BCA method from Thermo Scientific (23227, Barcelona, Spain). The samples were then placed in sample buffer (0.5 M Tris–HCl, pH 6.8; 10% glycerol; 2% (w/v) SDS; 5% (v/v) β-mercaptoethanol; and 0.05% bromophenol blue). After boiling for 5 min, 50 μg of protein was loaded and separated on a 10% SDS-polyacrylamide gel. The samples were then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories, 162-017, Barcelona, Spain) using a transblot apparatus (Bio-Rad, 16580229SP ) and blocked at room temperature for 1 h with 5% (w/v) non-fat milk in TTBS buffer (Tris-buffered saline (TBS) plus 0.5% (v/v) Tween-20). The membranes were incubated overnight at 4 °C with primary monoclonal antibodies directed against Nampt (Imgenex, IMX-6096, Nanterre, France), Bmal1 (LS-Bio, LS-, Vizcaya, Spain), acetyl-Bmal1 (Millipore, AB15396, Madrid, Spain), HmgcoAR (Santa Cruz, SC-33827, Nanterre, France) and anti-β-actin (Sigma-Aldrich, A2066-0.2 mL, Madrid, Spain) at a 1:1000 dilution in blocking solution. After washing with TTBS, the blots were incubated with a peroxidase-conjugated monoclonal anti-rabbit secondary antibody (Sigma-Aldrich, A1949, Madrid, Spain) at a 1:10,000 dilution at room temperature for 1.5 h. The blots were then washed thoroughly in TTBS followed by TBS. Immunoreactive proteins were visualized with an enhanced chemiluminescence substrate kit (ECL plus; Amersham Biosciences, GE Healthcare, RPN2132, Barcelona, Spain) according to the manufacturer’s instructions. Images were obtained with a GBOX Chemi XL 1.4 image system (Syngene, UK). Band quantification was performed with ImageJ software (NIH, USA). The results were expressed as relative intensity (Nampt/ β-actin, Bmal1/ β-actin, HmgcoAR/ β-actin and acetyl-Bmal1/ β-actin) and are relative to the loading control group. C16603
NAD quantification
NAD levels in the liver were quantified using an ELISA kit following the manufacturer’s instructions (Sigma-Aldrich, MAK037-1KT, Madrid, Spain).
Data and statistical analysis
The results are presented as the mean with the associated standard error (SE).The data were analyzed using a two-way ANOVA and Student t-test to determine the significant difference using SPSS statistical software (version 17.0 for Windows; SPSS, Inc.). P values < 0.05 were considered statistically significant.
Additional Information
: Ribas-Latre, A.Dietary proanthocyanidins modulate BMAL1 acetylation, Nampt expression and NAD levels in rat liver., 10954; doi: 10.1038/srep10954 (2015). How to cite this article 5 et al. Sci. Rep.