Changing Your Circadian Gene Expression with Polyphenols

This is a bit of a departure from my usual article. It is a paper that I recently wrote for a class that I’m taking for my Master’s in biology.  Thought I would share it in case anyone is interested…

Overview:
Resveratrol, EGCG, grape seed extract, passion flower, and other polyphenol supplements can increase the amplitude of core circadian genes — depending on the time of day that you take them.  Timing may be everything when it comes to the benefits from polyphenols.

 

 Introduction
All living things have core biological rhythms, which are set around the 24-hour period of dark and light. Life on Earth has evolved over the last 3.5 billion year with one constant: a 24-hour cycle of day and night.

Rhythms based on the 24-hour day are called circadian from the Latin word circa (about) and diem (day). (McClung 2006) Circadian rhythms are found in both plants and animals. Plant circadian examples include photosynthesis during the daylight hours, the opening and closing of flowers such as morning glories, and seasonal variations that respond to light and temperature. (Song, Ito, and Imaizumi 2010) Examples of animal circadian rhythms include sleep/wake cycles, body temperature fluctuations over the day, and blood pressure naturally rising in the early morning hours. (Thosar et al. 2018)

Plant Compounds Impact Our Human Circadian Rhythms
Recently, research has shown that many plant compounds interact with human circadian clock genes and can impact the amplitude or period of the transcription of the genes. Compiling the data from recent studies on polyphenols and circadian gene expression will show that the benefits of polyphenol consumption are derived, in part, from their impact on our circadian clock.

Plant Circadian Rhythms
Circadian rhythms, both in plants and animals, are endogenous and self-sustaining in stable environmental conditions where light and temperature remain fairly consistent. Even without the daily re-setting, or entrainment, of that rhythm, most organisms maintain an endogenous circadian period that is approximately, but usually not exactly, 24 hours. In the early 1900’s experiments showed that the leaf movements of plants kept in the dark varied just a little from being 24-hour cycles. The concept that endogenous cycles are not always exactly 24-hours is known as the organisms free-running time and applies to both plants an animals. (McClung 2006) Light, as a zeitgeber (German for time giver), entrains the core circadian rhythm each day, resetting a slightly longer or shorter free-running time back to 24-hours.

Plant circadian rhythms have been studied for hundreds of years. Charles Darwin wrote a book on plant movement in 1880.  In it, he postulates that heredity (genetics) is involved in the movement of plants. Some of the observations he made include the inherited movement of the hypocotyl arching and breaking through the ground as certain plants germinate. (Darwin 1880)

Groundbreaking genetic discoveries in the 1970’s and 80’s paved the way for understanding the genes that drive circadian rhythm.  While the first circadian gene to be cloned was the Per (period) gene from the Drosophila melanogaster, a fruit fly (Bargiello and Young 1984), the genetic basis for plant circadian function was not completely elucidated until the 1980’s. The circadian function of the Cab-1 gene was discovered in 1985 in peas and replicated in 1988 in wheat. (Nagy et al., 1988) A series of experiments in the ‘90’s and early 2000’s showed that there are three interlocking feedback loops in most plant circadian rhythms with the genes TOC1, CCA1, and LHY playing integral roles. (McClung 2006)

Human Circadian Rhythm
The human core circadian clock is governed by the feedback loop of the transcription of CLOCK and ARNTL (Bmal1) genes that rise during the day and are then repressed during the night by rising levels of the PER (period family) and CRY (cryptochrome family) of genes. When CRY and PER levels reach a certain level they inhibit their own transcription, forming the basis of the 24-hour oscillation of our molecular circadian pacemaker.  The region of the brain that controls the core circadian rhythm is called the suprachiasmatic nucleus (SCN) and is located in the hypothalamus. Light, specifically in the ~480nm blue wavelengths, hits the melanopsin-containing non-image forming photoreceptors in the retina, signaling to the SCN that it is daylight. This entrains the core clock genes each day to the 24-hour rhythm, and it also stops the production of melatonin, an antioxidant and signaling molecule that rises during the dark.(Xu and Lu 2018)

Research is increasingly showing how intertwined our circadian rhythm is with our health. Circadian dysfunction has been implicated in many chronic diseases including depression, bipolar disorder, heart disease (Thosar et al. 2018), certain cancers, Alzheimer’s disease, and other dementias. (Xu and Lu 2018)

Disruptions to our natural circadian rhythm are ubiquitous in modern life.  Artificial lighting at night, especially in the short blue wavelengths, disrupts the core circadian clock through activating melanopsin and signaling to the SCN that it is still daytime. Blue light in the evenings or at night from TVs, cell phones, computers, and CFL/LED bulbs creates havoc within our circadian system.

Our 24/7 society demands shift work, and we now have food readily available in any season and any time of the day. These two things come together in modern times to increase the risk of many chronic diseases that are dependent on metabolism. Feeding and fasting times regulate circadian metabolism, and mouse studies have shown that time of eating influences adiposity regardless of caloric intake. (Potter, Gregory D. M. et al. 2016) Not only does the time of day impact metabolism, but the overall number of hours in the feeding window, or time from the first food of the day to the last, also impacts metabolism. This was clearly shown by Hatori, et al. in a mouse study that restricted the mice to eat only during an 8-hour window during their normal active time at night. The time restricted feeding mice ate the same number of calories as the mice allowed to eat ad libitum, but they gained less fat and were protected from getting hyperinsulinemia and fatty liver. (Hatori I 2012)

While the body’s core circadian rhythm is governed by the oscillation of CLOCK, BMAL1 and PER, CRY in the SCN, there are also peripheral clocks governing circadian patterns in organs such as the liver, pancreas, and adipose tissue.  Twenty percent of proteins in the liver are under circadian control, varying in the time of day that they are expressed. (Ribas-Latre, Aleix, et al. 2015b.)

Polyphenols
While the term polyphenol is often used in health and nutrition articles, the formal definition of the term has been debated and changed over the years. Polyphenols are generally defined as phenolic molecules that contain one or more benzene rings linked to a hydroxyl group. Plants produce polyphenols in response to either UV radiation or as a defense against various different pathogens. (Beckman 2000, 101-110) Historically, polyphenol was a general term given to plant compounds capable of tanning (a tannin).

Large, epidemiological studies link the consumption of higher amounts of polyphenols to various health benefits including reduced risk of cardiovascular disease, cancer, and neurodegenerative diseases. Food sources that are high in polyphenols include tea, red wine, olive oil, and most fruits and vegetables. Tea is one of the most widely consumed sources of polyphenols, and drinking three or more cups a day has been found to reduce the risk of cardiovascular disease by 11%. (Vauzour et al. 2010)

Polyphenols can be categorized into several main types including flavonoids, phenolic acids, and phenolic amides.  Flavonoids, which often act as antioxidants, have a standard ring structure of C6-C3-C6 with the two C6 rings being phenols. They can be further categorized base on different ways of bonding with the ring structure, with subcategories including proanthocyanidins, flavonoids, flavones, and flavonols. (Tsao 2010, 1231-1246)

The category of flavonoids called proanthocyanidins includes several compounds that have been found to favorably affect human health. Proanthocyanidins, which are polymers of flavonols, include catechins, (Tsao 2010) epigallocatechin (e.g. EGCG from green tea), and grape seed extract. The most common sources of proanthocyanidins in the US diet are apples, chocolate, and grapes, with the 2 – 4-year-old age group eating the most proanthocyanidins.  (Gu et al. 2004)

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a non-flavonoid polyphenol known as a stilbenoid, which is produced by certain plants when they are attacked by fungus or another pathogen.  High levels are found in knotweed, grape skins, pine trees, berries such as blueberries, raspberries, cranberries, mulberries. (Tsao 2010) Resveratrol is a phytoalexin, which is a compound formed by a plant when under stress due to attack by pathogens such as fungus.  Red wine is known for its high resveratrol content, with resveratrol concentrated in the skin of grapes as a way of fighting off the fungus. The grape variety Vitis vinifera is well known for its high concentrations. (Jeandet, Bessis, and Gautheron 1991) Specific cultivars of grapes are higher in resveratrol and thus have a greater natural capacity to resist fungal infections. (Dercks and Creasy 1989)

Effects of Polyphenols on Circadian Rhythm
When looking at how polyphenols affect the expression of core circadian genes either in the SCN or in peripheral systems, there are two elements at play: the effect of the compounds and the timing of the compounds. A compound that causes a rise in one arm of the circadian clock could possibly be a negative at certain times and positive at other times.

Resveratrol
Oike and Kobori in 2008 found that 100um of resveratrol increased the expression of Per1, Per2, and Bmal1 genes. Although a cell study using rat cells, this was one of the first to show that gene expression can be altered by resveratrol. (Oike and Kobori, 2008)  In 2011, Pifferi, et al. used a primate model to determine the effects of resveratrol on circadian period.  The mouse lemurs were kept in the constant dark for two weeks, with a control group and a group fed resveratrol.  The resveratrol group had a shorter circadian free-running period and a lower body temperature compared to the control group. (Pifferi et al. 2011)

Miranda, et al. compared a rat model of obesity (given high-fat diet chow) with a group fed resveratrol plus the high-fat diet (HFD). They found that resveratrol targeted the clock related gene Rev-Erba in adipose tissue and it reversed the changes normally induced by HFD. (Miranda et al. 2013)

A mouse study by Sun, et al. in 2015 found that adding resveratrol to the obesity-inducing HFD attenuated the weight gain. The mice fed a high-fat diet with resveratrol (11-week study) had weight gain about halfway in between the regular high-fat diet group and the control mice. The high-fat diet obese mice had nearly flat daily circadian rhythms of Per2, Clock, and Bmal1; adding resveratrol to the HFD restored the circadian rhythm to that of mice in the control group on a normal chow diet. (Sun et al. 2015)

The timing of resveratrol consumption may be important to reap positive health effects.  Mouse study on resveratrol measured antioxidant effects based on time of day of dosing.  Resveratrol given during the active period (dark for mice) acted as a fairly strong antioxidant in a dose-dependent manner.  Doses ranged from 0.8 to 5 mg/kg, which converts to a physiological dose for humans.  Conversely, when resveratrol was given during the inactive period (light for mice) it actually became a pro-oxidant, increasing oxidative stress in a dose-dependent manner. (Gadacha et al. 2009)

Tea Polyphenols and EGCG
Mi, et al. examined the effects of EGCG, a phenolic component of green tea, on circadian rhythm in mice.  The study used a high-fat, high fructose diet (HFFD), which disrupts normal circadian rhythm by dampening or decreasing the amplitude of Clock, Bmal1, Cry1, Per2, and Per3 gene expression as well as increases triglycerides and total cholesterol.  Adding EGCG to the HFFD diet during the active phase (at night for mice) reversed the changes in lipid metabolism and clock gene expression that was found in HFFD only mice.  The EGCG was added at the beginning and middle of the active period.  Additionally, the group fed HFFD plus EGCG gained less weight than the HFFD group with similar food intake and calorie intake.

In 2017, Qi, et al. studied the effects of tea polyphenols on circadian disruption using a mouse model kept in constant darkness. The constant darkness group had higher insulin levels at certain time points, similar to other studies on circadian rhythm disruption and diabetes. In the group that was given tea polyphenols, there was a suppression of the dysregulation of insulin levels. When comparing the circadian gene expression of the group in constant darkness versus the group in constant dark plus tea polyphenol, the group without added polyphenols had decreased Clock and Bmal1 levels while the group kept in the dark but given polyphenols had circadian gene expression similar to that of the control group kept in a regular light / dark cycle.(Qi et al. 2017)

Passionflower Extract
Passionflower extract (passiflora incarnata) is an herbal folk remedy often used for anxiety and insomnia. Passionflower is a woody, climbing vine that that is native to the southeastern United States. A placebo-controlled study found that passionflower extract was as effective as oxazepam, a benzodiazepine, for the treatment of generalized anxiety disorder. (Akhondzadeh et al. 2001)

Toda et al. found that passionflower extract increases the amplitude of Per1, Per2, and Cry1 about 12 hours after treatment. Treatment at 0 hr showed that it affected the circadian genes over next 20 hours as well as changing the corticosterone rhythm. For the study, the floral parts of passionflower were extracted from dried flowers from France.  The flavonoids in the plants included isovitexin, isovitexin 2″-O-glucoside, schaftoside, isoschaftoside, and homoorientin. Homoorientin specifically was found to separately increase Per2 the most, although the other compounds added to the effect. (Toda et al. 2017)

Proanthocyanidins
Grape seeds are high in proanthocyanidins, and grape seed extract has been used to study the effects of proanthocyanidins on circadian gene expression.  A mouse study by Ribas-Latre, et al. looked at the effect of chronic consumption of grape seed proanthocyanidin extract (GSPE). The results showed increases in the gene expression of CLOCK and PER2 at a dosage of 25 and 50 mg/kg/day in both the liver and in white adipose tissue. Additionally, Nampt was also increased at those doses in white adipose tissue. Nampt expression correlates with nicotinamide adenine dinucleotide (NAD) levels, which activates Sirt1 and affects the expression of Bmal1. (Ribas-Latre 2015a)

A second study by Ribas-Latre, et al. looked at the timing of GSPE administration.  The rats were fed GCPE either at ZT0 (when the lights come on) or at ZT12 (lights off).  The protein expression in the liver was recorded, and it was found that grape seed extract given at ZT0 decreased the expression of Nampt. When given at ZT12, grape seed extract increased the expression of Nampt, which correlates to NAD levels.  NAD activates Sirt1, which is a sirtuin that acetylates Bmal1, thus affecting the expression of Bmal1.  Bmal1 forms a heterodimer with CLOCK, so increasing Bmal1 is only important if there is CLOCK available also. (Ribas-Latre et al. 2015b)  

A final study by Ribas-Latre, et al. examined the effect of GSPE on circadian gene expression using liver cells.  The liver is both under the influence of the core circadian clock genes in the SCN as well as maintaining a peripheral clock influenced by the timing of meals and diet composition. The study found that GSPE altered the expression of BMAL1, increasing the expression at 1 hour and 15 hours after application in comparison to control.  The study also examined the impact of melatonin on BMAL1 as well. The results showed that melatonin at 10umol/L had a very similar impact on BMAL1 expression as GSPE. The researchers further looked at the impact of blocking the melatonin receptor, MT1, and found that GSPE is not acting through the melatonin receptor. (Ribas-Latre et al. 2015)  

Nobiletin
Nobiletin is a polymethoxylated flavonoid found in citrus fruit rinds.  In 2016, He, et al. found that nobiletin increases the amplitude of core clock genes and decreases the effects of metabolic syndrome in a circadian-related manner.  The study found that nobiletin is acting on ROR to enhanced PER2 amplitude in peripheral tissue systems but not in the SCN.  In a mouse model of diabetes and diet-induced obesity (db/db mice fed HFD), nobiletin was effective in blocking the weight gain through decreasing the size of the fat mass and the size of the adipocytes. The nobiletin was administered at 8-10 ZT 200mg/kg every other day.  This blocking of weight gain was accomplished even though the amount of food the mice ate was the same as the obese mice. A second arm of the study looked at the effect of nobiletin vs. a diet-induced obesity model strain of mice that were bred to be deficient in the Clock gene.  The nobiletin group with deleted Clock gene had the same weight gain as the HFD obese mice, reinforcing the circadian rhythm impact of this flavonoid. (He et al. 2016)

Discussion
Plant polyphenols can have a remarkable effect on animal metabolism; part of their benefit may come from their ability to modulate circadian rhythm.  The studies on polyphenols in mice, rats, and primates clearly show that the compounds affect both metabolism and circadian function. More studies are needed to elucidate the role of plant polyphenols on human circadian function. Future studies of polyphenols should include the timing of consumption as well as the daily circadian cycle of the study participants.

The importance to human health of a robust circadian rhythm is becoming abundantly clear with the many studies linking circadian dysfunction with chronic health problems such as mood disorders, cancer, obesity, type 2 diabetes, and dementia. As it is doubtful that our society will go back to a time of only firelight at night, the ability to manipulate circadian gene function with natural polyphenols could have a large impact on the health of the whole human population.

The polyphenols discussed here, with the exception of nobiletin, are readily available as supplements. The timing of taking those supplements may affect the health benefits received from them. For example, passionflower extract, which increases the amplitude of PER1, PER2, and CRY1, should be timed to maximize the expression of the genes that rise during the evening and night. In fact, one of the uses of passionflower in herbal teas is for promoting sleep. Resveratrol, well known as an antioxidant, was shown to actually exhibit pro-oxidant properties when administered during the inactive period. This leads to the idea that resveratrol may be better to take during the day rather than in the evening to reap the benefits as an antioxidant.

The changing effects of polyphenols based on the timing may also explain some of the differing results in studies on them. For example, resveratrol supplement studies may show different results depending on the time of day that the participants took the supplement. Further studies into the timing of supplemental polyphenols could strengthen their protective effects against metabolic disorders also associated with circadian rhythm disruption.

 

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