Resveratrol regulates circadian clock genes in Rat-1 fibroblast cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 muM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

Sirt1与生物钟的相互调控

生物钟调控机制广泛存在于各种类型的细胞中,控制着细胞代谢的节律性变化.最近的研究发现,NAD+依赖的组蛋白去乙酰化酶Sirt1参与了生物钟调控过程,对维持正常的生物钟节律具有重要作用;另一方面,Sirt1的表达也受到生物钟系统的调控,呈现出昼夜节律性的表达.因此Sirt1能与生物钟进行相互调控,并且这一作用机制很可能广泛参与了不同类型细胞内的信号转导和能量代谢过程.本文总结了Sirt1与生物钟之间相互调控的一些研究进展,对它们之间的分子调控机制进行了概述.     

Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.     

Aging alters circadian and light-induced expression of clock genes in golden hamsters

Aging alters numerous aspects of circadian biology, including the amplitude of rhythms generated by the suprachiasmatic nuclei (SCN) of the hypothalamus, the site of the central circadian pacemaker in mammals, and the response of the pacemaker to environmental stimuli such as light. Although previous studies have described molecular correlates of these behavioral changes, to date only 1 study in rats has attempted to determine if there are age-related changes in the expression of genes that comprise the circadian clock itself. We used in situ hybridization to examine the effects of age on the circadian pattern of expression of a subset of the genes that comprise the molecular machinery of the circadian clock in golden hamsters. Here we report that age alters the 24-h expression profile of Clock and its binding partner Bmal1 in the hamster SCN. There is no effect of age on the 24-h profile of either Per1 or Per2 when hamsters are housed in constant darkness. We also found that light pulses, which induce smaller phase shifts in old animals than in young, lead to decreased induction of Per1, but not of Per2, in the SCN of old hamsters.     

Low-carbohydrate, high-protein diet affects rhythmic expression of gluconeogenic regulatory and circadian clock genes in mouse peripheral tissues

Recent studies have demonstrated that metabolic changes in mammals induce feedback regulation of the circadian clock. The present study evaluates the effects of a low-carbohydrate high-protein diet (HPD) on circadian behavior and peripheral circadian clocks in mice. Circadian rhythms of locomotor activity and core body temperature remained normal in mice fed with the HPD diet (HPD mice), suggesting that it did not affect the central clock in the hypothalamus. Two weeks of HPD feeding induced mild hypoglycemia without affecting body weight, although these mice consumed more calories than mice fed with a normal diet (ND mice). Plasma insulin levels were increased during the inactive phase in HPD mice, but increased twice, beginning and end of the active phase, in ND mice. Expression levels of the key gluconeogenic regulatory genes PEPCK and G6Pase were significantly induced in the liver and kidneys of HPD mice. The HPD appeared to induce peroxisome proliferator-activated receptor α (PPARα) activation, since mRNA expression levels of PPARα and its typical target genes, such as PDK4 and Cyp4A10, were significantly increased in the liver and kidneys. Circadian mRNA expression of clock genes, such as BMAL1, Cry1, NPAS2, and Rev-erbα, but not Per2, was significantly phase-advanced, and mean expression levels of BMAL1 and Cry1 mRNAs were significantly elevated, in the liver and kidneys of HPD mice. These findings suggest that a HPD not only affects glucose homeostasis, but that it also advances the molecular circadian clock in peripheral tissues.     

Activation of Sirt1 by resveratrol inhibits TNF-α induced inflammation in fibroblasts

Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1) has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9), interleukin-1beta (IL-1β), IL-6 and inducible nitric oxide synthase (iNOS) were induced by tumor necrosis factor alpha (TNF-α) in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR) and S6 ribosomal protein (S6RP) while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases.     

Sirt1 increases skeletal muscle precursor cell proliferation

It is important to understand the mechanisms that control muscle precursor cell (MPC) proliferation for the development of countermeasures to offset the deleterious effects of the aging-related loss of skeletal muscle mass (and myonuclei) and the impaired ability of old muscle to regrow and regenerate. Over-expression of the NAD+-dependent histone deacetylase Sirt1 increased MPC proliferation and cell cycle progression as evidenced by increased 5-bromo-2'-deoxyuridine (BrdU) incorporation, an increase in cell number, proliferating cell nuclear antigen expression, and the phosphorylation of retinoblastoma protein. Associated with the Sirt1-mediated increase in MPC cycle progression were the bidirectional decreases and increases in the expression of the cyclin-dependent kinase inhibitors p21(Waf/Cip1) and p27(Kip1), respectively. Based upon our recent observation that lowering oxygen (O2) in culture from ambient (20%) to estimated physiological levels (5%) increased MPC proliferation, we next measured Sirt1 protein at 5% and 20% O2. Interestingly, in addition to increased proliferation in MPCs cultured at 5% O2, Sirt1 expression increased, compared to 20% O2. Using O2 levels as a platform to modulate basal Sirt1 protein, activation of Sirt1 activity with resveratrol in 20% O2 increased MPC proliferation while inhibition of Sirt1 with nicotinamide in 5% O2 lowered proliferation. For the first time, Sirt1 has been shown to increase MPC proliferation. These findings could have clinical significance since MPC proliferation has important implications in regulating skeletal muscle growth, maintenance, and repair, and the aging-related loss of skeletal muscle mass.     

Resveratrol Regulates Circadian Clock Genes in Rat-1 Fibroblast Cells

Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 μM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ERA) gene also oscillates in a circadian fashion. In contrast, ERA-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ERA-positive breast cancer cells do not display circadian oscillation of ERA expression. Our findings suggest that estrogen signaling could be affected not only in ERA-negative breast cancer, but also in ERA-positive breast cancer due to lack of circadian availability of ERA. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.Key words: circadian rhythm, clock genes, estrogen receptor alpha (ERA), breast cancer cells, entrainment, serum shock     

干旱对水稻生物钟基因和干旱胁迫响应基因每日节律性变化的影响

内源生物钟的节律运动不仅调控植物的生长发育,而且在调控植物响应和适应环境过程中发挥重要的作用。为了解水稻(Oryza sativa L.)干旱胁迫响应基因和生物钟基因在干旱条件下每日表达变化情况,本文利用实时荧光定量PCR方法研究旱稻品种IRAT109在干旱胁迫下相关基因的表达变化。结果表明,干旱胁迫导致早晨生物钟基因OsPRRs、OsLHY和OsZTL1的表达量显著下降,振幅减弱;同时导致夜晚生物钟基因OsTOC1、OsGI和OsELF3整体表达量升高,振幅增强,但对OsFKF1基因影响不大。同样,大部分水稻干旱胁迫响应基因在干旱胁迫后整体表达量显著升高,但OsDST基因表达量下降;同时大部分抗逆基因周期性表达被扰乱,但OsCIPK12、OsCDPK7和OsDREB1A依然保持24 h内震荡。本研究结果表明干旱胁迫能影响生物钟元件的基因表达,这种互相影响改变了部分基因每日的震荡变化。     

Metformin affects the circadian clock and metabolic rhythms in a tissue-specific manner

Metformin is a commonly-used treatment for type 2 diabetes, whose mechanism of action has been linked, in part, to activation of AMP-activated protein kinase (AMPK). However, little is known regarding its effect on circadian rhythms. Our aim was to evaluate the effect of metformin administration on metabolism, locomotor activity and circadian rhythms. We tested the effect of metformin treatment in the liver and muscle of young lean, healthy mice, as obesity and diabetes disrupt circadian rhythms. Metformin led to increased leptin and decreased glucagon levels. The effect of metformin on liver and muscle metabolism was similar leading to AMPK activation either by liver kinase B1 (LKB1) and/or other kinases in the muscle. AMPK activation resulted in the inhibition of acetyl CoA carboxylase (ACC), the rate limiting enzyme in fatty acid synthesis. Metformin also led to the activation of liver casein kinase I α (CKIα) and muscle CKIε, known modulators of the positive loop of the circadian clock. This effect was mainly of phase advances in the liver and phase delays in the muscle in clock and metabolic genes and/or protein expression. In conclusion, our results demonstrate the differential effects of metformin in the liver and muscle and the critical role the circadian clock has in orchestrating metabolic processes.     

The Tilapia collagen peptide mixture TY001 protects against LPS-induced inflammation,disruption of glucose metabolism,and aberrant expression of circadian clock genes in mice

The Tilapia collagen peptide mixture TY001 has been shown to accelerate wound healing in streptozotocin-induced diabetic mice and to protect against streptozotocin-induced inflammation and elevation in blood glucose. The goals of the present study are to further study TY001 effects on lipopolysaccharide (LPS)-induced inflammation and metabolic syndrome. LPS is known to disrupt circadian clock to produce toxic effects, the effects of TY001 on rhythmic alterations of serum cytokines and hepatic clock gene expressions were examined. Mice were given TY001 (30 g/L, ≈ 40 g/kg) through the drinking water for 30 days, and on the 21^st day of TY001 supplementation, LPS (0.25 mg/kg, ip, daily) was given for 9 days to establish the inflammation model. Repeated LPS injections produced inflammation, impaired glucose metabolism, and suppressed the expression of circadian clock core genes Bmal1 and Clock; clock feedback gene Cry1, Cry2, Per1, and Per2; clock target gene Rev-erbα and RORα. TY001 prevented LPS-induced elevations of TNFα, IL-1β, IL-6, and IL-10 in the liver, along with improved histopathology. TY001 reduced LPS-elevated fasting blood glucose and increased LPS-reduced serum insulin levels, probably via increased glucose transporter GLUT2, enhanced insulin signaling p-Akt and p-IRS-1^Try612. Importantly, LPS-induced circadian elevations of serum TNFα and IL-1β and aberrant expression of circadian clock genes in the liver were ameliorated by TY001. Immunohistochemistry revealed that the LPS decreased Bmal1 and Clock protein in the liver, which was recovered by TY001. Taken together, TY001 is effective against LPS-induced inflammation, disruption of glucose metabolism and disruption of circadian clock gene expressions.

Abbreviations: TY001: Tilapia collagen peptide mixture; LPS: Lipopolysaccharide; TNFα: Tumor necrosis factor-α; IL-1β: Interleukin-1β; GLUT2: Glucose transporter 2