Regulation of Peripheral Molecular Clocks in Mammalian Tissues and In Vitro Skeletal Muscle Activation of AMP-Activated Protein Kinase via AICAR

Most organisms possess a common molecular machinery that governs cellular and tissue circadian rhythmicity through a roughly 24-hour transcription-translation feedback loop. It is estimated that up to 15 percent of human genes are influenced by the core clock machinery. It is likely, however, that the metabolic networks affected by the molecular clock differ according to body tissue. Recent evidence suggests that peripheral molecular clocks are governed to a greater extent by energy availability than by light and dark cycles. AMP-activated protein kinase (AMPK) acts as a cellular fuel gauge within the cell and is activated in response to exercise and fasting. AMPK can also be pharmacologically activated by 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR). AMPK likely serves as an intermediary between metabolism and the molecular clock due to its activation of the rate-limiting enzyme in Nicotinamide adenine dinucleotide (NAD) biosynthesis, Nicotinamide phosphoribosyltransferase (NAMPT), and its role in PER and CRY degradation. The NAD-dependent histone deacetylase SIRT 1 inhibits the BMAL1-CLOCK complex in a NAMPT-dependent manner.The complex interplay between metabolism and peripheral clocks mediated by AMPK is beginning to be unraveled. AMPK’s tissue-specific influence on the molecular clock in skeletal muscles and other mammalian tissues requires further elucidation as it may provide insight into the etiology and treatment of metabolic disease. [excerpt

Oscillations of the circadian clock protein, BMAL-1, align to daily cycles of mechanical stimuli: a novel means to integrate biological time within predictive in vitro model systems

PURPOSE: In vivo, the circadian clock drives 24-h rhythms in human physiology. Isolated cells in vitro retain a functional clockwork but lack necessary timing cues resulting in the rapid loss of tissue-level circadian rhythms. This study tests the hypothesis that repeated daily mechanical stimulation acts as a timing cue for the circadian clockwork. The delineation and integration of circadian timing cues into predictive in vitro model systems, including organ-on-a-chip (OOAC) devices, represent a novel concept that introduces a key component of in vivo physiology into predictive in vitro model systems. METHODS: Quiescent bovine chondrocytes were entrained for 3 days by daily 12-h bouts of cyclic biaxial tensile strain (10%, 0.33 Hz, Flexcell) before sampling during free-running conditions. The core clock protein, BMAL-1, was quantified from normalised Western Blot signal intensity and the temporal oscillations characterised by Cosinor linear fit with 24-h period. RESULTS: Following entrainment, the cell-autonomous oscillations of the molecular clock protein, BMAL-1, exhibited circadian (24 h) periodicity (p < 0.001) which aligned to the diurnal mechanical stimuli. A 6-h phase shift in the mechanical entrainment protocol resulted in an equivalent shift of the circadian clockwork. Thus, repeated daily mechanical stimuli synchronised circadian rhythmicity of chondrocytes in vitro. CONCLUSION: This work demonstrates that daily mechanical stimulation can act as a timing cue that is sufficient to entrain the peripheral circadian clock in vitro. This discovery may be exploited to induce and sustain circadian physiology within into predictive in vitro model systems, including OOAC systems. Integration of the circadian clock within these systems will enhance their potential to accurately recapitulate human diurnal physiology and hence augment their predictive value as drug testing platforms and as realistic models of human (patho)physiology. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s44164-022-00032-x

From implantation to birth: insight into molecular melatonin functions

Melatonin is a lipophilic hormone synthesized and secreted mainly in the pineal gland, acting as a neuroendocrine transducer of photoperiodic information during the night. In addition to this activity, melatonin has shown an antioxidant function and a key role as regulator of physiological processes related to human reproduction. Melatonin is involved in the normal outcome of pregnancy, beginning with the oocyte quality, continuing with embryo implantation, and finishing with fetal development and parturition. Melatonin has been shown to act directly on several reproductive events, including folliculogenesis, oocyte maturation, and corpus luteum (CL) formation. The molecular mechanism of action has been investigated through several studies which provide solid evidence on the connections between maternal melatonin secretion and embryonic and fetal development. Melatonin administration, reducing oxidative stress and directly acting on its membrane receptors, melatonin thyroid hormone receptors (MT1 and MT2), displays effects on the earliest phases of pregnancy and during the whole gestational period. In addition, considering the reported positive effects on the outcomes of compromised pregnancies, melatonin supplementation should be considered as an important tool for supporting fetal development, opening new opportunities for the management of several reproductive and gestational pathologies

Regulation of Pituitary Adenylate Cyclase-activating Polypeptide Gene Transcription by TTF-1, a Homeodomain-containing Transcription Factor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important hypophysiotrophic factor as well as a regulator for immune, reproductive, and neural tissues. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in the hypothalamic area and plays a transcription regulatory role for certain neurohormones. Based on the similarity of synthesis sites between PACAP and TTF-1 and, moreover, on the presence of conserved core TTF-1 binding motifs in the 5′-flanking region of the PACAP gene, we sought to uncover a regulatory role of TTF-1 in PACAP gene transcription. The TTF-1 homeodomain binds to six of the seven putative binding domains observed in the 5′-flanking region of the PACAP gene. In the C6 glioma cell-line, TTF-1 activates the PACAP promoter in a dose-dependent manner. This transactivation of PACAP by TTF-1 was totally removed when the core TTF-1 binding motif at −369 was deleted. RNase protection assays showed that TTF-1 and PACAP mRNAs have daily fluctuations in the rat hypothalamus. They both were at low levels during the day and high levels during the night. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased the PACAP mRNA level as well as TTF-1 protein content in the rat hypothalamus, suggesting that TTF-1 also regulates PACAP transcription in vivo. Moreover, the TTF-1 promoter was inhibited by molecular oscillators of CLOCK and BMAL-1. Taken together, these data suggest that TTF-1 plays an important regulatory role in the gene transcription for PACAP, which may be important for the generation of a daily rhythm of hypothalamic PACAP gene expression

Circadian clock, cell cycle and cancer

There are a few rhythms of our daily lives that we are under the influence. One of them is characterized by predictable changes over a 24-hour timescale called circadian clock. This cellular clock is coordinated by the suprachiasmatic nucleus in the anterior hypothalamus. The clock consist of an autoregulatory transcription-translation feedback loop compose of four genes/proteins; BMAL1, Clock, Cyrptochrome, and Period. BMAL 1 and Clock are transcriptional factors and Period and Cyrptochrome are their targets. Period and Cyrptochrome dimerize in the cytoplasm to enter the nucleus where they inhibit Clock/BMAL activity.It has been demonstrate that circadian clock plays an important role cellular proliferation, DNA damage and repair mechanisms, checkpoints, apoptosis and cancer

Clock-related regulation of mitochondrial physiology in skeletal muscle

Biological rhythms regulate numerous functional processes within organisms, including the expression of peroxisome proliferator-activated receptor-y coactivator 1-a (PGC-1a), a potent regulator of mitochondrial biogenesis. Homozygous Clock mutant mice are characterized by arrhythmic and suppressed expression of circadian genes within skeletal muscle, including PGC-1a. The present study sought to investigate mitochondrial physiology within these mutant animals, and to assess their adaptability to a chronic voluntary endurance training protocol. Our results indicate that Clock mutant mice exhibit decreased mitochondrial content, and this contributes to exercise intolerance in these mutant animals. Interestingly, endurance training ameliorates the decrement in mitochondrial content, as well as restores exercise capacity to levels evident in the wildtype mice. Thus, a functional CLOCK protein is necessary for optimal mitochondrial physiology, however Clock mutant mice retain the ability to adapt to chronic exercise

Crononutrición: efecto de la hora de la ingesta en el metabolismo de los nutrientes.

Nowadays the metabolic variations are one of the most suffered illness in the world. Therefore, the investigation of the study about the influence of intake time in the metabolism of a nutrient, it is gaining importance for the development and applications on new treatments as far as these diseases are concerned. This bibliographic review, through an in-depth bibliographic search in different databases, has allowed us to obtain several files, documents, articles and researches that have been used for the analysis, development and execution of the current article. The glucose molecule has more pronounced levels in the afternoon versus the morning, due to the decreased insulin activity through the day. Most lipids show their highest levels in the morning, except for triglycerides showing them in the afternoon. Regarding proteins, more study is needed for their knowledge in this appearance. More research is required to obtain a better conclusion. Even so, it can be concluded that the time of intake is a factor that affects the rhythmicity of metabolic processes, interfering and modifying the activity and response of nutrients.Las alteraciones metabólicas suponen hoy en día una de las afecciones más padecidas en todo el mundo. Es por ello, que la indagación en el estudio sobre la influencia de la hora de la ingesta en el metabolismo de un nutriente, es de gran importancia para el desarrollo y aplicación de nuevos tratamientos en lo que a estas enfermedades respecta. Mediante esta revisión bibliográfica, a través de la búsqueda bibliográfica profunda en diferentes bases de datos, se han obtenido diversos archivos, documentos, artículos y estudios que han servido para el análisis, desarrollo y ejecución del vigente artículo. La molécula de la glucosa presenta niveles más acentuados en la tarde versus la mañana, debido a la disminución de la actividad de la insulina con el avance del día. La mayoría de los lípidos presentan sus niveles más altos en la mañana, a excepción de los triglicéridos mostrándolos en la tarde. En cuanto a las proteínas se necesita más estudio para su conocimiento en este aspecto. Se requiere de más investigación para poder obtener una conclusión más exacta. Aun así, se puede concluir en que la hora de la ingesta es un factor que afecta en la ritmicidad de los procesos metabólicos, interfiriendo y alterando la actividad y respuesta de los nutrientes

Impact of physiological rhythms on energy homeostasis in rodents

Obesity and related metabolic disorders such as type 2 diabetes are a major health issue of our modern society. The brain has been identified to play an essential role in the pathogenesis of these diseases. Disruptions of the neuroendocrine system, such as the development of hypothalamic leptin resistance, are strongly correlated with the manifestation of diet-induced obesity (DIO). To date, the molecular mechanisms underlying these metabolic derangements are incompletely understood. Over the last decade, a close connection of energy metabolism and the circadian clock has been established, but the link between DIO and disruptions of physiological rhythms still needs further investigation. Therefore, the aim of this thesis was to gain new insights into neuroendocrine mechanisms that lead to the development of leptin resistance and the role of physiological rhythms in the disruption of energy metabolism. In this study, we investigated the implication of the adipocyte-derived hormone adiponectin in neuroendocrine control of energy metabolism. We detected expression of all investigated genes involved in the adiponectin signalling pathway in the hypothalamus of mice. Expression levels of adiponectin were reduced during states of food deprivation, potentially presenting a regulatory mechanism to counteract the anorexigenic traits that had been previously described for central adiponectin signalling and that were confirmed by us in this study, in order to prevent further reduction in body weight. In both fasted control mice as well as DIO mice, gene expression of the adiponectin receptor AdipoR1 was elevated, suggesting multiple regulatory mechanisms to maintain sufficient adiponectin signal transduction. The upregulation of AdipoR1 during DIO might be an attempt to support the beneficial effects of the hormone on metabolic health that have been reported for peripheral adiponectin. In line with this, we demonstrated that adiponectin holds insulin-sensitising, blood glucose-lowering and anti-inflammatory properties in control as well as DIO mice and that these effects are mediated via central signal transduction. We furthermore investigated the role of the WNT/β-catenin pathway in the neuroendocrine control of energy metabolism. Here, we found gene expression of members of the WNT pathway on all regulatory levels (ligands, intracellular pathway enzymes, target genes) in the hypothalamus of adult Djungarian hamsters, Phodopus sungorus, a seasonal rodent that exhibits profound annual changes in body weight and leptin sensitivity. Expression of all ligands as well as target genes was upregulated in hamsters acclimated to long day (LD) relative to short day (SD) conditions. Confirming our results from these transcriptional studies, we furthermore found increased phosphorylation of the WNT pathway co-receptor LRP-6, demonstrating elevated activation of canonical WNT signalling, in LD hamsters. These findings provide strong evidence for increased WNT signalling during LD compared with SD photoperiod. We found a 24-hour rhythm in the hypothalamic expression of WNT target genes, with decreasing levels during the light and increasing levels during the dark phase in both LD and SD hamsters. Moreover, leptin administration led to a further increase in LRP-6 activation in hamsters from both photoperiods. Taken together, we demonstrate a novel integration site for the leptin signal in the hypothalamus, potentially linking the WNT pathway to body weight regulation. Furthermore, our results suggest an important role of canonical WNT signalling in the seasonal as well as daily neuroendocrine control of energy metabolism in Djungarian hamsters. By examining whether hypothalamic leptin signalling and whole body metabolism are modulated by a daily rhythm, we detected a 24-hour rhythm of STAT3 phosphorylation, a marker for activated leptin signalling on a molecular level, in the hypothalamus of wild-type mice. Both basal as well as leptin-induced leptin sensitivity were highest at the end of the dark (active) phase and lowest at the end of the light (inactive) phase. Furthermore, we found that leptin sensitivity on a behavioural level followed the same rhythm, with mice showing a greater response to exogenous leptin at the end of the dark phase at Zeitgeber time (ZT) 0 compared with the end of the light phase at ZT12. Throughout the 24-hour cycle, mice displayed a robust rhythm in food intake, locomotor activity as well as oxygen consumption and energy expenditure, with reduced whole body metabolism during their inactive and increased metabolic rate during their active phase. In DIO mice that were subjected to high-fat diet (HFD) feeding, we found a disruption of the 24-hour rhythmic regulation of leptin pathway activation on a molecular level for both basal and leptin-induced leptin sensitivity. Intriguingly, we demonstrated that this hypothalamic leptin resistance is a temporary phenomenon that persists only at specific times during the day. Responsiveness to leptin was deteriorated during the second part of the dark and the first half of the light phase (ZT21 – ZT6), but identical to mice fed low-fat diet (LFD) at all other times on both the molecular and behavioural level. Furthermore, DIO mice displayed a disruption of the daily rhythms in food intake, locomotor activity, oxygen consumption and energy expenditure. We found that the daily caloric overconsumption observed in mice fed HFD was restricted to the phase when DIO mice were leptin resistant relative to mice fed LFD. In conclusion, these findings provide strong evidence for a crucial role of the 24-hour rhythm of leptin sensitivity in the control of energy metabolism. We furthermore demonstrated that mice with access to HFD exclusively during their leptin resistant phase (ZT21 – ZT3) displayed impairments in a variety of parameters that indicate metabolic health, such as compromised rhythms of locomotor activity, metabolic rate, and energy expenditure as well as increased circulating insulin levels. Restricting HFD exclusively to the leptin sensitive phase (ZT9 – ZT15), on the other hand, protected mice from the development of these severe metabolic impairments. To date it is still largely unknown whether HFD-induced development of metabolic diseases results from an increase in body fat content, diet composition or disrupted circadian rhythms. We observed these differences between TRF groups despite an identical reduction in body weight and plasma leptin levels in all TRF mice, suggesting that they are based on the time of food intake during the 24-hour rhythm of leptin sensitivity, but independent from factors such as body composition or HFD content. Nonetheless, all mice fed HFD displayed a reduction in the absolute values of average metabolic rate and energy expenditure relative to mice fed LFD, demonstrating that also the HFD itself affects energy metabolism. In conclusion, these results demonstrate that TRF is efficient in the reduction of body weight and the amelioration of metabolic health. However, our findings also highlight the importance of synchronising food intake with daily physiological rhythms to maintain metabolic health. Taken together, this thesis identifies novel pathways that are involved in the neuroendocrine regulation of energy metabolism and provides new insights into the connection between physiological rhythms and the development of metabolic diseases

Imaging Multidimensional Therapeutically Relevant Circadian Relationships

Circadian clocks gate cellular proliferation and, thereby, therapeutically target availability within proliferative pathways. This temporal coordination occurs within both cancerous and noncancerous proliferating tissues. The timing within the circadian cycle of the administration of drugs targeting proliferative pathways necessarily impacts the amount of damage done to proliferating tissues and cancers. Concurrently measuring target levels and associated key pathway components in normal and malignant tissues around the circadian clock provides a path toward a fuller understanding of the temporal relationships among the physiologic processes governing the therapeutic index of antiproliferative anticancer therapies. The temporal ordering among these relationships, paramount to determining causation, is less well understood using two- or three-dimensional representations. We have created multidimensional multimedia depictions of the temporal unfolding of putatively causative and the resultant therapeutic effects of a drug that specifically targets these ordered processes at specific times of the day. The systems and methods used to create these depictions are provided, as well as three example supplementary movies

The Diurnal Rhythm of Insulin Receptor Substrate-1 (IRS-1) and Kir4.1 in Diabetes: Implications for a Clock Gene Bmal1

Purpose: Diabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels. Methods: We assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA. Results: The IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1. Conclusions: Our studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1