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While it is generally known that metabolic disorders and circadian dysfunction are intertwined, how the two systems affect each other is not well understood, nor are the genetic factors that might exacerbate this pathological interaction. Blood chemistry is profoundly changed in metabolic disorders, and we have previously shown that serum factors change cellular clock properties. To investigate if circulating factors altered in metabolic disorders have circadian modifying effects, and whether these effects are of genetic origin, we measured circadian rhythms in U2OS cell in the presence of serum collected from diabetic, obese or control subjects. We observed that circadian period lengthening in U2OS cells was associated with serum chemistry that is characteristic of insulin resistance. Characterizing the genetic variants that altered circadian period length by genome‐wide association analysis, we found that one of the top variants mapped to the E3 ubiquitin ligase MARCH1 involved in insulin sensitivity. Confirming our data, the serum circadian modifying variants were also enriched in type 2 diabetes and chronotype variants identified in the UK Biobank cohort. Finally, to identify serum factors that might be involved in period lengthening, we performed detailed metabolomics and found that the circadian modifying variants are particularly associated with branched chain amino acids, whose levels are known to correlate with diabetes and insulin resistance. Overall, our multi‐omics data showed comprehensively that systemic factors serve as a path through which metabolic disorders influence circadian system, and these can be examined in human populations directly by simple cellular assays in common cultured cells. [ABSTRACT FROM AUTHOR]

The field of chronobiology has advanced significantly since ancient observations of natural rhythms. The intricate molecular architecture of circadian clocks, their hierarchical organization within the mammalian body, and their pivotal roles in organ physiology highlight the complexity and significance of these internal timekeeping mechanisms. In humans, circadian phenotypes exhibit considerable variability among individuals and throughout the individual's lifespan. A fundamental challenge in mechanistic studies of human chronobiology arises from the difficulty of conducting serial sampling from most organs. The concept of studying circadian clocks in vitro relies on the groundbreaking discovery by Ueli Schibler and colleagues that nearly every cell in the body harbours autonomous molecular oscillators. The advent of circadian bioluminescent reporters has provided a new perspective for this approach, enabling high‐resolution continuous measurements of cell‐autonomous clocks in cultured cells, following in vitro synchronization pulse. The work by Steven A. Brown has provided compelling evidence that clock characteristics assessed in primary mouse and human skin fibroblasts cultured in vitro represent a reliable estimation of internal clock properties in vivo. The in vitro approach for studying molecular human clocks in cultured explants and primary cells, pioneered by Steve Brown, represents an invaluable tool for assessing inter‐individual differences in circadian characteristics alongside comprehensive genetic, biochemical and functional analyses. In a broader context, this reliable and minimally invasive approach offers a unique perspective for unravelling the functional inputs and outputs of oscillators operative in nearly any human tissue in physiological contexts and across various pathologies. [ABSTRACT FROM AUTHOR]

In this study, we identified new lipid species associated with the loss of pancreatic β-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking β-cell prohibitin-2 (a model of monogenic diabetes) patients without previous history of diabetes but scheduled for pancreaticoduodenectomy resulting in the acute reduction of their β-cell mass (∼50%), and patients with type 2 diabetes (T2D). We found lysophosphatidylinositols (lysoPIs) were the main circulating lipid species altered in prediabetic mice. The changes were confirmed in the patients with acute reduction of their β-cell mass and in those with T2D. Increased lysoPIs significantly correlated with HbA1c (reflecting glycemic control), fasting glycemia, and disposition index, and did not correlate with insulin resistance or obesity in human patients with T2D. INS-1E β-cells as well as pancreatic islets isolated from nondiabetic mice and human donors exposed to exogenous lysoPIs showed potentiated glucose-stimulated and basal insulin secretion. Finally, addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. Overall, lysoPIs appear to be lipid species upregulated in the prediabetic stage associated with the loss of β-cells and that support the secretory function of the remaining β-cells. Article Highlights: Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with β-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E β-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of β-cells and supporting the function of the remaining β-cells. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.11.024 Byline: Yoni E. Gutkovich (a), Rachel Ofir (a), Yaniv M. Elkouby (a), Charna Dibner (a)(b), Aharon Gefen (a), Sarah Elias (a), Dale Frank (a) Keywords: Xenopus; XMeis3; Zic1; Pax3; FGF; Hindbrain; Primary neurons; Neural crest Abstract: In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development. Author Affiliation: (a) Department of Biochemistry, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion, Israel Institute of Technology, Haifa 31096, Israel (b) Department of Molecular Biology and NCCR Frontiers in Genetics, Sciences III, University of Geneva, 30, Quai Ernest Ansermet, CH-1211 Geneva-4, Switzerland Article History: Received 30 January 2009; Revised 16 November 2009; Accepted 16 November 2009

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2004.02.029 Byline: Charna Dibner, Sarah Elias, Rachel Ofir, Jacob Souopgui, Peggy J. Kolm, Hazel Sive, Tomas Pieler, Dale Frank Keywords: Xenopus laevis; Hindbrain; XMeis3; Retinoic acid; HoxD1 Abstract: In Xenopus embryos, proper hindbrain formation requires activities of both XMeis3 protein and retinoic acid (RA) signaling. In this study, we show that XMeis3 protein and RA signaling differentially interact to regulate hindbrain patterning. The knockdown of XMeis3 protein prevented RA-caudalizing activity from inducing hindbrain marker expression in both explants and embryos. In contrast, inhibition of RA signaling differentially modulated XMeis3 activity. Target genes that are jointly activated by either RA or XMeis3 activities could not be efficiently induced by XMeis3 when RA signaling was inhibited. However, transcription of an XMeis3 target gene that is not an RA target gene was hyper-induced in the absence of retinoid signaling. Target genes jointly induced by RA or XMeis3 protein were synergistically activated in the presence of both activities, while RA treatment inhibits the ability of XMeis3 to activate transcription of neural genes that are not RA targets. HoxD1, an RA direct-target gene was also identified as an XMeis3 direct-target gene. HoxD1 protein acts downstream of XMeis3 to induce hindbrain marker gene transcription. To pattern the hindbrain, RA requires functional XMeis3 protein activity. XMeis3 protein appears crucial for initial hindbrain induction, whereas RA signaling defines the spatial limits of hindbrain gene expression by modifying XMeis3 protein activity. Article History: Received 16 September 2003; Revised 11 February 2004; Accepted 25 February 2004

Abstract Background Fluorescent and bioluminescent time-lapse microscopy approaches have been successfully used to investigate molecular mechanisms underlying the mammalian circadian oscillator at the single cell level. However, most of the available software and common methods based on intensity-threshold segmentation and frame-to-frame tracking are not applicable in these experiments. This is due to cell movement and dramatic changes in the fluorescent/bioluminescent reporter protein during the circadian cycle, with the lowest expression level very close to the background intensity. At present, the standard approach to analyze data sets obtained from time lapse microscopy is either manual tracking or application of generic image-processing software/dedicated tracking software. To our knowledge, these existing software solutions for manual and automatic tracking have strong limitations in tracking individual cells if their plane shifts. Results In an attempt to improve existing methodology of time-lapse tracking of a large number of moving cells, we have developed a semi-automatic software package. It extracts the trajectory of the cells by tracking theirs displacements, makes the delineation of cell nucleus or whole cell, and finally yields measurements of various features, like reporter protein expression level or cell displacement. As an example, we present here single cell circadian pattern and motility analysis of NIH3T3 mouse fibroblasts expressing a fluorescent circadian reporter protein. Using Circadian Gene Express plugin, we performed fast and nonbiased analysis of large fluorescent time lapse microscopy datasets. Conclusions Our software solution, Circadian Gene Express (CGE), is easy to use and allows precise and semi-automatic tracking of moving cells over longer period of time. In spite of significant circadian variations in protein expression with extremely low expression levels at the valley phase, CGE allows accurate and efficient recording of large number of cell parameters, including level of reporter protein expression, velocity, direction of movement, and others. CGE proves to be useful for the analysis of widefield fluorescent microscopy datasets, as well as for bioluminescence imaging. Moreover, it might be easily adaptable for confocal image analysis by manually choosing one of the focal planes of each z-stack of the various time points of a time series. Availability CGE is a Java plugin for ImageJ; it is freely available at: http://bigwww.epfl.ch/sage/soft/circadian/.