Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Autonomous and self-sustained circadian oscillators displayed in human islet cells

Aims/hypothesis: Following on from the emerging importance of the pancreas circadian clock on islet function and the development of type 2 diabetes in rodent models, we aimed to examine circadian gene expression in human islets. The oscillator properties were assessed in intact islets as well as in beta cells. Methods: We established a system for long-term bioluminescence recording in cultured human islets, employing lentivector gene delivery of the core clock gene Bmal1 (also known as Arntl)-luciferase reporter. Beta cells were stably labelled using a rat insulin2 promoter fluorescent construct. Single-islet/cell oscillation profiles were measured by combined bioluminescence-fluorescence time-lapse microscopy. Results: Human islets synchronised in vitro exhibited self-sustained circadian oscillations of Bmal1-luciferase expression at both the population and single-islet levels, with period lengths of 23.6 and 23.9h, respectively. Endogenous BMAL1 and CRY1 transcript expression was circadian in synchronised islets over 48h, and antiphasic to REV-ERBα (also known as NR1D1), PER1, PER2, PER3 and DBP transcript circadian profiles. HNF1A and PDX1 exhibited weak circadian oscillations, in phase with the REV-ERBα transcript. Dispersed islet cells were strongly oscillating as well, at population and single-cell levels. Importantly, beta and non-beta cells revealed oscillatory profiles that were well synchronised with each other. Conclusions/interpretation: We provide for the first time compelling evidence for high-amplitude cell-autonomous circadian oscillators displayed in human pancreatic islets and in dispersed human islet cells. Moreover, these clocks are synchronised between beta and non-beta cells in primary human islet cell culture

Rhythmic Diurnal Gene Expression in Human Adipose Tissue From Individuals Who Are Lean, Overweight, and Type 2 Diabetic

OBJECTIVE Previous animal studies suggest a functional relationship between metabolism, type 2 diabetes, and the amplitude of daily rhythms in white adipose tissue (WAT). However, data interpretation is confounded by differences in genetic background and diet or limited sampling points. We have taken the novel approach of analyzing serial human WAT biopsies across a 24-h cycle in controlled laboratory conditions.RESEARCH DESIGN AND METHODS Lean (n = 8), overweight/obese (n = 11), or overweight/obese type 2 diabetic (n = 8) volunteers followed a strict sleep-wake and dietary regimen for 1 week prior to the laboratory study. They were then maintained in controlled light-dark conditions in a semirecumbent posture and fed hourly during wake periods. Subcutaneous WAT biopsies were collected every 6 h over 24 h, and gene expression was measured by quantitative PCR.RESULTS Lean individuals exhibited significant (P 0.05) of increased body weight or type 2 diabetes on rhythmic gene expression.CONCLUSIONS The robust nature of these rhythms and their relative phasing indicate that WAT now can be considered as a peripheral tissue suitable for the study of in vivo human rhythms. Comparison of data between subject groups clearly indicates that obesity and type 2 diabetes are not related to the amplitude of rhythmic WAT gene expression in humans maintained under controlled conditions. Diabetes 60:1577-1581, 201

Development of and Access to Products for Neglected Diseases

INTRODUCTION: Prior research on neglected disease drug development suggested inadequate funding was responsible for relatively few new approvals. In response, significantly more resources have been allocated towards development of drugs targeting neglected diseases. Our objective was to reassess drug development between 1975 and 1999, evaluate progress in neglected disease drug development since 2000, and explain how increased numbers of approvals are a necessary but insufficient condition to improving access. METHODS: To assess numbers of approvals targeting neglected diseases, we employed two distinct methodologies: First, to revisit numbers published in Trouiller et al. (2002) we used their method to count marketed new chemical entities (NCEs) between 1975 and 1999. Second, using the G-Finder report as a benchmark, we identified which diseases are currently considered "neglected" to tally approvals in the 1975-1999 and 2000-2009 periods. Searching PharmaProjects and IMS R&D Focus databases as well as websites from numerous drug regulatory agencies, we identified new drug approvals and indications. Also, we examined the World Health Organization's (WHO) Essential Drug List (EDL) to see which drugs and indications were on the list. FINDINGS: Upon recount, using Trouiller et al. methodology, we found that between 1975 and 1999 more NCEs (n = 32) targeting tropical diseases and tuberculosis were approved than reported in Trouiller et al. (n = 16). Using the G-Finder method of defining neglected diseases, we found 46 new drug approvals between 1975 and 1999. WHO included 85% of these drugs on the EDL. In the period 2000 to May 2009, despite much greater funding, only 26 new drugs and vaccines for neglected diseases were marketed. Of these, WHO placed 50% on the EDL. CONCLUSIONS: Product approvals for neglected diseases have increased, though progress has been uneven, with malaria appearing to benefit most in the short run from increased funding, while less success has been booked in other disease categories. Uneven progress suggests funding could be better targeted, particularly with regard to neglected diseases that have hitherto received scant attention. In addition, policymakers should focus on other aspects related to access. Besides drug development, there are the issues of EDL listing, architecture, availability, affordability, and adoption

MicroRNAs modulate core-clock gene expression in pancreatic islets during early postnatal life in rats.

Evidence continues to emerge detailing a fine-tuning of the regulation of metabolic processes and energy homeostasis by cell-autonomous circadian clocks. Pancreatic beta cell functional maturation occurs after birth and implies transcriptional changes triggered by a shift in the nutritional supply that occurs at weaning, enabling the adaptation of insulin secretion. So far, the developmental timing and exact mechanisms involved in the initiation of the circadian clock in the growing pancreatic islets have never been addressed. Circadian gene expression was measured by quantitative RT-PCR in islets of rats at different postnatal ages up to 3 months, and by in vitro bioluminescence recording in newborn (10-day-old) and adult (3-month-old) islets. The effect of the microRNAs miR-17-5p and miR-29b-3p on the expression of target circadian genes was assessed in newborn rat islets transfected with microRNA antisense or mimic oligonucleotides, and luciferase reporter assays were performed on the rat insulin-secreting cell line INS832/13 to determine a direct effect. The global regulatory network between microRNAs and circadian genes was computationally predicted. We found up to a sixfold-change in the 24 h transcriptional oscillations and overall expression of Clock, Npas2, Bmal1, Bmal2, Rev-erbα, Per1, Per2, Per3 and Cry2 between newborn and adult rat islets. Synchronisation of the clock machinery in cultured islet cells revealed a delayed cell-autonomous rhythmicity of about 1.5 h in newborn compared with adult rats. Computational predictions unveiled the existence of a complex regulatory network linking over 40 microRNAs displaying modifications in their expression profiles during postnatal beta cell maturation and key core-clock genes. In agreement with these computational predictions, we demonstrated that miR-17-5p and miR-29b-3p directly regulated circadian gene expression in the maturing islet cells of 10-day-old rats. These data show that the circadian clock is not fully operational in newborn islets and that microRNAs potently contribute to its regulation during postnatal beta cell maturation. Defects in this process may have long-term consequences on circadian physiology and pancreatic islet function, favouring the manifestation of metabolic diseases such as diabetes

XMeis3 Is Necessary for Mesodermal Hox Gene Expression and Function

Hox transcription factors provide positional information during patterning of the anteroposterior axis. Hox transcription factors can co-operatively bind with PBC-class co-factors, enhancing specificity and affinity for their appropriate binding sites. The nuclear localisation of these co-factors is regulated by the Meis-class of homeodomain proteins. During development of the zebrafish hindbrain, Meis3 has previously been shown to synergise with Hoxb1 in the autoregulation of Hoxb1. In Xenopus XMeis3 posteriorises the embryo upon ectopic expression. Recently, an early temporally collinear expression sequence of Hox genes was detected in Xenopus gastrula mesoderm (see intro. P3). There is evidence that this sequence sets up the embryo's later axial Hox expression pattern by time-space translation. We investigated whether XMeis3 is involved in regulation of this early mesodermal Hox gene expression. Here, we present evidence that XMeis3 is necessary for expression of Hoxd1, Hoxb4 and Hoxc6 in mesoderm during gastrulation. In addition, we show that XMeis3 function is necessary for the progression of gastrulation. Finally, we present evidence for synergy between XMeis3 and Hoxd1 in Hoxd1 autoregulation in mesoderm during gastrulation

Coupling of a Core Post-Translational Pacemaker to a Slave Transcription/Translation Feedback Loop in a Circadian System

Analysis of the cyanobacterial circadian biological clock reveals a complex interdependence between a transcription/translation feedback loop and a biochemical oscillator