Volodymyr Petrenko

A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells

To determine the impact of a functional human islet clock on insulin secretion and gene transcription.

Pancreatic α- and β-cellular clocks have distinct molecular properties and impact on islet hormone secretion and gene expression

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and β-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and β-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for β cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and β-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and β-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and β cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and β-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.

Glucose Homeostasis: Regulation by Peripheral Circadian Clocks in Rodents and Humans

Most organisms, including humans, have developed an intrinsic system of circadian oscillators, allowing the anticipation of events related to the rotation of Earth around its own axis. The mammalian circadian timing system orchestrates nearly all aspects of physiology and behavior. Together with systemic signals, emanating from the central clock that resides in the hypothalamus, peripheral oscillators orchestrate tissue-specific fluctuations in gene expression, protein synthesis, and posttranslational modifications, driving overt rhythms in physiology and behavior. There is increasing evidence on the essential roles of the peripheral oscillators, operative in metabolically active organs in the regulation of body glucose homeostasis. Here, we review some recent findings on the molecular and cellular makeup of the circadian timing system and its implications in the temporal coordination of metabolism in health and disease.

Process of cortical network formation and impact of early brain damage.

PURPOSE OF REVIEW The aim is to review mechanisms that are central to the formation of proper cortical circuitry and relevant to perinatal brain injury and premature birth. RECENT FINDINGS Clinical investigations using noninvasive imaging techniques suggest that impaired connectivity of cortical circuitry is associated with perinatal adverse conditions. Recent experimental and translational studies revealed developmental mechanisms that are critical for circuit formation and potentially at risk in the perinatal period. These include existence of last wave genesis, migration and integration of gamma-aminobutyric acid (GABA) interneurons in the perinatal period; maturation of GABA interneuron networks that are central to critical period plasticity; transient connections by subplate neurons that guide thalamocortical connectivity, and a perineuronal microglia network that maintains axonal growth and neuronal survival as well as executing synaptic pruning. In addition, recent work has demonstrated that birth plays a key role in triggering the maturation cascade of cortical circuits. SUMMARY Altered maturation of cortical circuits is an increasingly recognized aspect of perinatal injury and premature birth. Potential mechanisms are revealed but further translational studies are required to associate fine changes at the cellular and molecular level with imaging data in experimental models.

High-Resolution Recording of the Circadian Oscillator in Primary Mouse α- and β-Cell Culture

Circadian clocks have been developed in evolution as an anticipatory mechanism allowing for adaptation to the constantly changing light environment due to rotation of the Earth. This mechanism is functional in all light-sensitive organisms. There is a considerable body of evidence on the tight connection between the circadian clock and most aspects of physiology and metabolism. Clocks, operative in the pancreatic islets, have caught particular attention in the last years due to recent reports on their critical roles in regulation of insulin secretion and etiology of type 2 diabetes. While β-cell clocks have been extensively studied during the last years, α-cell clocks and their role in islet function and orchestration of glucose metabolism stayed unexplored, largely due to the difficulty to isolate α-cells, which represents a considerable technical challenge. Here, we provide a detailed description of an experimental approach for the isolation of separate mouse α- and β-cell population, culture of isolated primary α- and β-cells, and their subsequent long-term high-resolution circadian bioluminescence recording. For this purpose, a triple reporter ProGlucagon-Venus/RIP-Cherry/Per2:Luciferase mouse line was established, carrying specific fluorescent reporters for α- and β-cells, and luciferase reporter for monitoring the molecular clockwork. Flow cytometry fluorescence-activated cell sorting allowed separating pure α- and β-cell populations from isolated islets. Experimental conditions, developed by us for the culture of functional primary mouse α- and β-cells for at least 10 days, will be highlighted. Importantly, temporal analysis of freshly isolated α- and β-cells around-the-clock revealed preserved rhythmicity of core clock genes expression. Finally, we describe the setting to assess circadian rhythm in cultured α- and β-cells synchronized in vitro. The here-described methodology allows to analyze the functional properties of primary α- and β-cells under physiological or pathophysiological conditions and to assess the islet cellular clock properties.

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Lactoferrin during lactation reduces lipopolysaccharide‐induced brain injury

Lactoferrin (Lf), component of maternal milk, has antioxidant, anti-inflammatory and antimicrobial properties. Neuroprotective effects of Lf on the immature brain have been recently shown in rodent models of intrauterine growth restriction and cerebral hypoxia/ischemia. Here we postulated that Lf could also have beneficial effects on preterm inflammatory brain injury. Lf was supplemented in maternal food during lactation and lipopolysaccharide (LPS) was injected in subcortical white matter of rat pups at postnatal day 3 (P3). Effect of maternal Lf supplementation was investigated 24 h (P4), 4 (P7), or 21 days (P24) after LPS injection mainly on the striatum. Lateral ventricle and brain structures volumes were quantified. Microstructure was evaluated by diffusion tensor imaging, neurite orientation dispersion and density imaging as well as electron microscopy. Neurochemical profile was measured by (1) H-magnetic resonance spectroscopy. GFAP protein, proinflammatory cytokines mRNA expression microglial activation were assessed. Lf displayed neuroprotective effects as shown by reduced LPS-induced ventriculomegaly, brain tissue loss, and microstructural modifications, including myelination deficit. (1) H-MRS neurochemical profile was less altered through an antioxidant action of Lf. Despite the lack of effect on LPS-induced proinflammatory cytokines genes expression and on reactive gliosis, microglia was less activated under Lf treatment. In conclusion, Lf supplemented in food during lactation attenuated acute and long-term cerebral LPS-induced alterations. This provides a new evidence for a promising use of Lf as a preventive neuroprotective approach in preterm encephalopathy.

Multimodal MRI Imaging of Apoptosis-Triggered Microstructural Alterations in the Postnatal Cerebral Cortex

Prematurely born children often develop neurodevelopmental delay that has been correlated with reduced growth and microstructural alterations in the cerebral cortex. Much research has focused on apoptotic neuronal cell death as a key neuropathological features following preterm brain injuries. How scattered apoptotic death of neurons may contribute to microstructural alterations remains unknown. The present study investigated in a rat model the effects of targeted neuronal apoptosis on cortical microstructure using in vivo MRI imaging combined with neuronal reconstruction and histological analysis. We describe that mild, targeted death of layer IV neurons in the developing rat cortex induces MRI-defined metabolic and microstructural alterations including increased cortical fractional anisotropy. Delayed architectural modifications in cortical gray matter and myelin abnormalities in the subcortical white matter such as hypomyelination and microglia activation follow the acute phase of neuronal death and axonal degeneration. These results establish the link between mild cortical apoptosis and MRI-defined microstructure changes that are reminiscent to those previously observed in preterm babies.

Circadian orchestration of insulin and glucagon release

The circadian clock system represents an anticipatory mechanism, playing an essential role in the temporal coordination of metabolism in the physiological context, and in etiology ofmetabolic disorders. Cell-autonomous self-sustained circadian oscillators are functional in virtually all cells in the body. Such body-web of circadian clocks must be synchronized on a daily basis, with the lightdark cycle being the principle synchronizer or Zeitgeber (time-giver from German), which impacts on rhythmic systemic signals that are controlled by the master pacemaker in the suprachiasmatic nucleus (SCN) of the brain. Furthermore, oscillators operative in peripheral tissues and cells can be synchronized by a variety of signals, allowing their investigation in vitro. The circadian system plays a key role in the regulation of body metabolism, including glucose homeostasis via coordination of hormone secretion by the endocrine pancreas on one hand, and glucose utilization by skeletal muscle and adipose tissue on the other. A functional unit of the endocrine pancreas, the pancreatic islet of Langerhans, represents a 3-dimensional structure of distinct types of endocrine cells, tightly packed together in an organized and species-specific way. While intercellular oscillator coupling is indispensable for SCN neuron synchronization, peripheral clocks do not show coupling within organs. In line with these findings, mouse and human intact pancreatic islets and dispersed islet cells exhibit pronounced circadian oscillations, suggesting that physical interaction between neighboring cells within the 3-dimensional islet architecture is not crucial for the cellular oscillator function. Although fundamental aspects concerning the circadian control of transcriptional and functional regulation in whole pancreatic islets have recently been assessed, our knowledge regarding individual islet cell molecular clocks and their inputs and outputs is still limited. The bi-hormonal hypothesis proposed by Unger and Orci back in 1975 emphasizes that the perturbation of glucose homeostasis in the context of diabetes mellitus is stemming from altering the balance between the glucose regulating counter hormones insulin and glucagon, rather than from the pathology of insulin alone. Clearly, acute glucose sensing plays an essential role in the temporal separation of glucagon and insulin secretion by adjacent aand b-cells. Our recent parallel study of the circadian clockwork in glucagon-producing a-cells along with insulin producing b-cells, separated from each other following pancreatic islets isolation based on cell specific fluorescent reporters, revealed additional plausible mechanism for such temporal coordination. Strikingly, aand b-cellular clocks exhibited distinct circadian properties, including a phase difference between aand b-cell oscillators that was observed in vivo and in vitro, at population and at single-cell levels (Fig. 1,). Such phase coherence, which was kept upon islet cell synchronization in vitro with physiologically relevant stimuli such as adrenaline, is likely to be mediated by the distinct repertoire of receptors expressed on aand b-cell surfaces, as it is the case for the adrenergic receptors. Moreover, basal secretion profiles of glucagon and insulin by mixed and separated aand b-cell cultures revealed oscillatory profiles for both islet hormones, further suggesting that cell-autonomous islet oscillators are likely to contribute importantly to the orchestration of these temporal secretion patterns (Fig. 1,). Large-scale in vivo transcriptome analysis, conducted in separated aand b-cells, revealed that a high number of key islet genes exhibit oscillatory profiles in either one or both islet cell types. Rhythmically expressed transcripts with similar or distinct characteristics in aand b-cells comprised those encoding for glucose transporters, glucose metabolic enzymes, and regulators of granule trafficking and exocytosis, which may account for the cyclic pattern of islet hormone secretion as has been previously suggested by wholeislet analysis. Collectively, these findings elucidate that beyond acute control mechanisms of insulin or glucagon secretion, driven by feeding-fasting conditions and reflected by fast changes in blood glucose levels, there is an additional mechanism governed by cell-autonomous islet clocks. This latter regulatory machinery is coordinating the secretion of insulin and glucagon in a daytime-dependent manner, that ensures anticipation of changes related to rest-activity and feeding-fasting cycles, resulting in the optimal adaptation of both key glucose regulating islet hormones to the organismal needs. Notably, circadian regulation of basal insulin secretion by human pancreatic islets

Identification of Differential Transcriptional Patterns in Primary and Secondary Hyperparathyroidism

Context Hyperparathyroidism is associated with hypercalcemia and the excess of parathyroid hormone secretion; however, the alterations in molecular pattern of functional genes during parathyroid tumorigenesis have not been unraveled. We aimed at establishing transcriptional patterns of normal and pathological parathyroid glands (PGs) in sporadic primary (HPT1) and secondary hyperparathyroidism (HPT2). Objective To evaluate dynamic alterations in molecular patterns as a function of the type of PG pathology, a comparative transcript analysis was conducted in subgroups of healthy samples, sporadic HPT1 adenoma and hyperplasia, and HPT2. Design Normal, adenomatous, HPT1, and HPT2 hyperplastic PG formalin-fixed paraffin-embedded samples were subjected to NanoString analysis. In silico microRNA (miRNA) analyses and messenger RNA-miRNA network in PG pathologies were conducted. Individual messenger RNA and miRNA levels were assessed in snap-frozen PG samples. Results The expression levels of c-MET, MYC, TIMP1, and clock genes NFIL3 and PER1 were significantly altered in HPT1 adenoma compared with normal PG tissue when assessed by NanoString and quantitative reverse transcription polymerase chain reaction. RET was affected in HPT1 hyperplasia, whereas CaSR and VDR transcripts were downregulated in HPT2 hyperplastic PG tissue. CDH1, c-MET, MYC, and CaSR were altered in adenoma compared with hyperplasia. Correlation analyses suggest that c-MET, MYC, and NFIL3 exhibit collective expression level changes associated with HPT1 adenoma development. miRNAs, predicted in silico to target these genes, did not exhibit a clear tendency upon experimental validation. Conclusions The presented gene expression analysis provides a differential molecular characterization of PG adenoma and hyperplasia pathologies, advancing our understanding of their etiology.