Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.     

The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis

Recent studies have correlated metabolic diseases, such as metabolic syndrome and non-alcoholic fatty liver disease, with the circadian clock. However, whether such metabolic changes per se affect the circadian clock remains controversial. To address this, we investigated the daily mRNA expression profiles of clock genes in the liver of a dietary mouse model of non-alcoholic steatohepatitis (NASH) using a custom-made, high-precision DNA chip. C57BL/6J mice fed an atherogenic diet for 5 weeks developed hypercholesterolemia, oxidative stress, and NASH. DNA chip analyses revealed that the atherogenic diet had a great influence on the mRNA expression of a wide range of genes linked to mitochondrial energy production, redox regulation, and carbohydrate and lipid metabolism. However, the rhythmic mRNA expression of the clock genes in the liver remained intact. Most of the circadianly expressed genes also showed 24-h rhythmicity. These findings suggest that the biological clock is protected against such a metabolic derangement as NASH.     

Modification of gene expression by dietary antioxidants in radiation-induced apoptosis of mice splenocytes

The modification of radiation-induced apoptosis in splenocytes by a vitamin-containing dietary supplement was studied. For 45 days prior to irradiation at a lethal dose of 6 Gy, mice received a dietary supplement containing vitamins with antioxidant properties and microelements. The expression of TRPM-2 (a marker for programmed cell death), bcl-2 (the product of which has been shown to prevent apoptosis), superoxide dismutase, and catalase genes was studied at different time intervals after irradiation. Radiation-induced alterations in gene expression were different in the control and the antioxidant mixture-fed mice. The antioxidant mixture administration resulted in an inhibition of TRPM-2 expression both before and after irradiation. The bcl-2 mRNA content steadily increased after irradiation in splenocytes from antioxidant mixture-fed mice, while in the control group 2-h after irradiation only trace amount of bcl-2 mRNA was detected. In splenocytes from control mice, the expression of superoxide dismutase and catalase genes significantly decreased within 2-h after irradiation; whereas in mice receiving the antioxidant mixture, inhibition of catalase gene expression was not as prominent. The expression of superoxide dismutase gene was still high 24-h after irradiation. The antioxidant administration decreased the radiation-induced apoptosis and delayed internucleosomal fragmentation of DNA. Our data suggest that radiation-induced alteration of gene expression is, at least in part, determined by reactive oxygen species.     

Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.     

Gene expression profiling in the rhesus macaque: Experimental design considerations

The development of species-specific gene microarrays has greatly facilitated gene expression profiling in nonhuman primates. However, to obtain accurate and physiologically meaningful data from these microarrays, one needs to consider several factors when designing the studies. This article focuses on effective experimental design while the companion article focuses on methodology and data analysis. Biological cycles have a major influence on gene expression, and at least 10% of the expressed genes are likely to show a 24-h expression pattern. Consequently, the time of day when RNA samples are collected can influence detection of significant changes in gene expression levels. Similarly, when photoperiodic species such as the rhesus macaque are housed outdoors, some of their genes show differential expression according to the time of year. In addition, the sex-steroid environment of humans and many nonhuman primates changes markedly across the menstrual cycle, and so phase of the cycle needs to be considered when studying gene expression in adult females.     

Obesity and genetics regulate microRNAs in islets, liver, and adipose of diabetic mice

Type 2 diabetes results from severe insulin resistance coupled with a failure of β cells to compensate by secreting sufficient insulin. Multiple genetic loci are involved in the development of diabetes, although the effect of each gene on diabetes susceptibility is thought to be small. MicroRNAs (miRNAs) are noncoding 19–22-nucleotide RNA molecules that potentially regulate the expression of thousands of genes. To understand the relationship between miRNA regulation and obesity-induced diabetes, we quantitatively profiled approximately 220 miRNAs in pancreatic islets, adipose tissue, and liver from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mice. More than half of the miRNAs profiled were expressed in all three tissues, with many miRNAs in each tissue showing significant changes in response to genetic obesity. Furthermore, several miRNAs in each tissue were differentially responsive to obesity in B6 versus BTBR mice, suggesting that they may be involved in the pathogenesis of diabetes. In liver there were approximately 40 miRNAs that were downregulated in response to obesity in B6 but not BTBR mice, indicating that genetic differences between the mouse strains play a critical role in miRNA regulation. In order to elucidate the genetic architecture of hepatic miRNA expression, we measured the expression of miRNAs in genetically obese F2 mice. Approximately 10% of the miRNAs measured showed significant linkage (miR-eQTLs), identifying loci that control miRNA abundance. Understanding the influence that obesity and genetics exert on the regulation of miRNA expression will reveal the role miRNAs play in the context of obesity-induced type 2 diabetes.     

Differential gene expression in pancreatic tissues of streptozocin-induced diabetic rats and genetically-diabetic mice in response to hypoglycemic dipeptide cyclo (His-Pro) treatment

Diabetic studies are mostly interested in gene expression in the pancreas, the site of insulin secretion that regulates blood glucose levels. However, a single gene approach has been ruled out for many years in discovering new genes or the molecular networks involved in the induction process of diabetes. To understand the molecular mechanisms by which cyclo (His-Pro) (CHP) affects amelioration of diabetes mellitus, we performed gene expression profiling in the pancreatic tissues of two diabetic animal models, streptozocin (STZ)-induced diabetic rats (T1DM) and genetically-diabetic (C57BL/6J ob/ob) mice (T2DM). To understand the healing process of these diabetic rodents, we examined the effects of CHP on various gene expression in pancreatic tissues of both animal models. Our microarray analysis revealed that a total of 1,175 genes were down-regulated and 629 genes were up-regulated in response to STZ treatment, and the altered expression levels of numerous genes were restored to normal state upon CHP treatment. In particular, 476 genes showed significantly altered gene expression upon CHP treatment. In a functional classification, 7,198 genes were counted as differentially expressed in pancreatic tissues of STZ- and CHP-treated rats compared with control, whereas 1,534 genes were restored to normal states by CHP treatment. Microarray data demonstrated for the first time that overexpression of the genes encoding IL-1 receptor, lipid metabolic enzymes (e.g. Mte1, Ptdss1, and Sult2a1), myo-inositol oxygenase, glucagon, and somatostatin as well as down-regulation of olfactory receptor 984 and mitochondrial ribosomal protein, which are highly linked to T1DM etiology. In genetically-diabetic mice, 4,384 genes were altered in gene expression by more than 2-fold compared to the control mice, when counted differentially expressed. In genetically-diabetic mice, 4,384 genes altered in expression by higher than 2-fold were counted as differentially expressed genes in pancreatic tissues of CHP-treated mice. On the other hand, 2,140 genes were up-regulated and 2,244 genes were down-regulated by CHP treatment. The results of the microarray analysis revealed that up-regulation of IL-2, IL12a, and leptin receptor and down-regulation of PIK3 played important physiological roles in the onset of T2DM. In conclusion, we hypothesize that CHP accelerates alterations of gene expression in ameliorating diabetes and antagonizes those that induces the disease.     

Circadian Cycle-Dependent MeCP2 and Brain Chromatin Changes

Methyl CpG binding protein 2 (MeCP2) is a chromosomal protein of the brain, very abundant especially in neurons, where it plays an important role in the regulation of gene expression. Hence it has the potential to be affected by the mammalian circadian cycle. We performed expression analyses of mice brain frontal cortices obtained at different time points and we found that the levels of MeCP2 are altered circadianly, affecting overall organization of brain chromatin and resulting in a circadian-dependent regulation of well-stablished MeCP2 target genes. Furthermore, this data suggests that alterations of MeCP2 can be responsible for the sleeping disorders arising from pathological stages, such as in autism and Rett syndrome.     

An approximation to the temporal order in endogenous circadian rhythms of genes implicated in human adipose tissue metabolism

Although it is well established that human adipose tissue (AT) shows circadian rhythmicity, published studies have been discussed as if tissues or systems showed only one or few circadian rhythms at a time. To provide an overall view of the internal temporal order of circadian rhythms in human AT including genes implicated in metabolic processes such as energy intake and expenditure, insulin resistance, adipocyte differentiation, dyslipidemia, and body fat distribution. Visceral and subcutaneous abdominal AT biopsies (n=6) were obtained from morbid obese women (BMI≥40 kg/m(2) ). To investigate rhythmic expression pattern, AT explants were cultured during 24-h and gene expression was analyzed at the following times: 08:00, 14:00, 20:00, 02:00 h using quantitative real-time PCR. Clock genes, glucocorticoid metabolism-related genes, leptin, adiponectin and their receptors were studied. Significant differences were found both in achrophases and relative-amplitude among genes (P<0.05). Amplitude of most genes rhythms was high (>30%). When interpreting the phase map of gene expression in both depots, data indicated that circadian rhythmicity of the genes studied followed a predictable physiological pattern, particularly for subcutaneous AT. Interesting are the relationships between adiponectin, leptin, and glucocorticoid metabolism-related genes circadian profiles. Their metabolic significance is discussed. Visceral AT behaved in a different way than subcutaneous for most of the genes studied. For every gene, protein mRNA levels fluctuated during the day in synchrony with its receptors. We have provided an overall view of the internal temporal order of circadian rhythms in human adipose tissue.     

Expression of stathmin family genes in the murine uterus during early pregnancy

Stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics during cell-cycle progression, is abundantly expressed at embryo implantation sites in rats. Here, we characterized the expression of stathmin and its family genes in the murine uterus during the peri-implantation period. Stathmin protein was expressed in the glandular and luminal epithelium, blood vessels, and stromal cells on day 3 of pregnancy. On the day of implantation (day 5), stathmin was mainly localized in blood vessels in the endometrium. On day 7, intense stathmin expression was limited to capillary vessels and secondary decidual cells. Stathmin expression was higher at implantation sites than at uterine segments between implantation sites and increased during oil-induced decidualization. Although the artificially-induced deciduoma weights and number of implantation sites were similar between stathmin-knockout (KO) and wild-type (WT) mice, the stathmin-KO mice had fewer newborn pups (reduced by 30%). The expression of alkaline phosphatase, desmin, and cyclin D3 was attenuated in decidual zones of stathmin-KO mice. Messenger RNA level of the stathmin family gene, SCG10, was high at the time of decidualization in WT and stathmin-KO mice. In contrast, the others of stathmin family members, SCLIP and RB3 were highly expressed in stathmin-KO mice compared to WT mice. These results suggest that stathmin and stathmin family genes are expressed in the murine endometrium with enhanced expression in the implantation or the decidualization process.     

Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice

Adiponectin (ApN) and leptin are two adipocytokines that control fuel homeostasis, body weight, and insulin sensitivity. Their interplay is still poorly studied. These hormones are either undetectable or decreased in obese, diabetic ob/ob mice. We examined the effects of leptin treatment on ApN gene expression, protein production, secretion, and circulating levels of ob/ob mice. We also briefly tackled the influence of this treatment on resistin, another adipocytokine involved in obesity-related insulin resistance. Leptin-treated (T) obese mice (continuous sc infusion for 6 days) were compared with untreated lean (L), untreated obese (O), and untreated pair-fed obese (PF) mice. Blood was collected throughout the study. At day 3 or day 6, fat pads were either directly analyzed (mRNA, ApN content) or cultured for up to 24 h (ApN secretion). The direct effect of leptin was also studied in 3T3-F442A adipocytes. Compared with L mice, ApN content of visceral or subcutaneous fat and ApN secretion by adipose explants were blunted in obese mice. Accordingly, plasma ApN levels of O mice were decreased by 50%. Leptin treatment of ob/ob mice increased ApN mRNAs, ApN content, and secretion from the visceral depot by 50-80%. Leptin also directly stimulated ApN mRNAs and secretion from 3T3-F442A adipocytes. After 6 days of treatment, plasma ApN of ob/ob mice increased 2.5-fold, a rise that did not occur in PF mice. Plasma resistin of T mice was barely decreased. Leptin treatment, but not mere calorie restriction, corrects plasma ApN in obese mice by restoring adipose tissue ApN concentrations and secretion, at least in part, via a direct stimulation of ApN gene expression. Such a treatment only minimally affects circulating resistin. ApN restoration could, in concert with leptin, contribute to the metabolic effects classically observed during leptin administration.     

Daytime soybean transcriptome fluctuations during water deficit stress

Background

Since drought can seriously affect plant growth and development and little is known about how the oscillations of gene expression during the drought stress-acclimation response in soybean is affected, we applied Illumina technology to sequence 36 cDNA libraries synthesized from control and drought-stressed soybean plants to verify the dynamic changes in gene expression during a 24-h time course. Cycling variables were measured from the expression data to determine the putative circadian rhythm regulation of gene expression.

Results

We identified 4866 genes differentially expressed in soybean plants in response to water deficit. Of these genes, 3715 were differentially expressed during the light period, from which approximately 9.55 % were observed in both light and darkness. We found 887 genes that were either up- or down-regulated in different periods of the day. Of 54,175 predicted soybean genes, 35.52 % exhibited expression oscillations in a 24 h period. This number increased to 39.23 % when plants were submitted to water deficit. Major differences in gene expression were observed in the control plants from late day (ZT16) until predawn (ZT20) periods, indicating that gene expression oscillates during the course of 24 h in normal development. Under water deficit, dissimilarity increased in all time-periods, indicating that the applied stress influenced gene expression. Such differences in plants under stress were primarily observed in ZT0 (early morning) to ZT8 (late day) and also from ZT4 to ZT12. Stress-related pathways were triggered in response to water deficit primarily during midday, when more genes were up-regulated compared to early morning. Additionally, genes known to be involved in secondary metabolism and hormone signaling were also expressed in the dark period.

Conclusions

Gene expression networks can be dynamically shaped to acclimate plant metabolism under environmental stressful conditions. We have identified putative cycling genes that are expressed in soybean leaves under normal developmental conditions and genes whose expression oscillates under conditions of water deficit. These results suggest that time of day, as well as light and temperature oscillations that occur considerably affect the regulation of water deficit stress response in soybean plants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1731-x) contains supplementary material, which is available to authorized users.