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1.  Variability in Zucker diabetic fatty rats: differences in disease progression in hyperglycemic and normoglycemic animals 
Both obesity and chronic inflammation are often associated with insulin resistance and type 2 diabetes. The Zucker diabetic fatty (ZDF) rat (fa/fa) is an obese animal model frequently used in type 2 diabetes research. The current study determines whether chronic administration (from 5 weeks of age through 24 weeks of age) of salsalate, a salicylate with anti-inflammatory properties, would be effective in mitigating diabetes disease progression in ZDF rats. Although a trend existed for lower blood glucose in the salsalate-treated group, significant differences were obscured by high animal-level variability. However, even in the non-drug-treated group, not all ZDF rats became diabetic as expected. Therefore, animals were parsed into two groups, regardless of drug treatment: normoglycemic ZDF rats, which maintained blood glucose profiles identical to nondiabetic Zucker lean rats (ZLRs), and hyperglycemic ZDF rats, which exhibited progressive elevation in blood glucose. To ascertain the differences between ZDF rats that became hyperglycemic and those that did not, relevant physiological indices and expression levels of adiponectin, tumor necrosis factor-α, interleukin-6, and glucocorticoid-induced leucine zipper messenger RNAs in adipose tissue were measured at sacrifice. Plasma C-reactive protein concentrations and expression levels of cytokine and glucocorticoid-induced leucine zipper messenger RNAs suggested more prevalent chronic inflammation in hyperglycemic animals. Early elevation of the insulin-sensitizing adipokine, adiponectin, was present in both ZDF groups, with the rate of its age-related decline faster in hyperglycemic animals. The most marked difference between the two groups of ZDF animals was in insulin output. Although the two ZDF populations had very similar elevated plasma insulin concentrations for the first 10 weeks, after that time, plasma insulin decreased markedly in the animals that became hyperglycemic, whereas it remained high in the normoglycemic ZDF rats. Thus, hyperglycemic ZDF animals exhibit both insulin resistance and progressive beta cell failure, whereas normoglycemic ZDF rats exhibit a lesser degree of insulin resistance that does not progress to beta cell failure. In these respects, the normoglycemic ZDF rats appear to revert back to a phenotype that strongly resembles that of nondiabetic Zucker fatty rats from which they were derived.
doi:10.2147/DMSO.S69891
PMCID: PMC4234283  PMID: 25419150
type 2 diabetes; ZDF rats; animal models
2.  Tissue-Specific Gene Expression and Regulation in Liver and Muscle Following Chronic Corticosteroid Administration 
Although corticosteroids (CSs) affect gene expression in multiple tissues, the array of genes that are regulated by these catabolic steroids is diverse, highly tissue specific, and depends on their functions in the tissue. Liver has many important functions in performing and regulating diverse metabolic processes. Muscle, in addition to its mechanical role, is critical in maintaining systemic energy homeostasis and accounts for about 80% of insulin-directed glucose disposal. Consequently, a better understanding of CS pharmacogenomic effects in these tissues would provide valuable information regarding the tissue-specificity of transcriptional dynamics, and would provide insights into the underlying molecular mechanisms of action for both beneficial and detrimental effects.
We performed an integrated analysis of transcriptional data from liver and muscle in response to methylprednisolone (MPL) infusion, which included clustering and functional annotation of clustered gene groups, promoter extraction and putative transcription factor (TF) identification, and finally, regulatory closeness (RC) identification.
This analysis allowed the identification of critical transcriptional responses and CS-responsive functions in liver and muscle during chronic MPL administration, the prediction of putative transcriptional regulators relevant to transcriptional responses of CS-affected genes which are also potential secondary bio-signals altering expression levels of target-genes, and the exploration of the tissue-specificity and biological significance of gene expression patterns, CS-responsive functions, and transcriptional regulation.
The analysis provided an integrated description of the genomic and functional effects of chronic MPL infusion in liver and muscle.
doi:10.4137/GRSB.S13134
PMCID: PMC3956809  PMID: 24653645
liver; muscle; glucocorticoids; corticosteroids; gene expression; gene regulation; promoter analysis
3.  Evidence for a Glucocorticoid Receptor Beta Splice Variant in the Rat and Its Physiological Regulation in Liver 
Steroids  2012;78(2):312-320.
Glucocorticoids are important regulators of metabolism and immune function. Synthetic glucocorticoids are extensively used for immunosuppression/anti-inflammatory therapy. Since the glucocorticoid receptor (GR) is central to most hormone effects; its in vivo regulation will influence hormone/drug action. An alternative splice variant, GRβ, is present in humans and may function as a dominant negative regulator of GR transcriptional activity. Recently, a similar splice variant was reported in mouse, although the mechanism of alternative splicing differs from that in humans. We present evidence that a splice variant of GR with an alternative C-terminus also occurs in the rat by a mechanism of intron inclusion. A highly quantitative qRT-PCR assay for the simultaneous measurement of both splice variants in a single sample was developed in order to accurately measure their regulation. We used this assay to assess the tissue specific expression of both mRNAs, and demonstrate that GRα is predominant in all tissues. In addition, the regulation of both GRα and GRβ mRNA by various physiological factors in rat liver was assessed. GRα showed a robust circadian rhythm, which was entrained with the circadian oscillation of the endogenous hormone. Time series experiments showed that both corticosteroids and LPS but not insulin dosing resulted in the transient down-regulation of GRα mRNA. LPS treatment also resulted in down-regulation of GRβ expression. A modest up-regulation in GRβ expression was observed only in animals having chronically elevated plasma insulin concentrations. However the expression of GRβ was significantly lower than that of GRα in all cases.
doi:10.1016/j.steroids.2012.11.014
PMCID: PMC3552070  PMID: 23257260
glucocorticoids; glucocorticoid receptor; GRβ; qRTPCR
4.  Differential Muscle Gene Expression as a Function of Disease Progression in Goto-Kakizaki Diabetic Rats 
The Goto-Kakizaki (GK) rat, a polygenic non-obese model of type 2 diabetes, is a useful surrogate for study of diabetes-related changes independent of obesity. GK rats and appropriate controls were killed at 4, 8, 12, 16 and 20 weeks post-weaning and differential muscle gene expression along with body and muscle weights, plasma hormones and lipids, and blood cell measurements were carried out. Gene expression analysis identified 204 genes showing 2-fold or greater differences between GK and controls in at least 3 ages. Array results suggested increased oxidative capacity in GK muscles, as well as differential gene expression related to insulin resistance, which was also indicated by HOMA-IR measurements. In addition, potential new biomarkers in muscle gene expression were identified that could be either a cause or consequence of T2DM. Furthermore, we demonstrate here the presence of chronic inflammation evident both systemically and in the musculature, despite the absence of obesity.
doi:10.1016/j.mce.2011.02.016
PMCID: PMC3093670  PMID: 21356272
type 2 diabetes; skeletal muscle; inflammation; microarrays; gene expression
5.  Meta-Modeling of Methylprednisolone Effects on Glucose Regulation in Rats 
PLoS ONE  2013;8(12):e81679.
A retrospective meta-modeling analysis was performed to integrate previously reported data of glucocorticoid (GC) effects on glucose regulation following a single intramuscular dose (50 mg/kg), single intravenous doses (10, 50 mg/kg), and intravenous infusions (0.1, 0.2, 0.3 and 0.4 mg/kg/h) of methylprednisolone (MPL) in normal and adrenalectomized (ADX) male Wistar rats. A mechanistic pharmacodynamic (PD) model was developed based on the receptor/gene/protein-mediated GC effects on glucose regulation. Three major target organs (liver, white adipose tissue and skeletal muscle) together with some selected intermediate controlling factors were designated as important regulators involved in the pathogenesis of GC-induced glucose dysregulation. Assessed were dynamic changes of food intake and systemic factors (plasma glucose, insulin, free fatty acids (FFA) and leptin) and tissue-specific biomarkers (cAMP, phosphoenolpyruvate carboxykinase (PEPCK) mRNA and enzyme activity, leptin mRNA, interleukin 6 receptor type 1 (IL6R1) mRNA and Insulin receptor substrate-1 (IRS-1) mRNA) after acute and chronic dosing with MPL along with the GC receptor (GR) dynamics in each target organ. Upon binding to GR in liver, MPL dosing caused increased glucose production by stimulating hepatic cAMP and PEPCK activity. In adipose tissue, the rise in leptin mRNA and plasma leptin caused reduction of food intake, the exogenous source of glucose input. Down-regulation of IRS-1 mRNA expression in skeletal muscle inhibited the stimulatory effect of insulin on glucose utilization further contributing to hyperglycemia. The nuclear drug-receptor complex served as the driving force for stimulation or inhibition of downstream target gene expression within different tissues. Incorporating information such as receptor dynamics, as well as the gene and protein induction, allowed us to describe the receptor-mediated effects of MPL on glucose regulation in each important tissue. This advanced mechanistic model provides unique insights into the contributions of major tissues and quantitative hypotheses for the multi-factor control of a complex metabolic system.
doi:10.1371/journal.pone.0081679
PMCID: PMC3847111  PMID: 24312573
6.  Pharmacokinetic/Pharmacodynamic Modeling of Methylprednisolone Effects on iNOS mRNA Expression and Nitric Oxide During LPS-Induced Inflammation in Rats 
Pharmaceutical Research  2012;29(8):2060-2069.
Purpose
Increased expression of inducible nitric oxide synthase (iNOS) resulting in nitric oxide elevation represents an important component of inflammatory responses. We assess the effects of methylprednisolone (MPL) on these processes during endotoxin-induced acute inflammation and provide a mechanism-based model to quantitatively describe them.
Methods
Male Lewis rats were dosed with lipopolysaccharide (50 μg/kg LPS) alone or with methylprednisolone (10 and 50 mg/kg) and sacrificed at different time points. Plasma MPL, lung iNOS mRNA expression, plasma nitric oxide (NO) and other physiological factors were measured. Sodium nitrate (750 μmole/kg) was given to a separate cohort of rats to assess NO disposition kinetics. PK-PD modeling was performed with ADAPT 5.
Results
Disposition kinetics of plasma MPL and NO showed bi-exponential decline and were described by two-compartment models. LPS increased expression of iNOS mRNA in lung and increased plasma NO, while MPL dosing palliated this increase in a dose-dependent manner. These effects were well captured using tandem indirect response and precursor-pool models.
Conclusion
The model provides a quantitative assessment of the suppression of NO production by MPL and shows that the major effects are at the transcriptional level by reducing expression of iNOS mRNA.
doi:10.1007/s11095-012-0733-5
PMCID: PMC3400266  PMID: 22422321
corticosteroids; inflammation; iNOS; nitric oxide; PK-PD modeling
7.  Glucocorticoid Effects on Adiponectin Expression 
Vitamins and hormones  2012;90:163-186.
Maintenance of energy metabolism and glucose homeostasis is achieved by the regulatory effects of many hormones and their interactions. Glucocorticoids produced from adrenal cortex and adiponectin produced by adipose tissue play important roles in the production, distribution, storage, and utilization of energy substrates. Glucocorticoids are involved in the activation of a number of catabolic processes by affecting the expression of a plethora of genes, while adiponectin acts primarily as an insulin sensitizer. Both are regulated by a number of physiological and pharmacological factors. Although the effects of glucocorticoids on adiponectin expression have been extensively studied in different in vitro, animal and clinical study settings, no consensus has been reached. This report reviews the primary literature concerning the effects of glucocorticoids on adiponectin expression and identifies potential reasons for the contradictory results between different studies. In addition, methods to gain better insights pertaining to the regulation of adiponectin expression are discussed.
doi:10.1016/B978-0-12-398313-8.00007-5
PMCID: PMC3693220  PMID: 23017716
8.  Effects of High Fat Feeding on Liver Gene Expression in Diabetic Goto-Kakizaki Rats 
Effects of high fat diet (HFD) on obesity and, subsequently, on diabetes are highly variable and modulated by genetics in both humans and rodents. In this report, we characterized the response of Goto-Kakizaki (GK) rats, a spontaneous polygenic model for lean diabetes and healthy Wistar-Kyoto (WKY) controls, to high fat feeding from weaning to 20 weeks of age. Animals fed either normal diet or HFD were sacrificed at 4, 8, 12, 16 and 20 weeks of age and a wide array of physiological measurements were made along with gene expression profiling using Affymetrix gene array chips. Mining of the microarray data identified differentially regulated genes (involved in inflammation, metabolism, transcription regulation, and signaling) in diabetic animals, as well as the response of both strains to HFD. Functional annotation suggested that HFD increased inflammatory differences between the two strains. Chronic inflammation driven by heightened innate immune response was identified to be present in GK animals regardless of diet. In addition, compensatory mechanisms by which WKY animals on HFD resisted the development of diabetes were identified, thus illustrating the complexity of diabetes disease progression.
doi:10.4137/GRSB.S10371
PMCID: PMC3516129  PMID: 23236253
diabetes; high fat diet; gene expression; microarray
9.  Circadian Rhythms in Gene Expression: Relationship to Physiology, Disease, Drug Disposition and Drug Action 
Advanced drug delivery reviews  2010;62(9-10):904-917.
Circadian rhythms (24 h cycles) are observed in virtually all aspects of mammalian function from expression of genes to complex physiological processes. The master clock is present in the suprachiasmatic nucleus (SCN) in the anterior part of the hypothalamus and controls peripheral clocks present in other parts of the body. Components of this core clock mechanism regulate the circadian rhythms in genome-wide mRNA expression, which in turn regulate various biological processes. Disruption of circadian rhythms can be either the cause or the effect of various disorders including metabolic syndrome, inflammatory diseases and cancer. Furthermore, circadian rhythms in gene expression regulate both the action and disposition of various drugs and affect therapeutic efficacy and toxicity based on dosing time. Understanding the regulation of circadian rhythms in gene expression plays an important role in both optimizing the dosing time for existing drugs and in development of new therapeutics targeting the molecular clock.
doi:10.1016/j.addr.2010.05.009
PMCID: PMC2922481  PMID: 20542067
molecular clocks; metabolic disease; inflammation; cancer; drug targets; pharmacokinetics
10.  Adipose Tissue Deficiency and Chronic Inflammation in Diabetic Goto-Kakizaki Rats 
PLoS ONE  2011;6(2):e17386.
Type 2 diabetes (T2DM) is a heterogeneous group of diseases that is progressive and involves multiple tissues. Goto-Kakizaki (GK) rats are a polygenic model with elevated blood glucose, peripheral insulin resistance, a non-obese phenotype, and exhibit many degenerative changes observed in human T2DM. As part of a systems analysis of disease progression in this animal model, this study characterized the contribution of adipose tissue to pathophysiology of the disease. We sacrificed subgroups of GK rats and appropriate controls at 4, 8, 12, 16 and 20 weeks of age and carried out a gene array analysis of white adipose tissue. We expanded our physiological analysis of the animals that accompanied our initial gene array study on the livers from these animals. The expanded analysis included adipose tissue weights, HbA1c, additional hormonal profiles, lipid profiles, differential blood cell counts, and food consumption. HbA1c progressively increased in the GK animals. Altered corticosterone, leptin, and adiponectin profiles were also documented in GK animals. Gene array analysis identified 412 genes that were differentially expressed in adipose tissue of GKs relative to controls. The GK animals exhibited an age-specific failure to accumulate body fat despite their relatively higher calorie consumption which was well supported by the altered expression of genes involved in adipogenesis and lipogenesis in the white adipose tissue of these animals, including Fasn, Acly, Kklf9, and Stat3. Systemic inflammation was reflected by chronically elevated white blood cell counts. Furthermore, chronic inflammation in adipose tissue was evident from the differential expression of genes involved in inflammatory responses and activation of natural immunity, including two interferon regulated genes, Ifit and Iipg, as well as MHC class II genes. This study demonstrates an age specific failure to accumulate adipose tissue in the GK rat and the presence of chronic inflammation in adipose tissue from these animals.
doi:10.1371/journal.pone.0017386
PMCID: PMC3045458  PMID: 21364767
11.  Circadian signatures in rat liver: from gene expression to pathways 
BMC Bioinformatics  2010;11:540.
Background
Circadian rhythms are 24 hour oscillations in many behavioural, physiological, cellular and molecular processes that are controlled by an endogenous clock which is entrained to environmental factors including light, food and stress. Transcriptional analyses of circadian patterns demonstrate that genes showing circadian rhythms are part of a wide variety of biological pathways.
Pathway activity method can identify the significant pattern of the gene expression levels within a pathway. In this method, the overall gene expression levels are translated to a reduced form, pathway activity levels, via singular value decomposition (SVD). A given pathway represented by pathway activity levels can then be as analyzed using the same approaches used for analyzing gene expression levels. We propose to use pathway activity method across time to identify underlying circadian pattern of pathways.
Results
We used synthetic data to demonstrate that pathway activity analysis can evaluate the underlying circadian pattern within a pathway even when circadian patterns cannot be captured by the individual gene expression levels. In addition, we illustrated that pathway activity formulation should be coupled with a significance analysis to distinguish biologically significant information from random deviations. Next, we performed pathway activity level analysis on a rich time series of transcriptional profiling in rat liver. The over-represented five specific patterns of pathway activity levels, which cannot be explained by random event, exhibited circadian rhythms. The identification of the circadian signatures at the pathway level identified 78 pathways related to energy metabolism, amino acid metabolism, lipid metabolism and DNA replication and protein synthesis, which are biologically relevant in rat liver. Further, we observed tight coordination between cholesterol biosynthesis and bile acid biosynthesis as well as between folate biosynthesis, one carbon pool by folate and purine-pyrimidine metabolism. These coupled pathways are parts of a sequential reaction series where the product of one pathway is the substrate of another pathway.
Conclusions
Rather than assessing the importance of a single gene beforehand and map these genes onto pathways, we instead examined the orchestrated change within a pathway. Pathway activity level analysis could reveal the underlying circadian dynamics in the microarray data with an unsupervised approach and biologically relevant results were obtained.
doi:10.1186/1471-2105-11-540
PMCID: PMC2990769  PMID: 21040584

Results 1-11 (11)