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1.  Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens 
Physiological genomics  2007;30(3):282-299.
The transcriptional response of skeletal muscle to chronic corticosteroid exposure was examined over 168 h and compared with the response profiles observed following a single dose of corticosteroid. Male adrenalectomized Wistar rats were given a constant-rate infusion of 0.3 mg•kg−1•h−1 methylprednisolone for up to 7 days via subcutaneously implanted minipumps. Four control and forty drug-treated animals were killed at ten different time points during infusion. Liver total RNAs were hybridized to 44 individual Affymetrix REA230A gene chips. Previously, we described a filtration approach for identifying genes of interest in microarray data sets developed from tissues of rats treated with methylprednisolone (MPL) following acute dosing. Here, a similar approach involving a series of three filters was applied sequentially to identify genes of interest. These filters were designed to eliminate probe sets that were not expressed in the tissue, not regulated by the drug, or did not meet defined quality control standards. Filtering eliminated 86% of probe sets, leaving a remainder of 2,316 for further consideration. In a previous study, 653 probe sets were identified as MPL regulated following administration of a single (acute) dose of the drug. Comparison of the two data sets yielded 196 genes identified as regulated by MPL in both dosing regimens. Because of receptor downregulation, it was predicted that genes regulated by receptor-glucocorticoid response element interactions would exhibit tolerance in chronic profiles. However, many genes did not exhibit steroid tolerance, indicating that present perspectives on the mechanism of glucocorticoid action cannot entirely explain all temporal profiles.
doi:10.1152/physiolgenomics.00242.2006
PMCID: PMC4186702  PMID: 17473217
glucocorticoids; corticosteroids; Affymetrix gene chips; gene expression; time series
2.  Mathematical Modeling of Corticosteroid Pharmacogenomics in Rat Muscle following Acute and Chronic Methylprednisolone Dosing 
Molecular pharmaceutics  2008;5(2):328-339.
The pharmacogenomic effects of a corticosteroid (CS) were assessed in rat skeletal muscle using microarrays. Adrenalectomized (ADX) rats were treated with methylprednisolone (MPL) by either 50 mg/kg intravenous injection or 7-day 0.3 mg/kg/h infusion through subcutaneously implanted pumps. RNAs extracted from individual rat muscles were hybridized to Affymetrix Rat Genome Genechips. Data mining yielded 653 and 2316 CS-responsive probe sets following MPL bolus and infusion treatments. Of these, 196 genes were controlled by MPL under both dosing conditions. Cluster analysis revealed that 124 probe sets exhibited three typical expression dynamic profiles following acute dosing. Cluster A consisted of up-regulated probe sets which were grouped into five subclusters each exhibiting unique temporal patterns during the infusion. Cluster B comprised down-regulated probe sets which were divided into two subclusters with distinct dynamics during the infusion. Cluster C probe sets exhibited delayed down-regulation under both bolus and infusion conditions. Among those, 104 probe sets were further grouped into subclusters based on their profiles following chronic MPL dosing. Several mathematical models were proposed and adequately captured the temporal patterns for each subcluster. Multiple types of dosing regimens are needed to resolve common determinants of gene regulation as chronic exposure results in unexpected differences in gene expression compared to acute dosing. Pharmacokinetic/pharmacodynamic (PK/PD) modeling provides a quantitative tool for elucidating the complexities of CS pharmacogenomics in skeletal muscle.
doi:10.1021/mp700094s
PMCID: PMC4196382  PMID: 18271548
Microarray studies; pharmacokinetics; pharmacodynamics; mathematical models; computational biology
3.  Function-Based Discovery of Significant Transcriptional Temporal Patterns in Insulin Stimulated Muscle Cells 
PLoS ONE  2012;7(3):e32391.
Background
Insulin action on protein synthesis (translation of transcripts) and post-translational modifications, especially of those involving the reversible modifications such as phosphorylation of various signaling proteins, are extensively studied but insulin effect on transcription of genes, especially of transcriptional temporal patterns remains to be fully defined.
Methodology/Principal Findings
To identify significant transcriptional temporal patterns we utilized primary differentiated rat skeletal muscle myotubes which were treated with insulin and samples were collected every 20 min for 8 hours. Pooled samples at every hour were analyzed by gene array approach to measure transcript levels. The patterns of transcript levels were analyzed based on a novel method that integrates selection, clustering, and functional annotation to find the main temporal patterns associated to functional groups of differentially expressed genes. 326 genes were found to be differentially expressed in response to in vitro insulin administration in skeletal muscle myotubes. Approximately 20% of the genes that were differentially expressed were identified as belonging to the insulin signaling pathway. Characteristic transcriptional temporal patterns include: (a) a slow and gradual decrease in gene expression, (b) a gradual increase in gene expression reaching a peak at about 5 hours and then reaching a plateau or an initial decrease and other different variable pattern of increase in gene expression over time.
Conclusion/Significance
The new method allows identifying characteristic dynamic responses to insulin stimulus, common to a number of genes and associated to the same functional group. The results demonstrate that insulin treatment elicited different clusters of gene transcript profile supporting a temporal regulation of gene expression by insulin in skeletal muscle cells.
doi:10.1371/journal.pone.0032391
PMCID: PMC3291562  PMID: 22396763
4.  Transcriptome Signature of Virulent and Attenuated Pseudorabies Virus-Infected Rodent Brain 
Journal of Virology  2006;80(4):1773-1786.
Mammalian alphaherpesviruses normally establish latent infections in ganglia of the peripheral nervous system in their natural hosts. Occasionally, however, these viruses spread to the central nervous system (CNS), where they cause damaging, often fatal, infections. Attenuated alphaherpesvirus derivatives have been used extensively as neuronal circuit tracers in a variety of animal models. Their circuit-specific spread provides a unique paradigm to study the local and global CNS response to infection. Thus, we systematically analyzed the host gene expression profile after acute pseudorabies virus (PRV) infection of the CNS using Affymetrix GeneChip technology. Rats were injected intraocularly with one of three selected virulent and attenuated PRV strains. Relative levels of cellular transcripts were quantified from hypothalamic and cerebellar tissues at various times postinfection. The number of cellular genes responding to infection correlated with the extent of virus dissemination and relative virulence of the PRV strains. A total of 245 out of 8,799 probe sets, corresponding to 182 unique cellular genes, displayed increased expression ranging from 2- to more than 100-fold higher than in uninfected tissue. Over 60% thereof were categorized as immune, proinflammatory, and other cellular defense genes. Additionally, a large fraction of infection-induced transcripts represented cellular stress responses, including glucocorticoid- and redox-related pathways. This is the first comprehensive in vivo analysis of the global transcriptional response of the mammalian CNS to acute alphaherpesvirus infection. The differentially regulated genes reported here are likely to include potential diagnostic and therapeutic targets for viral encephalitides and other neurodegenerative or neuroinflammatory diseases.
doi:10.1128/JVI.80.4.1773-1786.2006
PMCID: PMC1367157  PMID: 16439534
5.  A Microarray Analysis of the Temporal Response of Liver to Methylprednisolone: A Comparative Analysis of Two Dosing Regimens 
Endocrinology  2007;148(5):2209-2225.
Microarray analyses were performed on livers from adrenalectomized male Wistar rats chronically infused with methylprednisolone (MPL) (0.3 mg/kg·h) using Alzet mini-osmotic pumps for periods ranging from 6 h to 7 d. Four control and 40 drug-treated animals were killed at 10 different times during drug infusion. Total RNA preparations from the livers of these animals were hybridized to 44 individual Affymetrix REA230A gene chips, generating data for 15,967 different probe sets for each chip. A series of three filters were applied sequentially. These filters were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug, or did not meet defined quality control standards. These filters eliminated 13,978 probe sets (87.5%) leaving a remainder of 1989 probe sets for further consideration. We previously described a similar dataset obtained from animals after administration of a single dose of MPL (50 mg/kg given iv). That study involved 16 time points over a 72-h period. A similar filtering schema applied to the single-bolus-dose data-set identified 1519 probe sets as being regulated by MPL. A comparison of datasets from the two different dosing regimens identified 358 genes that were regulated by MPL in response to both dosing regimens. Regulated genes were grouped into 13 categories, mainly on gene product function. The temporal profiles of these common genes were subjected to detailed scrutiny. Examination of temporal profiles demonstrates that current perspectives on the mechanism of glucocorticoid action cannot entirely explain the temporal profiles of these regulated genes.
doi:10.1210/en.2006-0790
PMCID: PMC4183266  PMID: 17303664
6.  Assessing the metabolic effects of prednisolone in healthy volunteers using urine metabolic profiling 
Genome Medicine  2012;4(11):94.
Background
Glucocorticoids, such as prednisolone, are widely used anti-inflammatory drugs, but therapy is hampered by a broad range of metabolic side effects including skeletal muscle wasting and insulin resistance. Therefore, development of improved synthetic glucocorticoids that display similar efficacy as prednisolone but reduced side effects is an active research area. For efficient development of such new drugs, in vivo biomarkers, which can predict glucocorticoid metabolic side effects in an early stage, are needed. In this study, we aim to provide the first description of the metabolic perturbations induced by acute and therapeutic treatments with prednisolone in humans using urine metabolomics, and to derive potential biomarkers for prednisolone-induced metabolic effects.
Methods
A randomized, double blind, placebo-controlled trial consisting of two protocols was conducted in healthy men. In protocol 1, volunteers received placebo (n = 11) or prednisolone (7.5 mg (n = 11), 15 mg (n = 13) or 30 mg (n = 12)) orally once daily for 15 days. In protocol 2, volunteers (n = 6) received placebo at day 0 and 75 mg prednisolone at day 1. We collected 24 h urine and serum samples at baseline (day 0), after a single dose (day 1) and after prolonged treatment (day 15) and obtained mass-spectrometry-based urine and serum metabolic profiles.
Results
At day 1, high-dose prednisolone treatment increased levels of 13 and 10 proteinogenic amino acids in urine and serum respectively, as well as levels of 3-methylhistidine, providing evidence for an early manifestation of glucocorticoid-induced muscle wasting. Prednisolone treatment also strongly increased urinary carnitine derivatives at day 1 but not at day 15, which might reflect adaptive mechanisms under prolonged treatment. Finally, urinary levels of proteinogenic amino acids at day 1 and of N-methylnicotinamide at day 15 significantly correlated with the homeostatic model assessment of insulin resistance and might represent biomarkers for prednisolone-induced insulin resistance.
Conclusion
This study provides evidence that urinary metabolomics represents a noninvasive way of monitoring the effect of glucocorticoids on muscle protein catabolism after a single dose and can derive new biomarkers of glucocorticoid-induced insulin resistance. It might, therefore, help the development of improved synthetic glucocorticoids.
Trial Registration
ClinicalTrials.gov NCT00971724
doi:10.1186/gm395
PMCID: PMC4064315  PMID: 23199229
3-methylhistidine; aminoaciduria; HOMA-IR; metabolomics; prednisolone; urine
7.  TXNIP Regulates Peripheral Glucose Metabolism in Humans  
PLoS Medicine  2007;4(5):e158.
Background
Type 2 diabetes mellitus (T2DM) is characterized by defects in insulin secretion and action. Impaired glucose uptake in skeletal muscle is believed to be one of the earliest features in the natural history of T2DM, although underlying mechanisms remain obscure.
Methods and Findings
We combined human insulin/glucose clamp physiological studies with genome-wide expression profiling to identify thioredoxin interacting protein (TXNIP) as a gene whose expression is powerfully suppressed by insulin yet stimulated by glucose. In healthy individuals, its expression was inversely correlated to total body measures of glucose uptake. Forced expression of TXNIP in cultured adipocytes significantly reduced glucose uptake, while silencing with RNA interference in adipocytes and in skeletal muscle enhanced glucose uptake, confirming that the gene product is also a regulator of glucose uptake. TXNIP expression is consistently elevated in the muscle of prediabetics and diabetics, although in a panel of 4,450 Scandinavian individuals, we found no evidence for association between common genetic variation in the TXNIP gene and T2DM.
Conclusions
TXNIP regulates both insulin-dependent and insulin-independent pathways of glucose uptake in human skeletal muscle. Combined with recent studies that have implicated TXNIP in pancreatic β-cell glucose toxicity, our data suggest that TXNIP might play a key role in defective glucose homeostasis preceding overt T2DM.
Vamsi Mootha, Leif Groop, and colleagues report that TXNIP regulates insulin-dependent and -independent pathways of glucose uptake in human skeletal muscle and that its expression is elevated in individuals with prediabetes and type 2 diabetes.
Editors' Summary
Background.
An epidemic of diabetes mellitus is threatening world health. 246 million people (6% of the world's population) already have diabetes and it is estimated that within 20 years, 380 million people will have this chronic disease, most of them in developing countries. Diabetes is characterized by high blood sugar (glucose) levels. It arises when the pancreas does not make enough insulin (type 1 diabetes) or when the body responds poorly to insulin (type 2 diabetes). Insulin, which is released in response to high blood glucose levels, instructs muscle, fat, and liver cells to take glucose (a product of food digestion) out of the bloodstream; cells use glucose as a fuel. Type 2 diabetes, which accounts for 90% of all cases of diabetes, is characterized by impaired glucose uptake by target tissues in response to insulin (this “insulin resistance” is one of the first signs of type 2 diabetes) and inappropriate glucose release from liver cells. Over time, the pancreas may also make less insulin. These changes result in poor glucose homeostasis (inadequate control of blood sugar levels), which can cause life-threatening complications such as kidney failure and heart attacks.
Why Was This Study Done?
If the world diabetes epidemic is to be halted, researchers need a better understanding of glucose homeostasis and need to identify which parts of this complex control system go awry in type 2 diabetes. This information might suggest ways to prevent type 2 diabetes developing in the first place and might reveal targets for drugs that could slow or reverse the disease process. In this study, the researchers have used multiple approaches to identify a new mediator of glucose homeostasis and to investigate whether this mediator is causally involved in the development of type 2 diabetes.
What Did the Researchers Do and Find?
The researchers took small muscle samples from people who did not have diabetes before and after increasing their blood insulin levels and used a technique called “microarray expression profiling” to identify genes whose expression was induced or suppressed by insulin. One of the latter genes was thioredoxin interacting protein (TXNIP), a gene whose expression is strongly induced by glucose yet suppressed by insulin. They next used previously published microarray expression data to show that TXNIP expression was consistently higher in the muscles of patients with diabetes or prediabetes (a condition in which blood glucose levels are slightly raised) than in normal individuals. The researchers then examined whether TXNIP expression was correlated with glucose uptake, again using previously published data. In people with no diabetes and those with prediabetes, as glucose uptake rates increased, TXNIP expression decreased but this inverse correlation was missing in people with diabetes. Finally, by manipulating TXNIP expression levels in insulin-responsive cells grown in the laboratory, the researchers found that TXNIP overexpression reduced basal and insulin-stimulated glucose uptake but that reduced TXNIP expression had the opposite effect.
What Do These Findings Mean?
These results provide strong evidence that TXNIP is a regulator of glucose homeostasis in people. Specifically, the researchers propose that TXNIP regulates glucose uptake in the periphery of the human body by acting as a glucose- and insulin-sensitive switch. They also suggest how it might be involved in the development of type 2 diabetes. Early in the disease process, a small insulin deficiency or slightly raised blood sugar levels would increase TXNIP expression in muscles and suppress glucose uptake by these cells. Initially, the pancreas would compensate for this by producing more insulin, but this compensation would eventually fail, allowing blood sugar levels to rise sufficiently to increase TXNIP expression in the pancreas. Previously published results suggest that this would induce the loss of insulin-producing cells in the pancreas, thus further reducing insulin production and glucose uptake in the periphery and, ultimately, resulting in type 2 diabetes. Although there are many unanswered questions about the exact role of TXNIP in glucose homeostasis, these results help to explain many of the changes in glucose control that occur early in the development of diabetes. Furthermore, they suggest that interventions designed to modulate the activity of TXNIP might break the vicious cycle that eventually leads to type 2 diabetes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040158.
The MedlinePlus encyclopedia has pages on diabetes
The US National Institute of Diabetes and Digestive and Kidney Diseases has information for patients on diabetes
Information on diabetes is available for patients and professionals from the US Centers for Disease Control and Prevention
The American Diabetes Association provides information on diabetes for patients
International Diabetes Federation has information on diabetes and a recent press release on the global diabetes epidemic
doi:10.1371/journal.pmed.0040158
PMCID: PMC1858708  PMID: 17472435
8.  Gene arrays and temporal patterns of drug response: corticosteroid effects on rat liver 
It was hypothesized that expression profiling using gene arrays can be used to distinguish temporal patterns of changes in gene expression in response to a drug in vivo, and that these patterns can be used to identify groups of genes regulated by common mechanisms. A corticosteroid, methylprednisolone (MPL), was administered intravenously to a group of 47 rats (Rattus rattus) that were sacrificed at 17 timepoints over 72 h after MPL administration. Plasma drug concentrations and hepatic glucocorticoid receptors were measured from each animal. In addition, RNAs prepared from individual livers were used to query Affymetrix genechips for mRNA expression patterns. Statistical analyses using Affymetrix and GeneSpring software were applied to the results. Cluster analysis revealed six major temporal patterns containing 196 corticosteroid-responsive probe sets representing 153 different genes. Four clusters showed increased expression with differences in lag-time, onset rate, and/or duration of transcriptional effect. A fifth cluster showed rapid reduction persisting for 18 h. The final cluster identified showed decreased expression followed by an extended period of increased expression. These results lend new insights into the diverse hepatic genes involved in the physiologic, therapeutic, and adverse effects of corticosteroids and suggest that a limited array of control processes account for the dynamics of their pharmacogenomic effects.
doi:10.1007/s10142-003-0090-x
PMCID: PMC4207265  PMID: 12928814
Corticosteroids; Glucocorticoids; Expression profiling; Cluster analysis
9.  Pioglitazone Enhances Mitochondrial Biogenesis and Ribosomal Protein Biosynthesis in Skeletal Muscle in Polycystic Ovary Syndrome 
PLoS ONE  2008;3(6):e2466.
Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P<0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1α expression (P<0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.
doi:10.1371/journal.pone.0002466
PMCID: PMC2413008  PMID: 18560589
10.  Pharmacodynamic/Pharmacogenomic Modeling of Insulin Resistance Genes in Rat Muscle After Methylprednisolone Treatment: Exploring Regulatory Signaling Cascades 
Corticosteroids (CS) effects on insulin resistance related genes in rat skeletal muscle were studied. In our acute study, adrenalectomized (ADX) rats were given single doses of 50 mg/kg methylprednisolone (MPL) intravenously. In our chronic study, ADX rats were implanted with Alzet mini-pumps giving zero-order release rates of 0.3 mg/kg/h MPL and sacrificed at various times up to 7 days. Total RNA was extracted from gastrocnemius muscles and hybridized to Affymetrix GeneChips. Data mining and literature searches identified 6 insulin resistance related genes which exhibited complex regulatory pathways. Insulin receptor substrate-1 (IRS-1), uncoupling protein 3 (UCP3), pyruvate dehydrogenase kinase isoenzyme 4 (PDK4), fatty acid translocase (FAT) and glycerol-3-phosphate acyltransferase (GPAT) dynamic profiles were modeled with mutual effects by calculated nuclear drug-receptor complex (DR(N)) and transcription factors. The oscillatory feature of endothelin-1 (ET-1) expression was depicted by a negative feedback loop. These integrated models provide testable quantitative hypotheses for these regulatory cascades.
PMCID: PMC2733097  PMID: 19787081
corticosteroid; glucocorticoid; microarrays; mathematical modeling; insulin resistance
11.  Muscle Mitochondrial ATP Synthesis and Glucose Transport/Phosphorylation in Type 2 Diabetes 
PLoS Medicine  2007;4(5):e154.
Background
Muscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM).
Methods and Findings
We measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with 31P/1H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux.
Conclusions
Patients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients.
Michael Roden and colleagues report that even patients with well-controlled insulin-resistant type 2 diabetes have altered mitochondrial function.
Editors' Summary
Background.
Diabetes mellitus is an increasingly common chronic disease characterized by high blood sugar (glucose) levels. In normal individuals, blood sugar levels are maintained by the hormone insulin. Insulin is released by the pancreas when blood glucose levels rise after eating (glucose is produced by the digestion of food) and “instructs” insulin-responsive muscle and fat cells to take up glucose from the bloodstream. The cells then use glucose as a fuel or convert it into glycogen, a storage form of glucose. In type 2 diabetes, the commonest type of diabetes, the muscle and fat cells become nonresponsive to insulin (a condition called insulin resistance) and consequently blood glucose levels rise. Over time, this hyperglycemia increases the risk of heart attacks, kidney failure, and other life-threatening complications.
Why Was This Study Done?
Insulin resistance is often an early sign of type 2 diabetes, sometimes predating its development by many years, so understanding its causes might provide clues about how to stop the global diabetes epidemic. One theory is that mitochondria—cellular structures that produce the energy (in the form of a molecule called ATP) needed to keep cells functioning—do not work properly in people with insulin resistance. Mitochondria change (metabolize) fatty acids into energy, and recent studies have revealed that fat accumulation caused by poorly regulated fatty acid metabolism blocks insulin signaling, thus causing insulin resistance. Other studies using magnetic resonance spectroscopy (MRS) to study mitochondrial function noninvasively in human muscle indicate that mitochondria are dysfunctional in people with insulin resistance by showing that ATP synthesis is impaired in such individuals. In this study, the researchers have examined both baseline and insulin-stimulated mitochondrial function in nonobese patients with well-controlled type 2 diabetes and in normal controls to discover more about the relationship between mitochondrial dysfunction and insulin resistance.
What Did the Researchers Do and Find?
The researchers determined the insulin sensitivity of people with type 2 diabetes and two sets of people (the “controls”) who did not have diabetes: one in which the volunteers were age-matched to the people with diabetes, and the other containing younger individuals (insulin resistance increases with age). To study insulin sensitivity in all three groups, the researchers used a “hyperinsulinemic–euglycemic clamp.” For this, after an overnight fast, the participants' insulin levels were kept high with a continuous insulin infusion while blood glucose levels were kept normal using a variable glucose infusion. In this situation, the glucose infusion rate equals glucose uptake by the body and therefore measures tissue sensitivity to insulin. Before and during the clamp, the researchers used MRS to measure glucose-6-phosphate (an indicator of how effectively glucose is taken into cells and phosphorylated), ATP synthesis, and the fat content of the participants' muscle cells. Insulin sensitivity was lower in the patients with diabetes than in the controls, but muscle lipid content was comparable and hyperinsulinemia increased glucose-6-phosphate levels similarly in all the groups. Patients with diabetes and the older controls had lower fasting ATP synthesis rates than the young controls and, whereas insulin stimulation increased ATP synthesis in all the controls, it had no effect in the patients with diabetes. In addition, fasting blood fatty acid levels were inversely related to basal ATP synthesis, whereas insulin sensitivity was directly related to insulin-stimulated ATP synthesis.
What Do These Findings Mean?
These findings indicate that the impairment of muscle mitochondrial ATP synthesis in fasting conditions and after insulin stimulation in people with diabetes is not due to impaired glucose transport/phosphorylation or fat deposition in the muscles. Instead, it seems to be determined by lipid availability and insulin sensitivity. These results add to the evidence suggesting that mitochondrial function is disrupted in type 2 diabetes and in insulin resistance, but also suggest that there may be abnormalities in glycogen synthesis. More work is needed to determine the exact nature of these abnormalities and to discover whether they can be modulated to prevent the development of insulin resistance and type 2 diabetes. For now, though, these findings re-emphasize the need for people with type 2 diabetes or insulin resistance to reduce their food intake to compensate for the reduced energy needs of their muscles and to exercise to increase the ATP-generating capacity of their muscles. Both lifestyle changes could improve their overall health and life expectancy.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040154.
The MedlinePlus encyclopedia has pages on diabetes
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information for patients on diabetes and insulin resistance
The US Centers for Disease Control and Prevention has information on diabetes for patients and professionals
American Diabetes Association provides information for patients on diabetes and insulin resistance
Diabetes UK has information for patients and professionals on diabetes
doi:10.1371/journal.pmed.0040154
PMCID: PMC1858707  PMID: 17472434
12.  The microRNA Signature in Response to Insulin Reveals Its Implication in the Transcriptional Action of Insulin in Human Skeletal Muscle and the Role of a Sterol Regulatory Element–Binding Protein-1c/Myocyte Enhancer Factor 2C Pathway 
Diabetes  2009;58(11):2555-2564.
OBJECTIVE
Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle.
RESEARCH DESIGN AND METHODS
We carried out comparative microRNA (miRNA) expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low-density arrays. Then, using DNA microarrays, we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a, in human muscle cells.
RESULTS
Insulin downregulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different downregulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that sterol regulatory element–binding protein (SREBP)-1c and myocyte enhancer factor 2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation.
CONCLUSIONS
This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscle and suggests a possible implication of these new modulators in insulin resistance.
doi:10.2337/db09-0165
PMCID: PMC2768160  PMID: 19720801
13.  11β-Hydroxysteroid Dehydrogenase Type 1 Regulates Glucocorticoid-Induced Insulin Resistance in Skeletal Muscle 
Diabetes  2009;58(11):2506-2515.
OBJECTIVE
Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11β-HSD1 inhibitors improve insulin sensitivity.
RESEARCH DESIGN AND METHODS
Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11β-HSD1 inhibition upon insulin signaling and action.
RESULTS
Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer307 insulin receptor substrate (IRS)-1. 11β-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11β-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer307 IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11β-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer307 IRS1 decreased and pThr308 Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression.
CONCLUSIONS
Prereceptor facilitation of glucocorticoid action via 11β-HSD1 increases pSer307 IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11β-HSD1 inhibition decreases pSer307 IRS1, increases pThr308 Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action.
doi:10.2337/db09-0525
PMCID: PMC2768185  PMID: 19675138
14.  Integration of microRNA changes in vivo identifies novel molecular features of muscle insulin resistance in type 2 diabetes 
Genome Medicine  2010;2(2):9.
Background
Skeletal muscle insulin resistance (IR) is considered a critical component of type II diabetes, yet to date IR has evaded characterization at the global gene expression level in humans. MicroRNAs (miRNAs) are considered fine-scale rheostats of protein-coding gene product abundance. The relative importance and mode of action of miRNAs in human complex diseases remains to be fully elucidated. We produce a global map of coding and non-coding RNAs in human muscle IR with the aim of identifying novel disease biomarkers.
Methods
We profiled >47,000 mRNA sequences and >500 human miRNAs using gene-chips and 118 subjects (n = 71 patients versus n = 47 controls). A tissue-specific gene-ranking system was developed to stratify thousands of miRNA target-genes, removing false positives, yielding a weighted inhibitor score, which integrated the net impact of both up- and down-regulated miRNAs. Both informatic and protein detection validation was used to verify the predictions of in vivo changes.
Results
The muscle mRNA transcriptome is invariant with respect to insulin or glucose homeostasis. In contrast, a third of miRNAs detected in muscle were altered in disease (n = 62), many changing prior to the onset of clinical diabetes. The novel ranking metric identified six canonical pathways with proven links to metabolic disease while the control data demonstrated no enrichment. The Benjamini-Hochberg adjusted Gene Ontology profile of the highest ranked targets was metabolic (P < 7.4 × 10-8), post-translational modification (P < 9.7 × 10-5) and developmental (P < 1.3 × 10-6) processes. Protein profiling of six development-related genes validated the predictions. Brain-derived neurotrophic factor protein was detectable only in muscle satellite cells and was increased in diabetes patients compared with controls, consistent with the observation that global miRNA changes were opposite from those found during myogenic differentiation.
Conclusions
We provide evidence that IR in humans may be related to coordinated changes in multiple microRNAs, which act to target relevant signaling pathways. It would appear that miRNAs can produce marked changes in target protein abundance in vivo by working in a combinatorial manner. Thus, miRNA detection represents a new molecular biomarker strategy for insulin resistance, where micrograms of patient material is needed to monitor efficacy during drug or life-style interventions.
doi:10.1186/gm130
PMCID: PMC2847700  PMID: 20353613
15.  Global Gene Expression Profiles of Subcutaneous Adipose and Muscle From Glucose-Tolerant, Insulin-Sensitive, and Insulin-Resistant Individuals Matched for BMI 
Diabetes  2011;60(3):1019-1029.
OBJECTIVE
To determine altered gene expression profiles in subcutaneous adipose and skeletal muscle from nondiabetic, insulin-resistant individuals compared with insulin-sensitive individuals matched for BMI.
RESEARCH DESIGN AND METHODS
A total of 62 nondiabetic individuals were chosen for extremes of insulin sensitivity (31 insulin-resistant and 31 insulin-sensitive subjects; 40 were European American and 22 were African American) and matched for age and obesity measures. Global gene expression profiles were determined and compared between ethnic groups and between insulin-resistant and insulin-sensitive participants individually and using gene-set enrichment analysis.
RESULTS
African American and European American subjects differed in 58 muscle and 140 adipose genes, including many inflammatory and metabolically important genes. Peroxisome proliferator–activated receptor γ cofactor 1A (PPARGC1A) was 1.75-fold reduced with insulin resistance in muscle, and fatty acid and lipid metabolism and oxidoreductase activity also were downregulated. Unexpected categories included ubiquitination, citrullination, and protein degradation. In adipose, highly represented categories included lipid and fatty acid metabolism, insulin action, and cell-cycle regulation. Inflammatory genes were increased in European American subjects and were among the top Kyoto Encyclopedia of Genes and Genomes pathways on gene-set enrichment analysis. FADS1, VEGFA, PTPN3, KLF15, PER3, STEAP4, and AGTR1 were among genes expressed differentially in both adipose and muscle.
CONCLUSIONS
Adipose tissue gene expression showed more differences between insulin-resistant versus insulin-sensitive groups than the expression of genes in muscle. We confirm the role of PPARGC1A in muscle and show some support for inflammation in adipose from European American subjects but find prominent roles for lipid metabolism in insulin sensitivity independent of obesity in both tissues.
doi:10.2337/db10-1270
PMCID: PMC3046820  PMID: 21266331
16.  Age and Tissue Specific Differences in the Development of Acute Insulin Resistance Following Injury 
The Journal of endocrinology  2009;203(3):365-374.
Injuries, hemorrhage, sepsis, burn, and critical illnesses all induce insulin resistance, and insulin resistance is strongly associated with advancing age. However, the effect of age on injury-induced insulin resistance is not well studied. We performed surgical trauma in male rats of three different ages (3-, 6- and 10-weeks old). Rats were either hemorrhaged to a mean arterial pressure of 35–40 mmHg and subsequently maintained at that pressure for up to 90 minutes, or maintained without hemorrhage as controls. Results indicate that insulin-induced intracellular signaling was diminished in liver and skeletal muscle of 6- and 10-week old rats following trauma and hemorrhage. In even younger rats, immediately post-weaning (approximately 3 weeks of age), insulin signaling was lost in liver, but not in skeletal muscle. Glucocorticoids can play a role in the chronic development of insulin resistance. Our results demonstrate that corticosterone levels were increased in 6- and 10-week old animals following hemorrhage, but little change was measured in 3-week old animals. Blockade of glucocorticoid synthesis prevented the development of insulin resistance in skeletal muscle, but not in liver of 6-and 10-week old rats. Moreover, skeletal muscle glucocorticoid receptor levels increased dramatically between 3 and 6 weeks of age. These results indicate that trauma and hemorrhage-induced hepatic insulin resistance occurs at all ages tested. However, there is no development of insulin resistance following trauma and hemorrhage in skeletal muscle of post-weaning rats. In skeletal muscle of 6- and 10-week old rats, inhibition of glucocorticoid levels prevents the development of insulin resistance.
doi:10.1677/JOE-09-0269
PMCID: PMC2929648  PMID: 19752148
insulin resistance; hemorrhage; glucocorticoid receptor; liver; skeletal muscle
17.  High-fat diet and glucocorticoid treatment cause hyperglycemia associated with adiponectin receptor alterations 
Background
Adiponectin is the most abundant plasma protein synthesized for the most part in adipose tissue, and it is an insulin-sensitive hormone, playing a central role in glucose and lipid metabolism. In addition, it increases fatty acid oxidation in the muscle and potentiates insulin inhibition of hepatic gluconeogenesis. Two adiponectin receptors have been identified: AdipoR1 is the major receptor expressed in skeletal muscle, whereas AdipoR2 is mainly expressed in liver. Consumption of high levels of dietary fat is thought to be a major factor in the promotion of obesity and insulin resistance. Excessive levels of cortisol are characterized by the symptoms of abdominal obesity, hypertension, glucose intolerance or diabetes and dyslipidemia; of note, all of these features are shared by the condition of insulin resistance. Although it has been shown that glucocorticoids inhibit adiponectin expression in vitro and in vivo, little is known about the regulation of adiponectin receptors. The link between glucocorticoids and insulin resistance may involve the adiponectin receptors and adrenalectomy might play a role not only in regulate expression and secretion of adiponectin, as well regulate the respective receptors in several tissues.
Results
Feeding of a high-fat diet increased serum glucose levels and decreased adiponectin and adipoR2 mRNA expression in subcutaneous and retroperitoneal adipose tissues, respectively. Moreover, it increased both adipoR1 and adipoR2 mRNA levels in muscle and adipoR2 protein levels in liver. Adrenalectomy combined with the synthetic glucocorticoid dexamethasone treatment resulted in increased glucose and insulin levels, decreased serum adiponectin levels, reduced adiponectin mRNA in epididymal adipose tissue, reduction of adipoR2 mRNA by 7-fold in muscle and reduced adipoR1 and adipoR2 protein levels in muscle. Adrenalectomy alone increased adiponectin mRNA expression 3-fold in subcutaneous adipose tissue and reduced adipoR2 mRNA expression 2-fold in liver.
Conclusion
Hyperglycemia as a result of a high-fat diet is associated with an increase in the expression of the adiponectin receptors in muscle. An excess of glucocorticoids, rather than their absence, increase glucose and insulin and decrease adiponectin levels.
doi:10.1186/1476-511X-10-11
PMCID: PMC3031255  PMID: 21244702
18.  Glucocorticoid regulation of insulin receptor and substrate IRS-1 tyrosine phosphorylation in rat skeletal muscle in vivo. 
Journal of Clinical Investigation  1993;91(5):2020-2030.
To test the hypothesis that glucocorticoid-induced insulin resistance might originate from abnormalities in insulin receptor signaling, we investigated the effects of glucocorticoids on in vivo tyrosine phosphorylation of the insulin receptor and the insulin receptor substrate IRS-1 in rat skeletal muscle. Male Sprague-Dawley rats were treated with cortisone (100 mg/kg for 5 d) and compared to pair-fed controls. Cortisone treatment of rats resulted in both hyperglycemia and hyperinsulinemia. Anesthetized animals were injected with 10 U/kg insulin via cardiac puncture and, after 2 min, hindlimb muscles were removed, snap-frozen, and homogenized in SDS. Protein tyrosine phosphorylation was studied by immunoblotting with phosphotyrosine antibody. Insulin receptors and substrate IRS-1 were identified and quantified with specific antibodies. Cortisone treatment increased the amount of insulin receptor protein by 36%, but decreased the total level of receptor tyrosine phosphorylation (69 +/- 4% of control, P < 0.05). The decreased level of receptor phosphorylation was explained by a reduced number of receptors containing phosphorylated tyrosine residues (64.6 +/- 5% of control, P < 0.05). Glucocorticoid excess decreased skeletal muscle IRS-1 content by 50%, but did not significantly alter the total level of IRS-1 tyrosine phosphorylation. The apparent M(r) of IRS-1 was reduced by approximately 10 kD. Treatment with protein phosphatase-2A reduced IRS-1 M(r) in control but not in glucocorticoid-treated muscle indicating that the lower M(r) likely results from lower phosphoserine and/or phosphothreonine content. To investigate the role of hyperinsulinemia in the glucocorticoid response, rats were made insulin-deficient with streptozotocin (100 mg/kg, i.p.). Subsequent treatment with cortisone for 5 d had no effects on insulin levels, tyrosine phosphorylation of insulin receptors or IRS-1, or the M(r) of IRS-1. In conclusion, glucocorticoid-treated skeletal muscle is characterized by: (a) decreased total tyrosine phosphorylation of insulin receptors as a result of a reduction in the pool of receptors undergoing tyrosine phosphorylation; (b) decreased IRS-1 content and reduced serine and/or threonine phosphorylation of IRS-1. Glucocorticoid-induced hyperinsulinemia appears to be essential for the development of these alterations.
Images
PMCID: PMC288200  PMID: 7683695
19.  Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis 
Molecular Cancer  2005;4:1.
Background
We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined.
Results
Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH.
Conclusions
The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.
doi:10.1186/1476-4598-4-1
PMCID: PMC545049  PMID: 15644144
colon cancer; soy; whey; gene expression profiling; neuro-endocrine; microarray; rat
20.  Pharmacodynamic Modeling of Acute and Chronic Effects of Methylprednisolone on Hepatic Urea Cycle Genes in Rats* 
Corticosteroids (CS) regulate many enzymes at both mRNA and protein levels. This study used microarrays to broadly assess regulation of various genes related to the greater urea cycle and employs pharmacokinetic/pharmacodynamic (PK/PD) modeling to quantitatively analyze and compare the temporal profiles of these genes during acute and chronic exposure to methylprednisolone (MPL). One group of adrenalectomized male Wistar rats received an intravenous bolus dose (50 mg/kg) of MPL, whereas a second group received MPL by a subcutaneous infusion (Alzet osmotic pumps) at a rate of 0.3 mg/kg/hr for seven days. The rats were sacrificed at various time points over 72 hours (acute) or 168 hours (chronic) and livers were harvested. Total RNA was extracted and Affymetrix® gene chips (RG_U34A for acute and RAE 230A for chronic) were used to identify genes regulated by CS. Besides five primary urea cycle enzymes, many other genes related to the urea cycle showed substantial changes in mRNA expression. Some genes that were simply up- or down-regulated after acute MPL showed complex biphasic patterns upon chronic infusion indicating involvement of secondary regulation. For the simplest patterns, indirect response models were used to describe the nuclear steroid-bound receptor mediated increase or decrease in gene transcription (e.g. tyrosine aminotransferase, glucocorticoid receptor). For the biphasic profiles, involvement of a secondary biosignal was assumed (e.g. ornithine decarboxylase, CCAAT/enhancer binding protein) and more complex models were derived. Microarrays were used successfully to explore CS effects on various urea cycle enzyme genes. PD models presented in this report describe testable hypotheses regarding molecular mechanisms and quantitatively characterize the direct or indirect regulation of various genes by CS.
PMCID: PMC2733100  PMID: 19787073
urea cycle; corticosteroids; methylprednisolone; pharmacodynamics; genomics
21.  Pharmacogenomic Responses of Rat Liver to Methylprednisolone: An Approach to Mining a Rich Microarray Time Series 
The AAPS journal  2005;7(1):E156-E194.
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparations from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
doi:10.1208/aapsj070117
PMCID: PMC2607485  PMID: 16146338
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
22.  Pharmacogenomic responses of rat liver to methylprednisolone: An approach to mining a rich microarray time series 
The AAPS Journal  2005;7(1):E156-E194.
A data set was generated to examine global changes in gene expression in rat liver over time in response to a single bolus dose of methylprednisolone. Four control animals and 43 drug-treated animals were humanely killed at 16 different time points following drug administration. Total RNA preparation from the livers of these animals were hybridized to 47 individual Affymetrix RU34A gene chips, generating data for 8799 different probe sets for each chip. Data mining techniques that are applicable to gene array time series data sets in order to identify drug-regulated changes in gene expression were applied to this data set. A series of 4 sequentially applied filters were developed that were designed to eliminate probe sets that were not expressed in the tissue, were not regulated by the drug treatment, or did not meet defined quality control standards. These filters eliminated 7287 probe sets of the 8799 total (82%) from further consideration. Application of judiciously chosen filters is an effective tool for data mining of time series data sets. The remaining data can then be further analyzed by clustering and mathematical modeling techniques.
doi:10.1208/aapsj070117
PMCID: PMC2607485  PMID: 16146338
Data mining; gene arrays; glucocorticoids; mathematical modeling; pharmacogenomics
23.  Whole Blood Gene Expression Profiles in Insulin Resistant Latinos with the Metabolic Syndrome 
PLoS ONE  2013;8(12):e84002.
Although insulin resistance in skeletal muscle is well-characterized, the role of circulating whole blood in the metabolic syndrome phenotype is not well understood. We set out to test the hypothesis that genes involved in inflammation, insulin signaling and mitochondrial function would be altered in expression in the whole blood of individuals with metabolic syndrome. We further wanted to examine whether similar relationships that we have found previously in skeletal muscle exist in peripheral whole blood cells. All subjects (n=184) were Latino descent from the Arizona Insulin Resistance registry. Subjects were classified based on the metabolic syndrome phenotype according to the National Cholesterol Education Program’s Adult Treatment Panel III. Of the 184 Latino subjects in the study, 74 were classified with the metabolic syndrome and 110 were without. Whole blood gene expression profiling was performed using the Agilent 4x44K Whole Human Genome Microarray. Whole blood microarray analysis identified 1,432 probes that were altered in expression ≥1.2 fold and P<0.05 after Benjamini-Hochberg in the metabolic syndrome subjects. KEGG pathway analysis revealed significant enrichment for pathways including ribosome, oxidative phosphorylation and MAPK signaling (all Benjamini-Hochberg P<0.05). Whole blood mRNA expression changes observed in the microarray data were confirmed by quantitative RT-PCR. Transcription factor binding motif enrichment analysis revealed E2F1, ELK1, NF-kappaB, STAT1 and STAT3 significantly enriched after Bonferroni correction (all P<0.05). The results of the present study demonstrate that whole blood is a useful tissue for studying the metabolic syndrome and its underlying insulin resistance although the relationship between blood and skeletal muscle differs.
doi:10.1371/journal.pone.0084002
PMCID: PMC3866261  PMID: 24358323
24.  Transcriptomic profiles of skeletal muscle tissue following an euglycemic-hyperinsulinemic clamp in insulin-resistant obese subjects 
Genes & Nutrition  2012;8(1):91-98.
Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Muscle is mainly responsible for insulin-stimulated glucose clearance from the bloodstream. Thus, regulation of gene expression in muscle tissue may be involved in the pathogenesis of insulin resistance. The objective was to investigate gene expression and metabolic pathways alterations in skeletal muscle tissue following an euglycemic-hyperinsulinemic clamp in obese insulin-resistant subjects. We carried out a transcriptome comparison of skeletal muscle tissue before and after a 3-h euglycemic-hyperinsulinemic clamp following 8-week supplementation with n-3 polyunsaturated fatty acid (PUFA) (1.8 g/day) with or without a supplement of fish gelatin (FG) (25 % of daily protein intake) in 16 obese insulin-resistant subjects. Results indicate that approximately 5 % (1932) of expressed transcripts were significantly changed after the clamp in both n-3 PUFA and n-3 PUFA + FG supplementation periods. Of these differentially expressed transcripts, 1394 genes associated with enzymes, transcription and translation regulators, transporters, G protein-coupled receptors, cytokines, and ligand-dependent nuclear receptors were modified. Metabolic pathways that were significantly modified included liver X receptor/retinoid X receptors (RXR) activation, vitamin D receptor/RXR activation, interleukin (IL)-8, acute phase response, IL10, triggering receptor expressed on myeloid cells 1, peroxisome proliferator-activated receptor, G-beta/gamma and hepatocyte growth factor and IL6 signaling. Taken together, results suggest that mainly inflammatory and transcription factors are modified following clamp in obese insulin-resistant subjects. Overall, understanding the changes in metabolic pathways due to insulin may be a potential target for the management of insulin resistance.
doi:10.1007/s12263-012-0298-2
PMCID: PMC3534998  PMID: 22566203
Diabetes; Obesity; Microarray; Gene expression; Metabolic pathways
25.  CORTICOSTEROIDS AND MUSCLE WASTING ROLE OF TRANSCRIPTION FACTORS, NUCLEAR COFACTORS, AND HYPERACETYLATION 
Purpose of review
The purpose of this review is to discuss novel insight into mechanisms of glucocorticoid-regulated muscle wasting, in particular the role of transcription factors and nuclear cofactors. In addition, novel strategies that may become useful in the treatment or prevention of glucocorticoid-induced muscle wasting are reviewed.
Recent findings
Studies suggest that glucocorticoid-induced upregulation of the transcription factors FOXO1 and C/EBPβ and downregulation of MyoD and myogenin are involved in glucocorticoid-induced muscle wasting. In addition, glucocorticoid-induced hyperacetylation caused by increased expression of the nuclear cofactor p300 and its histone acetyl transferase activity and decreased expression and activity of histone deacetylases (HDACs) plays an important role in glucocorticoid-induced muscle proteolysis and wasting. Other mechanisms may also be involved in glucocorticoid-induced muscle wasting, including insulin resistance and store-operated calcium entry. Novel potential strategies to prevent or treat glucocorticoid-induced muscle wasting include the use of small molecule HDAC activators, dissociated glucocorticoid receptor agonists, and 11β-hydroxysteroid dehydrogenase type 1 inhibitors.
Summary
An increased understanding of molecular mechanisms regulating glucocorticoid-induced muscle wasting will help develop new strategies to prevent and treat this debilitating condition.
doi:10.1097/MCO.0b013e32833a5107
PMCID: PMC2911625  PMID: 20473154
Muscle wasting; glucocorticoids; FOXO1; C/EBPβ; MyoD; myogenin; p300; histone deacetylases

Results 1-25 (983565)