Endotrophin neutralization through targeted antibody treatment protects from renal fibrosis in a podocyte ablation model.

OBJECTIVE: Renal fibrosis is a hallmark for chronic kidney disease (CKD), and often leads to end stage renal disease (ESRD). However, limited interventions are available clinically to ameliorate or reverse renal fibrosis. METHODS: Herein, we evaluated whether blockade of endotrophin through neutralizing antibodies protects from renal fibrosis in the podocyte insult model (the "POD-ATTAC" mouse). We determined the therapeutic effects of endotrophin targeted antibody through assessing renal function, renal inflammation and fibrosis at histological and transcriptional levels, and podocyte regeneration. RESULTS: We demonstrated that neutralizing endotrophin antibody treatment significantly ameliorates renal fibrosis at the transcriptional, morphological, and functional levels. In the antibody treatment group, expression of pro-inflammatory and pro-fibrotic genes was significantly reduced, normal renal structures were restored, collagen deposition was decreased, and proteinuria and renal function were improved. We further performed a lineage tracing study confirming that podocytes regenerate as de novo podocytes upon injury and loss, and blockade of endotrophin efficiently enhances podocyte-specific marker expressions. CONCLUSION: Combined, we provide pre-clinical evidence supporting neutralizing endotrophin as a promising therapy for intervening with renal fibrosis in CKD, and potentially in other chronic fibro-inflammatory diseases.

The association of enteric neuropathy with gut phenotypes in acute and progressive models of Parkinson's disease.

Parkinson's disease (PD) is associated with neuronal damage in the brain and gut. This work compares changes in the enteric nervous system (ENS) of commonly used mouse models of PD that exhibit central neuropathy and a gut phenotype. Enteric neuropathy was assessed in five mouse models: peripheral injection of MPTP; intracerebral injection of 6-OHDA; oral rotenone; and mice transgenic for A53T variant human α-synuclein with and without rotenone. Changes in the ENS of the colon were quantified using pan-neuronal marker, Hu, and neuronal nitric oxide synthase (nNOS) and were correlated with GI function. MPTP had no effect on the number of Hu+ neurons but was associated with an increase in Hu+ nuclear translocation (P < 0.04). 6-OHDA lesioned mice had significantly fewer Hu+ neurons/ganglion (P < 0.02) and a reduced proportion of nNOS+ neurons in colon (P < 0.001). A53T mice had significantly fewer Hu+ neurons/area (P < 0.001) and exhibited larger soma size (P < 0.03). Treatment with rotenone reduced the number of Hu+ cells/mm2 in WT mice (P < 0.006) and increased the proportion of Hu+ translocated cells in both WT (P < 0.02) and A53T mice (P < 0.04). All PD models exhibited a degree of enteric neuropathy, the extent and type of damage to the ENS, however, was dependent on the model.

Chronic isolation stress is associated with increased colonic and motor symptoms in the A53T mouse model of Parkinson's disease.

BACKGROUND: Chronic stress exacerbates motor deficits and increases dopaminergic cell loss in several rodent models of Parkinson's disease (PD). However, little is known about effects of stress on gastrointestinal (GI) dysfunction, a common non-motor symptom of PD. We aimed to determine whether chronic stress exacerbates GI dysfunction in the A53T mouse model of PD and whether this relates to changes in α-synuclein distribution. METHODS: Chronic isolation stress was induced by single-housing WT and homozygote A53T mice between 5 and 15 months of age. GI and motor function were compared with mice that had been group-housed. KEY RESULTS: Chronic isolation stress increased plasma corticosterone and exacerbated deficits in colonic propulsion and whole-gut transit in A53T mice and also increased motor deficits. However, our results indicated that the novel environment-induced defecation response, a common method used to evaluate colorectal function, was not a useful test to measure exacerbation of GI dysfunction, most likely because of the reported reduced level of anxiety in A53T mice. A53T mice had lower corticosterone levels than WT mice under both housing conditions, but single-housing increased levels for both genotypes. Enteric neuropathy was observed in aging A53T mice and A53T mice had a greater accumulation of alpha-synuclein (αsyn) in myenteric ganglia under both housing conditions. CONCLUSIONS & INFERENCES: Chronic isolation stress exacerbates PD-associated GI dysfunction, in addition to increasing motor deficits. However, these changes in GI symptoms are not directly related to corticosterone levels, worsened enteric neuropathy, or enteric αsyn accumulation.

Endothelial Cell-Targeted Deletion of PPARγ Blocks Rosiglitazone-Induced Plasma Volume Expansion and Vascular Remodeling in Adipose Tissue.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor γ (PPARγ) agonists that represent an effective class of insulin-sensitizing agents; however, clinical use is associated with weight gain and peripheral edema. To elucidate the role of PPARγ expression in endothelial cells (ECs) in these side effects, EC-targeted PPARγ knockout (Pparg ΔEC) mice were placed on a high-fat diet to promote PPARγ agonist-induced plasma volume expansion, and then treated with the TZD rosiglitazone. Compared with Pparg-floxed wild-type control (Pparg f/f) mice, Pparg ΔEC treated with rosiglitazone are resistant to an increase in extracellular fluid, water content in epididymal and inguinal white adipose tissue, and plasma volume expansion. Interestingly, histologic assessment confirmed significant rosiglitazone-mediated capillary dilation within white adipose tissue of Pparg f/f mice, but not Pparg ΔEC mice. Analysis of ECs isolated from untreated mice in both strains suggested the involvement of changes in endothelial junction formation. Specifically, compared with cells from Pparg f/f mice, Pparg ΔEC cells had a 15-fold increase in focal adhesion kinase, critically important in EC focal adhesions, and >3-fold significant increase in vascular endothelial cadherin, the main component of focal adhesions. Together, these results indicate that rosiglitazone has direct effects on the endothelium via PPARγ activation and point toward a critical role for PPARγ in ECs during rosiglitazone-mediated plasma volume expansion.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice.

AIMS: Since 2006, DPP-4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP-4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity. METHODS: We have compared the binding properties of the most commonly used inhibitors and have investigated the relationship between their inhibitory potency at the level of the enzyme and their acute glucose-lowering efficacy. RESULTS: Firstly, using a combination of published crystal structures and in-house data, we demonstrated that the binding site utilized by all of the DPP-4 inhibitors assessed was the same as that used by neuropeptide Y, supporting the hypothesis that DPP-4 inhibitors are able to competitively inhibit endogenous substrates for the enzyme. Secondly, we ascertained that the enzymatic cleft of DPP-4 is a relatively large cavity which displays conformational flexibility to accommodate structurally diverse inhibitor molecules. Finally, we found that for all inhibitors, irrespective of their chemical structure, the inhibition of plasma DPP-4 enzyme activity correlates directly with acute plasma glucose lowering in mice. CONCLUSION: The common binding site utilized by different DPP-4 inhibitors enables similar competitive inhibition of the cleavage of the endogenous DPP-4 substrates. Furthermore, despite chemical diversity and a range of binding potencies observed amongst the DPP-4 inhibitors, a direct relationship between enzyme inhibition in the plasma and glucose lowering is evident in mice for each member of the classes studied.

Novel, highly potent systemic glucokinase activators for the treatment of Type 2 Diabetes Mellitus.

Glucokinase (GK, hexokinase IV) is a unique hexokinase that plays a central role in mammalian glucose homeostasis. Glucose phosphorylation by GK in the pancreatic ß-cell is the rate-limiting step that controls glucose-stimulated insulin secretion. Similarly, GK-mediated glucose phosphorylation in hepatocytes plays a major role in increasing hepatic glucose uptake and metabolism and possibly lowering hepatic glucose output. Small molecule GK activators (GKAs) have been identified that increase enzyme activity by binding to an allosteric site. GKAs offer a novel approach for the treatment of Type 2 Diabetes Mellitus (T2DM) and as such have garnered much attention. We now report the design, synthesis, and biological evaluation of a novel series of 2,5,6-trisubstituted indole derivatives that act as highly potent GKAs. Among them, Compound 1 was found to possess high in vitro potency, excellent physicochemical properties, and good pharmacokinetic profile in rodents. Oral administration of Compound 1 at doses as low as 0.03mg/kg led to robust blood glucose lowering efficacy in 3week high fat diet-fed mice.

Discovery of orally active hepatoselective glucokinase activators for treatment of Type II Diabetes Mellitus.

Systemically acting glucokinase activators (GKA) have been demonstrated in clinical trials to effectively lower blood glucose in patients with type II diabetes. However, mechanism-based hypoglycemia is a major adverse effect that limits the therapeutic potential of these agents. We hypothesized that the predominant mechanism leading to hypoglycemia is GKA-induced excessive insulin secretion from pancreatic ß-cells at (sub-)euglycemic levels. We further hypothesized that restricting GK activation to hepatocytes would maintain glucose-lowering efficacy while significantly reducing hypoglycemic risk. Here we report the discovery of a novel series of carboxylic acid substituted GKAs based on pyridine-2-carboxamide. These GKAs exhibit preferential distribution to the liver versus the pancreas in mice. SAR studies led to the identification of a potent and orally active hepatoselective GKA, compound 6. GKA 6 demonstrated robust glucose lowering efficacy in high fat diet-fed mice at doses ⩾10mpk, with ⩾70-fold liver:pancreas distribution, minimal effects on plasma insulin levels, and significantly reduced risk of hypoglycemia.

Downstream signaling pathways in mouse adipose tissues following acute in vivo administration of fibroblast growth factor 21.

FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation.

Pegylated Fgf21 rapidly normalizes insulin-stimulated glucose utilization in diet-induced insulin resistant mice.

Fibroblast growth factor 21 (FGF21) is a novel hormone-like polypeptide that when administered exogenously, has been shown to have beneficial effects on food intake, body weight, and metabolism. The in vivo mechanisms of action for its positive metabolic effects remain to be fully elucidated. It has been shown that PEGylation of human FGF21 at specific and preferred sites confer superior metabolic pharmacology. We therefore hypothesized that low doses of PEGylated (30K PEG on position Q108) FGF21 (PEG30-Q108) would improve insulin action, independent of any effect on food intake or body weight. We identified a dose (0.25mg/kg) that had no effect on food intake or body weight, yet did show beneficial metabolic effects. Four groups of 12 weeks, high-fat fed, insulin resistant mice were studied: mice dosed subcutaneously once with vehicle or 0.25mg/kg of PEG30-Q108 24h before the experiment, or mice dosed 4 times over 2 weeks with vehicle or PEG30-Q108. Conscious, unrestrained mice were fasted for 5h and underwent a hyperinsulinemic-euglycemic clamp. Both PEG30-Q108 treatments significantly lowered fasting insulin compared to vehicle, with no difference in food intake or body weight. Insulin-stimulated whole body glucose utilization was normalized to that of lean mice with both PEG30-Q108 treatments compared to vehicle. This accounted for all of the enhanced insulin action, as there was no improvement in insulin's ability to suppress endogenous glucose production. In line with these findings, neither PEG30-Q108 treatment lowered hepatic triglycerides. These results demonstrate the profound ability of PEG30-Q108 to increase whole body insulin sensitivity.

FGF21 suppresses hepatic glucose production through the activation of atypical protein kinase Cι/λ.

Fibroblast growth factor 21 (FGF21) has been identified as a potent and robust metabolic regulator. Administration of recombinant FGF21 protein to rodents and rhesus monkeys exerts strong anti-diabetic effects. Previous studies have demonstrated that FGF21 inhibits glucose output in the rat H4IIE hepatoma cell line. We performed pharmacological studies to investigate the mechanisms by which FGF21 regulates glucose production in these cells. We found that both insulin and FGF21 suppressed gene expression of G6Pase and PEPCK. Accordingly, glucose production was inhibited. The FGF21 effects were phosphoinositide 3-kinase (PI3K)-dependent, and, unlike insulin, Akt-independent. Additionally, we found that FGF21 induced PKCι/λ phosphorylation in a PI3K-dependent manner; and that a non-isoform selective PKC inhibitor blocked FGF21 inhibition of glucose production, while an inhibitor of classical and novel PKC isoforms had no effect on FGF21 inhibitory activity. Furthermore, hepatic PKCι/λ phosphorylation was upregulated in FGF21-treated diabetic db/db mice.These data support the proposition that FGF21 inhibits hepatic glucose production by the PI3K-dependent activation of PKCι/λ.

A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP) and contributes to the development of type 2 diabetes (T2D). Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC) promoter (AH-G6PC cells). Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4) mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD) of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in our cell-based druggable genome siRNA screen hold the potential to serve as novel pharmacological targets for the treatment of T2D.

Novel transcriptome profiling analyses demonstrate that selective peroxisome proliferator-activated receptor γ (PPARγ) modulators display attenuated and selective gene regulatory activity in comparison with PPARγ full agonists.

Selective peroxisome proliferator-activated receptor γ (PPARγ) modulators (SPPARγMs) have been actively pursued as the next generation of insulin-sensitizing antidiabetic drugs, because the currently marketed PPARγ full agonists, pioglitazone and rosiglitazone, have been reported to produce serious adverse effects among patients with type 2 diabetes mellitus. We conducted extensive transcriptome profiling studies to characterize and to contrast the activities of 70 SPPARγMs and seven PPARγ full agonists. In both 3T3-L1 adipocytes and adipose tissue from db/db mice, the SPPARγMs generated attenuated and selective gene-regulatory responses, in comparison with full agonists. More importantly, SPPARγMs regulated the expression of antidiabetic efficacy-associated genes to a greater extent than that of adverse effect-associated genes, whereas PPARγ full agonists regulated both gene sets proportionally. Such SPPARγM selectivity demonstrates that PPARγ ligand regulation of gene expression can be fine-tuned, and not just turned on and off, to achieve precise control of complex cellular and physiological functions. It also provides a potential molecular basis for the superior therapeutic window previously observed with SPPARγMs versus full agonists. On the basis of our profiling results, we introduce two novel, gene expression-based scores, the γ activation index and the selectivity index, to aid in the detection and characterization of novel SPPARγMs. These studies provide new insights into the gene-regulatory activity of SPPARγMs as well as novel quantitative indices to facilitate the identification of PPARγ ligands with robust insulin-sensitizing activity and improved tolerance among patients with type 2 diabetes, compared with presently available PPARγ agonist drugs.

FGF21 analogs of sustained action enabled by orthogonal biosynthesis demonstrate enhanced antidiabetic pharmacology in rodents.

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

Benzimidazolones: a new class of selective peroxisome proliferator-activated receptor γ (PPARγ) modulators.

A series of benzimidazolone carboxylic acids and oxazolidinediones were designed and synthesized in search of selective PPARγ modulators (SPPARγMs) as potential therapeutic agents for the treatment of type II diabetes mellitus (T2DM) with improved safety profiles relative to rosiglitazone and pioglitazone, the currently marketed PPARγ full agonist drugs. Structure-activity relationships of these potent and highly selective SPPARγMs were studied with a focus on their unique profiles as partial agonists or modulators. A variety of methods, such as X-ray crystallographic analysis, PPARγ transactivation coactivator profiling, gene expression profiling, and mutagenesis studies, were employed to reveal the differential interactions of these new analogues with PPARγ receptor in comparison to full agonists. In rodent models of T2DM, benzimidazolone analogues such as (5R)-5-(3-{[3-(5-methoxybenzisoxazol-3-yl)benzimidazol-1-yl]methyl}phenyl)-5-methyloxazolidinedione (51) demonstrated efficacy equivalent to that of rosiglitazone. Side effects, such as fluid retention and heart weight gain associated with PPARγ full agonists, were diminished with 51 in comparison to rosiglitazone based on studies in two independent animal models.

Pharmacokinetic and pharmacodynamic properties of the glucokinase activator MK-0941 in rodent models of type 2 diabetes and healthy dogs.

Glucokinase activators (GKAs) are small-molecule agents that enhance glucose sensing by pancreatic ß cells and glucose metabolism by hepatocytes. There is strong interest in these agents as potential therapies for type 2 diabetes. Here, we report key pharmacokinetic and pharmacodynamic findings from preclinical studies of the GKA 3-[[6-(ethylsulfonyl)-3-pyridinyl]oxy]-5-[(1S)-2-hydroxy-1-methylethoxy]-N-(1-methyl-1H-pyrazol-3-yl)benzamide (MK-0941). Incubated in vitro with recombinant human glucokinase, 1 µM MK-0941 lowered the S(0.5) of this enzyme for glucose from 6.9 to 1.4 mM and increased the maximum velocity of glucose phosphorylation by 1.5-fold. In 2.5 and 10 mM glucose, the EC(50) values for activation of GK by MK-0941 were 0.240 and 0.065 µM, respectively. Treatment of isolated rat islets of Langerhans and hepatocytes with 10 µM MK-0941 increased insulin secretion by 17-fold and glucose uptake up to 18-fold, respectively. MK-0941 exhibited strong glucose-lowering activity in C57BL/6J mice maintained on a high-fat diet (HFD), db/db mice, HFD plus low-dose streptozotocin-treated mice, and nondiabetic dogs. In both mice and dogs, oral doses of MK-0941 were rapidly absorbed and rapidly cleared from the blood; plasma levels reached maximum within 1 h and fell thereafter with a half-life of ~2 h. During oral glucose tolerance testing in dogs, MK-0941 reduced total area-under-the-curve postchallenge (0-2 h) plasma glucose levels by up to 48% compared with vehicle-treated controls. When administered twice daily to mice for 16 days, and once daily to the dog for 4 days, MK-0941 remained efficacious on successive days. These findings support further investigation of MK-0941 as a potential therapeutic agent for treatment of type 2 diabetes.

cSSMD: assessing collective activity for addressing off-target effects in genome-scale RNA interference screens.

MOTIVATION: Off-target activity commonly exists in RNA interference (RNAi) screens and often generates false positives. Existing analytic methods for addressing the off-target effects are demonstrably inadequate in RNAi confirmatory screens. RESULTS: Here, we present an analytic method assessing the collective activity of multiple short interfering RNAs (siRNAs) targeting a gene. Using this method, we can not only reduce the impact of off-target activities, but also evaluate the specific effect of an siRNA, thus providing information about potential off-target effects. Using in-house RNAi screens, we demonstrate that our method obtains more reasonable and sensible results than current methods such as the redundant siRNA activity (RSA) method, the RNAi gene enrichment ranking (RIGER) method, the frequency approach and the t-test. CONTACT: xiaohua_zhang@merck.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities.

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.

The use of SSMD-based false discovery and false nondiscovery rates in genome-scale RNAi screens.

In genome-scale RNA interference (RNAi) screens, it is critical to control false positives and false negatives statistically. Traditional statistical methods for controlling false discovery and false nondiscovery rates are inappropriate for hit selection in RNAi screens because the major goal in RNAi screens is to control both the proportion of short interfering RNAs (siRNAs) with a small effect among selected hits and the proportion of siRNAs with a large effect among declared nonhits. An effective method based on strictly standardized mean difference (SSMD) has been proposed for statistically controlling false discovery rate (FDR) and false nondiscovery rate (FNDR) appropriate for RNAi screens. In this article, the authors explore the utility of the SSMD-based method for hit selection in RNAi screens. As demonstrated in 2 genome-scale RNAi screens, the SSMD-based method addresses the unmet need of controlling for the proportion of siRNAs with a small effect among selected hits, as well as controlling for the proportion of siRNAs with a large effect among declared nonhits. Furthermore, the SSMD-based method results in reasonably low FDR and FNDR for selecting inhibition or activation hits. This method works effectively and should have a broad utility for hit selection in RNAi screens with replicates.

Preliminary report: pharmacologic 11beta-hydroxysteroid dehydrogenase type 1 inhibition increases hepatic fat oxidation in vivo and expression of related genes in rats fed an obesogenic diet.

This study aimed to explore in a model of diet-induced steatosis the impact of pharmacologic 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) inhibition, under conditions of unchanged ingestive behavior, on liver fat oxidation. Male Sprague-Dawley rats were fed an obesogenic diet and were continuously treated or not with an 11beta-HSD1 inhibitor (Compound A, 3 mg/[kg d]; Merck Research Laboratories, Rahway, NJ), after which liver expression of oxidative genes and in vivo hepatic fat oxidation were quantified. Treatment with Compound A reduced liver triglyceride concentration (-28%), increased hepatic expression of several genes coding for enzymes of mitochondrial and peroxisomal beta-oxidation, and concomitantly enhanced in vivo liver fat oxidation (+38%). The study demonstrates, under conditions that avoided changes in food intake seen in gene knockout or higher-dose pharmacologic models, the efficacy of 11beta-HSD1 inhibition to up-regulate hepatic fat oxidation gene expression, which functionally translates into enhanced hepatic lipid oxidation in vivo.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by nitroalkene fatty acids: importance of nitration position and degree of unsaturation.

Nitroalkene fatty acids are potent endogenous ligand activators of PPARgamma-dependent transcription. Previous studies with the naturally occurring regioisomers of nitrolinoleic acid revealed that the isomers are not equivalent with respect to PPARgamma activation. To gain further insight into the structure-activity relationships between nitroalkenes and PPARgamma, we examined additional naturally occurring nitroalkenes derived from oleic acid, 9-nitrooleic acid (E-9-NO2-18:1 [1]) and 10-nitrooleic acid (E-10-NO2-18:1 [2]), and several synthetic nitrated enoic fatty acids of variable carbon chain length, double bonds, and nitration site. At submicromolar concentrations, E-12-NO2 derivatives were considerably more potent than isomers nitrated at carbons 5, 6, 9, 10, and 13, and chain length (16 versus 18) or number of double bonds (1 versus 2) was of little consequence for PPARgamma activation. Interestingly, at higher concentrations (>2 microM) the nitrated enoic fatty acids (E-9-NO2-18:1 [1], E-9-NO2-16:1 [3], E-10-NO2-18:1 [2], and E-12-NO2-18:1 [7]) deviated significantly from the saturable pattern of PPARgamma activation observed for nitrated 1,4-dienoic fatty acids (E-9-NO2-18:2, E-10-NO2-18:2, E-12-NO2-18:2, and E-13-NO2-18:2).