Rat PRDM9 shapes recombination landscapes, duration of meiosis, gametogenesis, and age of fertility.

BACKGROUND: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear. RESULTS: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants. CONCLUSIONS: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.

Systems genetic analysis of brown adipose tissue function.

Brown adipose tissue (BAT) has been suggested to play an important role in lipid and glucose metabolism in rodents and possibly also in humans. In the current study, we used genetic and correlation analyses in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), to identify genetic determinants of BAT function. Linkage analyses revealed a quantitative trait locus (QTL) associated with interscapular BAT mass on chromosome 4 and two closely linked QTLs associated with glucose oxidation and glucose incorporation into BAT lipids on chromosome 2. Using weighted gene coexpression network analysis (WGCNA) we identified 1,147 gene coexpression modules in the BAT from BXH/HXB rats and mapped their module eigengene QTLs. Through an unsupervised analysis, we identified modules related to BAT relative mass and function. The Coral4.1 coexpression module is associated with BAT relative mass (includes Cd36 highly connected gene), and the Darkseagreen coexpression module is associated with glucose incorporation into BAT lipids (includes Hiat1, Fmo5, and Sort1 highly connected transcripts). Because multiple statistical criteria were used to identify candidate modules, significance thresholds for individual tests were not adjusted for multiple comparisons across modules. In summary, a systems genetic analysis using genomic and quantitative transcriptomic and physiological information has produced confirmation of several known genetic factors and significant insight into novel genetic components functioning in BAT and possibly contributing to traits characteristic of the metabolic syndrome.

Autocrine effects of transgenic resistin reduce palmitate and glucose oxidation in brown adipose tissue.

Resistin has been originally identified as an adipokine that links obesity to insulin resistance in mice. In our previous studies in spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin (Retn) transgene specifically in adipose tissue (SHR-Retn), we have observed an increased lipolysis and serum free fatty acids, ectopic fat accumulation in muscles, and insulin resistance. Recently, brown adipose tissue (BAT) has been suggested to play an important role in the pathogenesis of metabolic disturbances. In the current study, we have analyzed autocrine effects of transgenic resistin on BAT glucose and lipid metabolism and mitochondrial function in the SHR-Retn vs. nontransgenic SHR controls. We observed that interscapular BAT isolated from SHR-Retn transgenic rats compared with SHR controls showed a lower relative weight (0.71 ± 0.05 vs. 0.91 ± 0.08 g/100 g body wt, P < 0.05), significantly reduced both basal and insulin stimulated incorporation of palmitate into BAT lipids (658 ± 50 vs. 856 ± 45 and 864 ± 47 vs. 1,086 ± 35 nmol/g/2 h, P ≤ 0.01, respectively), and significantly decreased palmitate oxidation (37.6 ± 4.5 vs. 57 ± 4.1 nmol/g/2 h, P = 0.007) and glucose oxidation (277 ± 34 vs. 458 ± 38 nmol/g/2 h, P = 0.001). In addition, in vivo microPET imaging revealed significantly reduced (18)F-FDG uptake in BAT induced by exposure to cold in SHR-Retn vs. control SHR (232 ± 19 vs. 334 ± 22 kBq/ml, P < 0.05). Gene expression profiles in BAT identified differentially expressed genes involved in skeletal muscle and connective tissue development, inflammation and MAPK and insulin signaling. These results provide evidence that autocrine effects of resistin attenuate differentiation and activity of BAT and thus may play a role in the pathogenesis of insulin resistance in the rat.

Effects of mtDNA in SHR-mtF344 versus SHR conplastic strains on reduced OXPHOS enzyme levels, insulin resistance, cardiac hypertrophy, and systolic dysfunction.

Common inbred strains of the laboratory rat can be divided into four major mitochondrial DNA (mtDNA) haplotype groups represented by the BN, F344, LEW, and SHR strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. F344 mtDNA by comparing the SHR vs. SHR-mt(F344) conplastic strains that are genetically identical except for their mitochondrial genomes. Altogether 13 amino acid substitutions in protein coding genes, seven single nucleotide polymorphisms in tRNA genes, and 12 single nucleotide changes in rRNA genes were detected in F344 mtDNA compared with SHR mtDNA. Analysis of oxidative phosphorylation system (OXPHOS) in heart left ventricles (LV), muscle, and liver revealed reduced activity and content of several respiratory chain complexes in SHR-mt(F344) conplastic rats compared with the SHR strain. Lower function of OXPHOS in LV of conplastic rats was associated with significantly increased relative ventricular mass and reduced fractional shortening that was independent of blood pressure. In addition, conplastic rats exhibited reduced sensitivity of skeletal muscles to insulin action and impaired glucose tolerance. These results provide evidence that inherited alterations in mitochondrial genome, in the absence of variation in the nuclear genome and other confounding factors, predispose to insulin resistance, cardiac hypertrophy and systolic dysfunction.

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.

Nonsynonymous variants in mt-Nd2, mt-Nd4, and mt-Nd5 are linked to effects on oxidative phosphorylation and insulin sensitivity in rat conplastic strains.

Common inbred strains of the laboratory rat can be divided into four different mitochondrial DNA haplotype groups represented by the SHR, BN, LEW, and F344 strains. In the current study, we investigated the metabolic and hemodynamic effects of the SHR vs. LEW mitochondrial genomes by comparing the SHR to a new SHR conplastic strain, SHR-mt(LEW); these strains are genetically identical except for their mitochondrial genomes. Complete mitochondrial DNA (mtDNA) sequence analysis comparing the SHR and LEW strains revealed gene variants encoding amino acid substitutions limited to a single mitochondrial enzyme complex, NADH dehydrogenase (complex I), affecting subunits 2, 4, and 5. Two of the variants in the mt-Nd4 subunit gene are located close to variants known to be associated with exercise intolerance and diabetes mellitus in humans. No variants were found in tRNA or rRNA genes. These variants in mt-Nd2, mt-Nd4, and mt-Nd5 in the SHR-mt(LEW) conplastic strain were linked to reductions in oxidative and nonoxidative glucose metabolism in skeletal muscle. In addition, SHR-mt(LEW) conplastic rats showed increased serum nonesterified fatty acid levels and resistance to insulin stimulated incorporation of glucose into adipose tissue lipids. These results provide evidence that inherited variation in mitochondrial genes encoding respiratory chain complex I subunits, in the absence of variation in the nuclear genome and other confounding factors, can influence glucose and lipid metabolism when expressed on the nuclear genetic background of the SHR strain.

CD36 overexpression predisposes to arrhythmias but reduces infarct size in spontaneously hypertensive rats: gene expression profile analysis.

CD36 fatty acid translocase plays a key role in supplying heart with its major energy substrate, long-chain fatty acids (FA). Previously, we found that the spontaneously hypertensive rat (SHR) harbors a deletion variant of Cd36 gene that results in reduced transport of long-chain FA into cardiomyocytes and predisposes the SHR to cardiac hypertrophy. In the current study, we analyzed the effects of mutant Cd36 on susceptibility to ischemic ventricular arrhythmias and myocardial infarction in adult SHR-Cd36 transgenic rats with wild-type Cd36 compared with age-matched SHR controls. Using an open-chest model of coronary artery occlusion, we found that SHR-Cd36 transgenic rats showed profound arrhythmogenesis resulting in significantly increased duration of tachyarrhythmias (207 ± 48 s vs. 55 ± 21 s, P < 0.05), total number of premature ventricular complexes (2,623 ± 517 vs. 849 ± 250, P < 0.05) and arrhythmia score (3.86 ± 0.18 vs. 3.13 ± 0.13, P < 0.001). On the other hand, transgenic SHR compared with SHR controls showed significantly reduced infarct size (52.6 ± 4.3% vs. 72.4 ± 2.9% of area at risk, P < 0.001). Similar differences were observed in isolated perfused hearts, and the increased susceptibility of transgenic SHR to arrhythmias was abolished by reserpine, suggesting the involvement of catecholamines. To further search for possible molecular mechanisms of altered ischemic tolerance, we compared gene expression profiles in left ventricles dissected from 6-wk-old transgenic SHR vs. age-matched controls using Illumina-based sequencing. Circadian rhythms and oxidative phosphorylation were identified as the top KEGG pathways, while circadian rhythms, VDR/RXR activation, IGF1 signaling, and HMGB1 signaling were the top IPA canonical pathways potentially important for Cd36-mediated effects on ischemic tolerance. It can be concluded that transgenic expression of Cd36 plays an important role in modulating the incidence and severity of ischemic and reperfusion ventricular arrhythmias and myocardial infarct size induced by coronary artery occlusion. The proarrhythmic effect of Cd36 transgene appears to be dependent on adrenergic stimulation.

Age-related autocrine diabetogenic effects of transgenic resistin in spontaneously hypertensive rats: gene expression profile analysis.

Increased circulating levels of resistin have been proposed as a possible link between obesity and insulin resistance; however, many of the potential metabolic effects of resistin remain to be investigated, including systemic versus local resistin action. We investigated potential autocrine effects of resistin on lipid and glucose metabolism in 2- and 16-mo-old transgenic spontaneously hypertensive rats (SHR) expressing a nonsecreted form of mouse resistin under control of the aP2 promoter. To search for possible molecular mechanisms, we compared gene expression profiles in adipose tissue in 6-wk-old transgenic SHR versus control rats, before development of insulin resistance, by digital transcriptional profiling using high-throughput sequencing. Both young and old transgenic rats showed moderate expression of the resistin transgene in adipose tissue but had serum resistin levels similar to control SHR and undetectable levels of transgenic resistin in the circulation. Young transgenic rats exhibited mild glucose intolerance. In contrast, older transgenic rats displayed marked glucose intolerance in association with near total resistance of adipose tissue to insulin-stimulated glucose incorporation into lipids (6 ± 2 vs. 77 ± 19 nmol glucose·g(-1)·2 h(-1), P < 0.00001). Ingenuity Pathway Analysis of differentially expressed genes revealed calcium signaling, Nuclear factor-erythroid 2-related factor-2 (NRF2)-mediated oxidative stress response, and actin cytoskeletal signaling canonical pathways as those most significantly affected. Analysis using DAVID software revealed oxidative phosphorylation, glutathione metabolism, pyruvate metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling as top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. These results suggest that with increasing age autocrine effects of resistin in fat tissue may predispose to diabetes in part by impairing insulin action in adipose tissue.

Succinimidyl oleate, established inhibitor of CD36/FAT translocase inhibits complex III of mitochondrial respiratory chain.

The functional role of CD36 protein detected in mitochondrial fractions in long chain fatty acid (LCFA) oxidation is unclear due to conflicting results obtained in Cd36 knockout mice and experiments using sulfo-N-succinimidyl oleate (SSO) for inhibition of CD36 mediated LCFA transport. We investigated effect of SSO on mitochondrial respiration and found that SSO substantially inhibits not only LCFA oxidation, but also oxidation of flavoprotein- and NADH-dependent substrates and generation of mitochondrial membrane potential. Experiments in rat liver, heart and kidney mitochondria demonstrated a direct effect on mitochondrial respiratory chain with the most pronounced inhibition of the complex III (IC(50) 4microM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.

Increased liver oxidative stress and altered PUFA metabolism precede development of non-alcoholic steatohepatitis in SREBP-1a transgenic spontaneously hypertensive rats with genetic predisposition to hepatic steatosis.

The temporal relationship of hepatic steatosis and changes in liver oxidative stress and fatty acid (FA) composition to the development of non-alcoholic steatohepatitis (NASH) remain to be clearly defined. Recently, we developed an experimental model of hepatic steatosis and NASH, the transgenic spontaneously hypertensive rat (SHR) that overexpresses a dominant positive form of the human SREBP-1a isoform in the liver. These rats are genetically predisposed to hepatic steatosis at a young age that ultimately progresses to NASH in older animals. Young transgenic SHR versus SHR controls exhibited simple hepatic steatosis which was associated with significantly increased hepatic levels of oxidative stress markers, conjugated dienes, and TBARS, with decreased levels of antioxidative enzymes and glutathione and lower concentrations of plasma alpha- and gamma-tocopherol. Transgenic rats exhibited increased plasma levels of saturated FA, decreased levels of n-3 and n-6 polyunsaturated FA (PUFA), and increased n-6/n-3 PUFA ratios. These results are consistent with the hypothesis that excess fat accumulation in the liver in association with increased oxidative stress and disturbances in the metabolism of saturated and unsaturated fatty acids may precede and contribute to the primary pathogenesis of NASH.

Correction: Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.

[This corrects the article DOI: 10.1371/journal.pone.0164206.].

Transgenic overexpression of glutathione S-transferase µ-type 1 reduces hypertension and oxidative stress in the stroke-prone spontaneously hypertensive rat.

BACKGROUND: Combined congenic breeding and microarray gene expression profiling previously identified glutathione S-transferase µ-type 1 (Gstm1) as a positional and functional candidate gene for blood pressure (BP) regulation in the stroke-prone spontaneously hypertensive (SHRSP) rat. Renal Gstm1 expression in SHRSP rats is significantly reduced when compared with normotensive Wistar Kyoto (WKY) rats. As Gstm1 plays an important role in the secondary defence against oxidative stress, significantly lower expression levels may be functionally relevant in the development of hypertension. The aim of this study was to investigate the role of Gstm1 in BP regulation and oxidative stress by transgenic overexpression of the Gstm1 gene. METHOD: Two independent Gstm1 transgenic SHRSP lines were generated by microinjecting SHRSP embryos with a linear construct controlled by the EF-1α promoter encoding WKY Gstm1 cDNA [SHRSP-Tg(Gstm1)1 and SHRSP-Tg(Gstm1)2]. RESULTS: Transgenic rats exhibit significantly reduced BP and pulse pressure when compared with SHRSP [systolic: SHRSP 205.2 ±â€Š3.7 mmHg vs. SHRSP-Tg(Gstm1)1 175.5 ±â€Š1.6 mmHg and SHRSP-Tg(Gstm1)2 172 ±â€Š3.2 mmHg, P < 0.001; pulse pressure: SHRSP 58.4 ±â€Š0.73 mmHg vs. SHRSP-Tg(Gstm1)1 52.7 ±â€Š0.19 mmHg and SHRSP-Tg(Gstm1)2 40.7 ±â€Š0.53 mmHg, P < 0.001]. Total renal and aortic Gstm1 expression in transgenic animals was significantly increased compared with SHRSP [renal relative quantification (RQ): SHRSP-Tg(Gstm1)1 1.95 vs. SHRSP 1.0, P < 0.01; aorta RQ: SHRSP-Tg(Gstm1)1 2.8 vs. SHRSP 1.0, P < 0.05]. Renal lipid peroxidation (malondialdehyde: protein) and oxidized : reduced glutathione ratio levels were significantly reduced in both transgenic lines when compared with SHRSP [malondialdehyde: SHRSP 0.04 ±â€Š0.009 µmol/l vs. SHRSP-Tg(Gstm1)1 0.024 ±â€Š0.002 µmol/l and SHRSP-Tg(Gstm1)2 0.021 ±â€Š0.002 µmol/l; (oxidized : reduced glutathione ratio): SHRSP 5.19 ±â€Š2.26 µmol/l vs. SHRSP-Tg(Gstm1)1 0.17 ±â€Š0.11 µmol/l and SHRSP-Tg(Gstm1)2 0.47 ±â€Š0.22 µmol/l]. Transgenic SHRSP rats containing the WKY Gstm1 gene demonstrate significantly lower BP, reduced oxidative stress and improved levels of renal Gstm1 expression. CONCLUSION: These data support the hypothesis that reduced renal Gstm1 plays a role in the development of hypertension.

Nrf2-Mediated Antioxidant Defense and Peroxiredoxin 6 Are Linked to Biosynthesis of Palmitic Acid Ester of 9-Hydroxystearic Acid.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with promising antidiabetic and anti-inflammatory properties that are formed in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, quantitative trait locus mapping, and correlation analyses in rat BXH/HXB recombinant inbred strains, as well as response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs. Comprehensive analysis of WAT samples identified ∼160 regioisomers, documenting the complexity of this lipid class. The linkage analysis highlighted several members of the nuclear factor, erythroid 2 like 2 (Nrf2)-mediated antioxidant defense system (Prdx6, Mgst1, Mgst3), lipid-handling proteins (Cd36, Scd6, Acnat1, Acnat2, Baat), and the family of flavin containing monooxygenases (Fmo) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically. Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein-driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues.

Salsalate ameliorates metabolic disturbances by reducing inflammation in spontaneously hypertensive rats expressing human C-reactive protein and by activating brown adipose tissue in nontransgenic controls.

Chronic low-grade inflammation plays an important role in the pathogenesis of insulin resistance. In the current study, we tested the effects of salsalate, a non-steroidal anti-inflammatory drug, in an animal model of inflammation and metabolic syndrome using spontaneously hypertensive rats (SHR) that transgenically express human C-reactive protein (SHR-CRP rats). We treated 15-month-old male transgenic SHR-CRP rats and nontransgenic SHR with salsalate (200 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP and SHR rats were fed a standard diet without salsalate. In the SHR-CRP transgenic strain, salsalate treatment decreased circulating concentrations of the inflammatory markers TNF-α and MCP-1, reduced oxidative stress in the liver and kidney, increased sensitivity of skeletal muscles to insulin action and improved tolerance to glucose. In SHR controls with no CRP-induced inflammation, salsalate treatment reduced body weight, decreased concentrations of serum free fatty acids and total and HDL cholesterol and increased palmitate oxidation and incorporation in brown adipose tissue. Salsalate regulated inflammation by affecting the expression of genes from MAPK signalling and NOD-like receptor signalling pathways and lipid metabolism by affecting hepatic expression of genes that favour lipid oxidation from PPAR-α signalling pathways. These findings suggest that salsalate has metabolic effects beyond suppressing inflammation.

Downregulation of Plzf Gene Ameliorates Metabolic and Cardiac Traits in the Spontaneously Hypertensive Rat.

The spontaneously hypertensive rat (SHR), one of the most widely used model of essential hypertension, is predisposed to left ventricular hypertrophy, myocardial fibrosis, and metabolic disturbances. Recently, quantitative trait loci influencing blood pressure, left ventricular mass, and heart interstitial fibrosis were genetically isolated within a minimal congenic subline that contains only 7 genes, including mutant Plzf (promyelocytic leukemia zinc finger) candidate gene. To identify Plzf as a quantitative trait gene, we targeted Plzf in the SHR using the transcription activator-like effector nuclease technique and obtained SHR line harboring targeted Plzf gene with a premature stop codon. Because the Plzf targeted allele is semilethal, morphologically normal heterozygous rats were used for metabolic and hemodynamic analyses. SHR-Plzf+/- heterozygotes versus SHR wild-type controls exhibited reduced body weight and relative weight of epididymal fat, lower serum and liver triglycerides and cholesterol, and better glucose tolerance. In addition, SHR-Plzf+/- rats exhibited significantly increased sensitivity of adipose and muscle tissue to insulin action when compared with wild-type controls. Blood pressure was comparable in SHR versus SHR-Plzf+/-; however, there was significant amelioration of cardiomyocyte hypertrophy and cardiac fibrosis in SHR-Plzf+/- rats. Gene expression profiles in the liver and expression of selected genes in the heart revealed differentially expressed genes that play a role in metabolic pathways, PPAR (peroxisome proliferator-activated receptor) signaling, and cell cycle regulation. These results provide evidence for an important role of Plzf in regulation of metabolic and cardiac traits in the rat and suggest a cross talk between cell cycle regulators, metabolism, cardiac hypertrophy, and fibrosis.

Effects of Metformin on Tissue Oxidative and Dicarbonyl Stress in Transgenic Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein.

Inflammation and oxidative and dicarbonyl stress play important roles in the pathogenesis of type 2 diabetes. Metformin is the first-line drug of choice for the treatment of type 2 diabetes because it effectively suppresses gluconeogenesis in the liver. However, its "pleiotropic" effects remain controversial. In the current study, we tested the effects of metformin on inflammation, oxidative and dicarbonyl stress in an animal model of inflammation and metabolic syndrome, using spontaneously hypertensive rats that transgenically express human C-reactive protein (SHR-CRP). We treated 8-month-old male transgenic SHR-CRP rats with metformin (5 mg/kg/day) mixed as part of a standard diet for 4 weeks. A corresponding untreated control group of male transgenic SHR-CRP rats were fed a standard diet without metformin. In a similar fashion, we studied a group of nontransgenic SHR treated with metformin and an untreated group of nontransgenic SHR controls. In each group, we studied 6 animals. Parameters of glucose and lipid metabolism and oxidative and dicarbonyl stress were measured using standard methods. Gene expression profiles were determined using Affymetrix GeneChip Arrays. Statistical significance was evaluated by two-way ANOVA. In the SHR-CRP transgenic strain, we found that metformin treatment decreased circulating levels of inflammatory response marker IL-6, TNFα and MCP-1 while levels of human CRP remained unchanged. Metformin significantly reduced oxidative stress (levels of conjugated dienes and TBARS) and dicarbonyl stress (levels of methylglyoxal) in left ventricles, but not in kidneys. No significant effects of metformin on oxidative and dicarbonyl stress were observed in SHR controls. In addition, metformin treatment reduced adipose tissue lipolysis associated with human CRP. Possible molecular mechanisms of metformin action-studied by gene expression profiling in the liver-revealed deregulated genes from inflammatory and insulin signaling, AMP-activated protein kinase (AMPK) signaling and gluconeogenesis pathways. It can be concluded that in the presence of high levels of human CRP, metformin protects against inflammation and oxidative and dicarbonyl stress in the heart, but not in the kidney. Accordingly, these cardioprotective effects of metformin might be especially effective in diabetic patients with high levels of CRP.

Genetic Variation in Renal Expression of Folate Receptor 1 (Folr1) Gene Predisposes Spontaneously Hypertensive Rats to Metabolic Syndrome.

Metabolism of homocysteine and other sulfur amino acids is closely associated with metabolism of folates. In this study, we analyzed the possible role of folates and sulfur amino acids in the development of features of the metabolic syndrome in the BXH/HXB recombinant inbred strains derived from the spontaneously hypertensive rat (SHR) and Brown Norway progenitors. We mapped a quantitative trait locus for cysteine concentrations to a region of chromosome 1 that contains a cis-acting expression quantitative trait locus regulating mRNA levels of folate receptor 1 (Folr1) in the kidney. Sequence analysis revealed a deletion variant in the Folr1 promoter region of the SHR. Transfection studies demonstrated that the SHR-promoter region of Folr1 is less effective in driving luciferase reporter gene expression than the Brown Norway promoter region of Folr1. Results in the SHR.BN-chr.1 congenic strain confirmed that the SHR variant in Folr1 cosegregates with markedly reduced renal expression of Folr1 and renal folate reabsorption, decreased serum levels of folate, increased serum levels of cysteine and homocysteine, increased adiposity, ectopic fat accumulation in liver and muscle, reduced muscle insulin sensitivity, and increased blood pressure. Transgenic rescue experiments performed by expressing a Folr1 transgene in the SHR ameliorated most of the metabolic disturbances. These findings are consistent with the hypothesis that inherited variation in the expression of Folr1 in the kidney influences the development of the metabolic syndrome and constitutes a previously unrecognized genetic mechanism that may contribute to increased risk for diabetes mellitus and cardiovascular disease.

Targeting of the Plzf Gene in the Rat by Transcription Activator-Like Effector Nuclease Results in Caudal Regression Syndrome in Spontaneously Hypertensive Rats.

Recently, it has been found that spontaneous mutation Lx (polydactyly-luxate syndrome) in the rat is determined by deletion of a conserved intronic sequence of the Plzf (Promyelocytic leukemia zinc finger protein) gene. In addition, Plzf is a prominent candidate gene for quantitative trait loci (QTLs) associated with cardiac hypertrophy and fibrosis in the spontaneously hypertensive rat (SHR). In the current study, we tested the effects of Plzf gene targeting in the SHR using TALENs (transcription activator-like effector nucleases). SHR ova were microinjected with constructs pTAL438/439 coding for a sequence-specific endonuclease that binds to target sequence in the first coding exon of the Plzf gene. Out of 43 animals born after microinjection, we detected a single male founder. Sequence analysis revealed a deletion of G that resulted in frame shift mutation starting in codon 31 and causing a premature stop codon at position of amino acid 58. The Plzftm1Ipcv allele is semi-lethal since approximately 95% of newborn homozygous animals died perinatally. All homozygous animals exhibited manifestations of a caudal regression syndrome including tail anomalies and serious size reduction and deformities of long bones, and oligo- or polydactyly on the hindlimbs. The heterozygous animals only exhibited the tail anomalies. Impaired development of the urinary tract was also revealed: one homozygous and one heterozygous rat exhibited a vesico-ureteric reflux with enormous dilatation of ureters and renal pelvis. In the homozygote, this was combined with a hypoplastic kidney. These results provide evidence for the important role of Plzf gene during development of the caudal part of a body-column vertebrae, hindlimbs and urinary system in the rat.

Fumaric acid esters can block pro-inflammatory actions of human CRP and ameliorate metabolic disturbances in transgenic spontaneously hypertensive rats.

Inflammation and oxidative stress have been implicated in the pathogenesis of metabolic disturbances. Esters of fumaric acid, mainly dimethyl fumarate, exhibit immunomodulatory, anti-inflammatory, and anti-oxidative effects. In the current study, we tested the hypothesis that fumaric acid ester (FAE) treatment of an animal model of inflammation and metabolic syndrome, the spontaneously hypertensive rat transgenically expressing human C-reactive protein (SHR-CRP), will ameliorate inflammation, oxidative stress, and metabolic disturbances. We studied the effects of FAE treatment by administering Fumaderm, 10 mg/kg body weight for 4 weeks, to male SHR-CRP. Untreated male SHR-CRP rats were used as controls. All rats were fed a high sucrose diet. Compared to untreated controls, rats treated with FAE showed significantly lower levels of endogenous CRP but not transgenic human CRP, and amelioration of inflammation (reduced levels of serum IL6 and TNFα) and oxidative stress (reduced levels of lipoperoxidation products in liver, heart, kidney, and plasma). FAE treatment was also associated with lower visceral fat weight and less ectopic fat accumulation in liver and muscle, greater levels of lipolysis, and greater incorporation of glucose into adipose tissue lipids. Analysis of gene expression profiles in the liver with Affymetrix arrays revealed that FAE treatment was associated with differential expression of genes in pathways that involve the regulation of inflammation and oxidative stress. These findings suggest potentially important anti-inflammatory, anti-oxidative, and metabolic effects of FAE in a model of inflammation and metabolic disturbances induced by human CRP.

Germline transgenesis in rabbits by pronuclear microinjection of Sleeping Beauty transposons.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for a variety of inherited and acquired human diseases. In addition, the rabbit is the smallest livestock animal that is used to transgenically produce pharmaceutical proteins in its milk. Here we describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in rabbits by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection into the pronuclei of fertilized oocytes of synthetic mRNA encoding the SB100X hyperactive transposase together with plasmid DNA carrying a transgene construct flanked by binding sites for the transposase. The translation of the transposase mRNA is followed by enzyme-mediated excision of the transgene cassette from the plasmids and its permanent genomic insertion to produce stable transgenic animals. Generation of a germline-transgenic founder animal by using this protocol takes ∼2 months. Transposon-mediated transgenesis compares favorably in terms of both efficiency and reliable transgene expression with classic pronuclear microinjection, and it offers comparable efficacies (numbers of transgenic founders obtained per injected embryo) to lentiviral approaches, without limitations on vector design, issues of transgene silencing, and the toxicity and biosafety concerns of working with viral vectors.