Vladimír Landa

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Identification of renal Cd36 as a determinant of blood pressure and risk for hypertension

To identify renally expressed genes that influence risk for hypertension, we integrated expression quantitative trait locus (QTL) analysis of the kidney with genome-wide correlation analysis of renal expression profiles and blood pressure in recombinant inbred strains derived from the spontaneously hypertensive rat (SHR). This strategy, together with renal transplantation studies in SHR progenitor, transgenic and congenic strains, identified deficient renal expression of Cd36 encoding fatty acid translocase as a genetically determined risk factor for spontaneous hypertension.

Pharmacogenetic evidence that Cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor γ and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-typeCd36. In congenic and transgenic SHR with wild-typeCd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutantCd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor γ.

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.—Katter, K., Geurts, A. M., Hoffmann, O., Mátés, L., Landa, V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bősze, Z., Rülicke, T., Izsvák, Z. Transposon‐mediated transgenesis, transgenic rescue, and tissue‐specific gene expression in rodents and rabbits. FASEB J. 27, 930–941 (2013). www.fasebj.org

Germline transgenesis in pigs by cytoplasmic microinjection of Sleeping Beauty transposons

The pig has emerged as an important large animal model in biomedical and pharmaceutical research. We describe a protocol for high-efficiency germline transgenesis and sustained transgene expression in pigs by using the Sleeping Beauty (SB) transposon system. The protocol is based on co-injection of a plasmid encoding the SB100X hyperactive transposase, together with a second plasmid carrying a transgene flanked by binding sites for the transposase, into the cytoplasm of porcine zygotes. The transposase mediates excision of the transgene cassette from the plasmid vector and its permanent insertion into the genome to produce stable transgenic animals. This method compares favorably in terms of both efficiency and reliable transgene expression to classic pronuclear microinjection or somatic cell nuclear transfer (SCNT), and it offers comparable efficacies to lentiviral approaches, without limitations on vector design, issues of transgene silencing and the toxicity and biosafety concerns of working with viral vectors. Microinjection of the vectors into zygotes and transfer of the embryos to recipient animals can be performed in 1 d; generation of germline-transgenic lines by using this protocol takes ∼1 year.

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.

Effects of Human C-Reactive Protein on Pathogenesis of Features of the Metabolic Syndrome

Major controversy exists as to whether increased C-reactive protein (CRP) contributes to individual components of the metabolic syndrome or is just a secondary response to inflammatory disease processes. We measured blood pressure and metabolic phenotypes in spontaneously hypertensive rats (SHRs) in which we transgenically expressed human CRP in the liver under control of the apolipoprotein E promoter. In transgenic SHRs, serum levels of human CRP approximated the endogenous levels of CRP normally found in the rat. Systolic and diastolic blood pressures measured by telemetry were 10 to 15 mm Hg greater in transgenic SHRs expressing human CRP than in SHR controls (P<0.01). During oral glucose tolerance testing, transgenic SHRs exhibited hyperinsulinemia compared with controls (insulin area under the curve: 36±7 versus 8±2 nmol/L per 2 hours, respectively; P<0.05). Transgenic SHRs also exhibited resistance to insulin stimulated glycogenesis in skeletal muscle (174±18 versus 278±32 nmol of glucose per gram per 2 hours; P<0.05), hypertriglyceridemia (0.84±0.05 versus 0.64±0.03 mmol/L; P<0.05), reduced serum adiponectin (2.4±0.3 versus 4.3±0.6 mmol/L; P<0.05), and microalbuminuria (200±35 versus 26±5 mg of albumin per gram of creatinine, respectively; P<0.001). Transgenic SHRs had evidence of inflammation and oxidative tissue damage with increased serum levels of interleukin 6 (36.4±5.2 versus 18±1.7 pg/mL; P<0.005) and increased hepatic and renal thiobarbituric acid reactive substances (1.2±0.09 versus 0.8±0.07 and 1.5±0.1 versus 1.1±0.05 nmol/L per milligram of protein, respectively; P<0.01), suggesting that oxidative stress may be mediating adverse effects of increased human CRP. These findings are consistent with the hypothesis that increased CRP is more than just a marker of inflammation and can directly promote multiple features of the metabolic syndrome.

Folate deficiency is associated with oxidative stress, increased blood pressure, and insulin resistance in spontaneously hypertensive rats.

BACKGROUND The role of folate deficiency and associated hyperhomocysteinemia in the pathogenesis of metabolic syndrome is not fully established. In the current study, we analyzed the role of folate deficiency in pathogenesis of the metabolic syndrome in the spontaneously hypertensive rat (SHR). METHODS Metabolic and hemodynamic traits were assessed in SHR/Ola rats fed either folate-deficient or control diet for 4 weeks starting at the age of 3 months. RESULTS Compared to SHRs fed a folate-replete diet, SHRs fed a folate-deficient diet showed significantly reduced serum folate (104 ± 5 vs. 11 ± 1 nmol/L, P < 0.0005) and urinary folate excretion (4.3 ± 0.6 vs. 1.2 ± 0.1 nmol/16 h, P < 0.0005) together with a near 3-fold increase in plasma total homocysteine concentration (4.5 ± 0.1 vs 13.1 ± 0.7 μmol/L, P < 0.0005), ectopic fat accumulation in liver, and impaired glucose tolerance. Folate deficiency also increased systolic blood pressure by approximately 15 mm Hg (P < 0.01). In addition, the low-folate diet was accompanied by significantly reduced activity of antioxidant enzymes and increased concentrations of lipoperoxidation products in liver, renal cortex, and heart. CONCLUSIONS These findings demonstrate that the SHR model is susceptible to the adverse metabolic and hemodynamic effects of low dietary intake of folate. The results are consistent with the hypothesis that folate deficiency can promote oxidative stress and multiple features of the metabolic syndrome that are associated with increased risk for diabetes and cardiovascular disease.

A New Transgenic Rat Model of Hepatic Steatosis and the Metabolic Syndrome

Fatty liver is extremely common in insulin-resistant patients with either obesity or lipodystrophy and it has been proposed that hepatic steatosis be considered an additional feature of the metabolic syndrome. Although insulin resistance can promote fatty liver, excessive hepatic accumulation of fat can also promote insulin resistance and could contribute to the pathogenesis of the metabolic syndrome. We sought to create a new nonobese rat model with hypertension, hepatic steatosis, and the metabolic syndrome by transgenic overexpression of a sterol-regulatory element-binding protein (SREBP-1a) in the spontaneously hypertensive rat (SHR). SREBPs are transcription factors that activate the expression of multiple genes involved in the hepatic synthesis of cholesterol, triglycerides, and fatty acids. The new transgenic strain of SHR overexpressing a dominant-positive form of human SREBP-1a under control of the phosphoenolpyruvate carboxykinase (PEPCK) promoter exhibited marked hepatic steatosis with major biochemical features of the metabolic syndrome, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia. Both oxidative and nonoxidative skeletal muscle glucose metabolism were significantly impaired in the SHR transgenic strain and glucose tolerance deteriorated as the animals aged. The SHR transgenic strain also exhibited reduced body weight and reduced adipose tissue stores; however, the level of hypertension in the transgenic SHR was similar to that in the nontransgenic SHR control. The transgenic SHR overexpressing SREBP-1a represents a nonobese rat model of fatty liver, disordered glucose and lipid metabolism, and hypertension that may provide new opportunities for studying the pathogenesis and treatment of the metabolic syndrome associated with hepatic steatosis.

Identification of Mutated Srebf1 as a QTL Influencing Risk for Hepatic Steatosis in the Spontaneously Hypertensive Rat

Approximately 30% of patients with hypertension have hepatic steatosis, and it has recently been proposed that fatty liver be considered a feature of the metabolic syndrome. Obesity, diet, and level of physical activity are likely factors modulating risk for hepatic steatosis, however genetic factors could also influence susceptibility or resistance to fatty liver in hypertensive or normotensive subjects. In genetic studies in spontaneously hypertensive rats (SHRs) and Brown Norway (BN) rats, we discovered that a variant form of sterol regulatory element binding transcription factor 1 (Srebf1 gene, SREBP-1 protein) underlies a quantitative trait locus (QTL) influencing hepatic cholesterol levels in response to a high cholesterol diet. Compared with the BN allele of Srebf1, the SHR allele of Srebf1 includes variants in the promoter and coding regions that are linked to hepatic deficiency of SREBP-1 mRNA and protein, reduced expression of the SREBP-1 target gene stearoyl-CoA desaturase 1, reduced promoter activity for SREBP-1c, and relative protection from dietary induced accumulation of liver cholesterol. Genetic correction of reduced SREBP-1 activity by derivation of congenic and transgenic strains of SHR increased hepatic cholesterol levels, thereby confirming Srebf1 as a QTL influencing hepatic lipid metabolism in the rat. The Srebf1 variant regulating hepatic cholesterol did not appear to affect blood pressure. These findings (1) are consistent with the results of association studies indicating that common polymorphisms affecting SREBP-1 may influence cholesterol synthesis in humans and (2) indicate that variation in Srebf1 may influence risk for hepatic steatosis.