Human apolipoprotein B transgenic SHR/NDmcr-cp rats show exacerbated kidney dysfunction

Nephropathy frequently co-occurs with metabolic syndrome in humans. Metabolic syndrome is a cluster of metabolic diseases including obesity, diabetes, hypertension, and dyslipidemia, and some previous studies revealed that dyslipidemia contributes to the progression of kidney dysfunction. To establish a new nephropathy model with metabolic syndrome, we produced human apolipoprotein B (apoB) transgenic (Tg.) SHR/NDmcr-cp (SHR-cp/cp) rats, in which dyslipidemia is exacerbated more than in an established metabolic syndrome model, SHR-cp/cp rats. Human apoB Tg. SHR-cp/cp rats showed obesity, hyperinsulinemia, hypertension, and severe hyperlipidemia. They also exhibited exacerbated early-onset proteinuria, accompanied by increased kidney injury and increased oxidative and inflammatory markers. Histological analyses revealed the characteristic features of human apoB Tg. SHR-cp/cp rats including prominent glomerulosclerosis with lipid accumulation. Our newly established human apoB Tg. SHR-cp/cp rat could be a useful model for the nephropathy in metabolic syndrome and for understanding the interaction between dyslipidemia and renal dysfunction in metabolic syndrome.     

Transgenic expression of CD36 in the spontaneously hypertensive rat is associated with amelioration of metabolic disturbances but has no effect on hypertension

Spontaneously hypertensive rats (SHR/NIH strain) harbor a deletion variant in the Cd36 fatty acid transporter and display defective fatty acid metabolism, insulin resistance and hypertension. Transgenic rescue of Cd36 in SHR ameliorates insulin resistance and improves dyslipidemia. However, the role of Cd36 in blood pressure regulation remains controversial due to inconsistent blood pressure effects that were observed with transgenic expression of Cd36 on the SHR background. In the current studies, we developed two new SHR transgenic lines, which express wild type Cd36 under the control of the universal Ef-1 alpha promoter, and examined the effects of transgenic expression of wild type Cd36 on selected metabolic and cardiovascular phenotypes. Transgenic expression of Cd36 in the new lines was associated with significantly decreased serum fatty acids, amelioration of insulin resistance and glucose intolerance but failed to induce any consistent changes in blood pressure as measured by radiotelemetry. The current findings confirm the genetic association of defective Cd36 with disordered insulin action and fatty acid metabolism in the SHR/NIH strain and suggest that Cd36 is linked to other gene(s) on rat chromosome 4 that regulate blood pressure.     

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 gamma 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-type Cd36. In congenic and transgenic SHR with wild-type Cd36, 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 mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Evaluation of insulin resistance linkage to rat chromosome 4 in SHR of a Japanese colony

The spontaneously hypertensive rat (SHR) is a model of human insulin resistance syndrome. Quantitative trait loci for cellular defects in glucose and fatty acid metabolism have been mapped to an overlapping region of rat chromosome (RNO) RNO4 in SHR of the National Institute of Health colony, where a deletion in the Cd36 gene has been implicated as the causative mutation of insulin resistance. The present study has examined the potential presence of RNO4 linkage to a series of metabolic phenotypes in F(2) progeny derived from SHR of a Japanese colony (SHR/Izm) without the Cd36 mutation. Our data demonstrate that 'major' insulin resistance gene(s) are unlikely to exist on RNO4 in SHR/Izm and in vitro phenotypes measured in isolated adipocytes do not cosegregate in the F(2) population studied. Thus, it seems to be difficult to explain the underlying genetic mechanisms of insulin resistance by a single major gene on RNO4.     

Role of FAT/CD36 in novel PKC isoform activation in heart of spontaneously hypertensive rats

Disruption to the sensitive balance of long-chain fatty acids and glucose in the heart could cause cardiovascular diseases. Searching for a possible role of novel protein kinase C (nPKC) in heart with disrupted energy balance, we compared the insulin-resistant spontaneously hypertensive rats (SHR), which carry a nonfunctional variant of the fatty acid transporter FAT/CD36, with the less insulin-resistant congenic strain SHR-4 that is genetically identical except for a segment on chromosome 4 including a wild-type gene for a functional FAT/CD36. We analyzed expression of the nPKC-δ and -ε isoforms plus triacylglycerols (TAG) content in the myocardium of both FAT/CD36 strains and after a high sucrose diet (HSD). Two weeks before killing, males of both strains were randomly divided into two groups and fed either a standard laboratory chow or an HSD. PKC was determined by Western blotting in particulate and cytosolic fractions from left ventricles. The SHR-4 rats exhibited lower serum levels of insulin and free fatty acids than did SHR rats and higher amounts of PKC-ε in the heart particulate fraction. HSD caused accumulation of heart TAG in SHR but not in SHR-4. HSD increased PKC-δ and decreased PKC-ε expression in particulate fraction from left ventricles of SHR-4 while having no effects in SHR. These results demonstrate that reduced insulin resistance in SHR-4 rats with wild-type FAT/CD36 is associated with the insulin signaling pathway involving nPKCs.     

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 (IC₅₀ 4 μM SSO). The results presented here show that SSO is a potent and irreversible inhibitor of mitochondrial respiratory chain.     

Chronic hydrogen-rich saline treatment reduces oxidative stress and attenuates left ventricular hypertrophy in spontaneous hypertensive rats

In hypertensive animals and patients, oxidative stress represents the primary risk factor for progression of left ventricular hypertrophy. Recently, it has been demonstrated that hydrogen, as a novel antioxidant, can selectively reduce hydroxyl radicals and peroxynitrite anion to exert therapeutic antioxidant activity. In the current study, we explored the effect of chronic treatment with hydrogen-rich saline (HRS) on left ventricular hypertrophy in spontaneously hypertensive rats (SHR). The 8-week-old male SHR and age-matched Wistar-Kyoto rats (WKY) were randomized into HRS-treated (6 ml/kg/day for 3 months, i.p.) and vehicle-treated groups. HRS treatment had no significant effect on blood pressure, but it effectively attenuated left ventricular hypertrophy in SHR. HRS treatment abated oxidative stress, restored the activity of antioxidant enzymes including GPx, GST, catalase, and SOD, suppressed NADPH oxidase activity and downregulated Nox2 and Nox4 expression in left ventricles of SHR. HRS treatment suppressed pro-inflammatory cytokines including IL-1β, IL-6, TNF-α, and MCP-1, and inhibited NF-κB activation through preventing IκBα degradation in left ventricles of SHR. HRS treatment preserved mitochondrial function through restoring electron transport chain enzyme activity, repressing ROS formation, and enhancing ATP production in left ventricles of SHR. Moreover, HRS treatment suppressed ACE expression and locally reduced angiotensin II generation in left ventricles of SHR. In conclusion, HRS treatment attenuates left ventricular hypertrophy through abating oxidative stress, suppressing inflammatory process, preserving mitochondrial function, in which suppression of HRS on angiotensin II in left ventricles locally might be involved.     

Does Cd36 gene play a key role in disturbed glucose and fatty acid metabolism in Prague hypertensive hypertriglyceridemic rats?

Close links between hypertension, hypertriglyceridemia, insulin resistance and other symptoms of metabolic syndrome was demonstrated in humans and experimental animals. Quantitative trait loci for defects in glucose and fatty acid metabolism, hypertriglyceridemia and hypertension were mapped in spontaneously hypertensive rats (SHR) on chromosome 4 and defective Cd36 gene was identified in this region. Here we investigated the polymorphism of Cd36 gene in Prague hereditary hypertriglyceridemic (HTG) rats, which represent another model of genetic hypertension and metabolic syndrome. These animals were compared with NIH-derived SHR and two different normotensive control strains (WKY, LEW). In spite of the fact that HTG and SHR rats had similar metabolic disturbances, genotype analysis of PCR products has shown that Cd36 mutation was not present in HTG rats. In conclusion, we have revealed that defective Cd36 is probably a candidate gene for disorded fatty-acid metabolism, glucose intolerance and insulin resistance in NIH-derived SHR, but other genes might play a role in pathogenesis of metabolic syndrome in Prague hereditary hypertriglyceridemic rats. This is in accordance with the absence of defective Cd36 gene in original SHR from Japan.     

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.     

Promotion of artemisinin content in <Emphasis Type="Italic">Artemisia annua</Emphasis> by overexpression of multiple artemisinin biosynthetic pathway genes

Artemisinin, isolated from an annual herbaceous plant Artemisia annua L., is an effective antimalarial compound. However, artemisinin is accumulated in small amounts (0.01–0.1% leaf dry weight) in A. annua, resulting in constant high artemisinin price. Although metabolic engineering of partial artemisinin metabolic pathway in yeast achieved great success, artemisinin from A. annua is still the important business resource. Here, we report on the generation of transgenic plants with simultaneously overexpressing four artemisinin biosynthetic pathway genes, amorpha-4,11-diene synthase gene (ADS), amorpha-4,11-diene 12-monooxygenase gene (CYP71AV1), cytochrome P450 reductase gene (CPR), and aldehyde dehydrogenase 1 gene (ALDH1) via Agrobacterium-mediated transformation. The qRT-PCR analysis demonstrated that the introduced four genes of the transgenic lines were all highly expressed. Through high-performance liquid chromatography analysis, the artemisinin contents were increased markedly in transformants, with the highest being 3.4-fold higher compared with non-converter. These results indicate that overexpression of multiple artemisinin biosynthetic pathway genes is a promising approach to improve artemisinin yield in A. annua.     

Chronic hypoxia-induced alterations in mitochondrial energy metabolism are not reversible in rat heart ventricles

Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.     

Changes in cardiac substrate transporters and metabolic proteins mirror the metabolic shift in patients with aortic stenosis

In the hypertrophied human heart, fatty acid metabolism is decreased and glucose utilisation is increased. We hypothesized that the sarcolemmal and mitochondrial proteins involved in these key metabolic pathways would mirror these changes, providing a mechanism to account for the modified metabolic flux measured in the human heart. Echocardiography was performed to assess in vivo hypertrophy and aortic valve impairment in patients with aortic stenosis (n = 18). Cardiac biopsies were obtained during valve replacement surgery, and used for western blotting to measure metabolic protein levels. Protein levels of the predominant fatty acid transporter, fatty acid translocase (FAT/CD36) correlated negatively with levels of the glucose transporters, GLUT1 and GLUT4. The decrease in FAT/CD36 was accompanied by decreases in the fatty acid binding proteins, FABPpm and H-FABP, the β-oxidation protein medium chain acyl-coenzyme A dehydrogenase, the Krebs cycle protein α-ketoglutarate dehydrogenase and the oxidative phosphorylation protein ATP synthase. FAT/CD36 and complex I of the electron transport chain were downregulated, whereas the glucose transporter GLUT4 was upregulated with increasing left ventricular mass index, a measure of cardiac hypertrophy. In conclusion, coordinated downregulation of sequential steps involved in fatty acid and oxidative metabolism occur in the human heart, accompanied by upregulation of the glucose transporters. The profile of the substrate transporters and metabolic proteins mirror the metabolic shift from fatty acid to glucose utilisation that occurs in vivo in the human heart.     

Putative candidate genes for blood pressure control in the SHR.BN-RNO8 congenic substrains

The SHR-Lx congenic strain carrying a differential segment of chromosome 8 of BN and PD origin was recently shown to exhibit a significant decrease in blood pressure as compared to the SHR strain. There were two positional candidate genes for blood pressure control mapped to the differential segment: the rat kidney epithelial potassium channel gene (Kcnj1) and brain dopamine receptor 2 gene (Drd2). Bot these genes were separated into SHR.BN-RNO8 congenic substrains. In this communication, we are presenting the assignment of two further putative candidate genes, which might be involved in blood pressure control to the BN/PD differential segment of the SHR-Lx congenic strain. These are: the gene coding for smooth muscle cell specific protein 22 (Sm22) defined by the D8Mcw1 marker and neuronal nicotinic acetylcholine receptor gene cluster, defined by the D8Bord1 marker. Moreover, the glutamate receptor gene Grik4 which also maps to the differential segment of the SHR-Lx should be taken into account. The genetic separation of all these putative candidate genes of blood pressure control is being performed by recombinations and subsequent selection using (SHR×SHR-Lx) intercross population.     

Transmural distribution of glucose metabolizing enzymes across the left and the right ventricle heart walls in three different mammalian species

The transmural distribution of five glucose metabolizing enzymes (hexokinase; glucose-6-phosphate dehydrogenase; phosphofructokinase; aldolase; and lactate dehydrogenase) were explored in the left and in the right ventricle wall of rat, ox and pig hearts. The levels of most of these enzyme activities were different in the different animal species and (within the same species) in the two ventricles. Most of these enzyme activities were found to be non-uniformly distributed across the left (but not across the right) ventricle wall. Differences in the transmural distribution of enzyme activities were detected among the three examined mammalian species.