Dissecting the role of Folr1 and Folh1 genes in the pathogenesis of metabolic syndrome in spontaneously hypertensive rats.

Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.

Metformin attenuates myocardium dicarbonyl stress induced by chronic hypertriglyceridemia.

Reactive dicarbonyls stimulate production of advanced glycation endproducts, increase oxidative stress and inflammation and contribute to the development of vascular complications. We measured concentrations of dicarbonyls - methylglyoxal (MG), glyoxal (GL) and 3-deoxyglucosone (3-DG) - in the heart and kidney of a model of metabolic syndrome - hereditary hypertriglyceridemic rats (HHTg) and explored its modulation by metformin. Adult HHTg rats were fed a standard diet with or without metformin (300 mg/kg b.w.) and dicarbonyl levels and metabolic parameters were measured. HHTg rats had markedly elevated serum levels of triacylglycerols (p<0.001), FFA (p<0.01) and hepatic triacylglycerols (p<0.001) along with increased concentrations of reactive dicarbonyls in myocardium (MG: p<0.001; GL: p<0.01; 3-DG: p<0.01) and kidney cortex (MG: p<0.01). Metformin treatment significantly reduced reactive dicarbonyls in the myocardium (MG: p<0.05, GL: p<0.05, 3-DG: p<0.01) along with increase of myocardial concentrations of reduced glutathione (p<0.01) and glyoxalase 1 mRNA expression (p<0.05). Metformin did not have any significant effect on dicarbonyls, glutathione or on glyoxalase 1 expression in kidney cortex. Chronically elevated hypertriglyceridemia was associated with increased levels of dicarbonyls in heart and kidney. Beneficial effects of metformin on reactive dicarbonyls and glyoxalase in the heart could contribute to its cardioprotective effects.

Mutant Wars2 gene in spontaneously hypertensive rats impairs brown adipose tissue function and predisposes to visceral obesity.

Brown adipose tissue (BAT) plays an important role in lipid and glucose metabolism in rodents and possibly also in humans. Identification of genes responsible for BAT function would shed light on underlying pathophysiological mechanisms of metabolic disturbances. Recent linkage analysis in the BXH/HXB recombinant inbred (RI) strains, derived from Brown Norway (BN) and spontaneously hypertensive rats (SHR), identified two closely linked quantitative trait loci (QTL) associated with glucose oxidation and glucose incorporation into BAT lipids in the vicinity of Wars2 (tryptophanyl tRNA synthetase 2 (mitochondrial)) gene on chromosome 2. The SHR harbors L53F WARS2 protein variant that was associated with reduced angiogenesis and Wars2 thus represents a prominent positional candidate gene. In the current study, we validated this candidate as a quantitative trait gene (QTG) using transgenic rescue experiment. SHR-Wars2 transgenic rats with wild type Wars2 gene when compared to SHR, showed more efficient mitochondrial proteosynthesis and increased mitochondrial respiration, which was associated with increased glucose oxidation and incorporation into BAT lipids, and with reduced weight of visceral fat. Correlation analyses in RI strains showed that increased activity of BAT was associated with amelioration of insulin resistance in muscle and white adipose tissue. In summary, these results demonstrate important role of Wars2 gene in regulating BAT function and consequently lipid and glucose metabolism.

Visfatin is actively secreted in vitro from U-937 macrophages, but only passively released from 3T3-L1 adipocytes and HepG2 hepatocytes.

Visfatin is a multi-functional molecule that can act intracellularly and extracellularly as an adipokine, cytokine and enzyme. One of the main questions concerning visfatin is the mechanism of its secretion; whether, how and from which cells visfatin is released. The objective of this in vitro study was to observe the active secretion of visfatin from 3T3-L1 preadipocytes and adipocytes, HepG2 hepatocytes, U-937, THP-1 and HL-60 monocytes and macrophages. The amount of visfatin in media and cell lysate was always related to the intracellular enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to exclude the passive release of visfatin. Visfatin was not found in media of 3T3-L1 preadipocytes. In media of 3T3-L1 adipocytes and HepG2 hepatocytes, the ratio of visfatin to the amount of GAPDH was identical to cell lysates. Hence, it is likely that these cells do not actively secrete visfatin in a significant manner. However, we found that significant producers of visfatin are differentiated macrophages and that the amount of secreted visfatin depends on used cell line and it is affected by the mode of differentiation. Results show that 3T3-L1 adipocytes and HepG2 hepatocytes released visfatin only passively during the cell death. U-937 macrophages secrete visfatin in the greatest level from all of the tested cell lines.

Hepatotoxic effects of fenofibrate in spontaneously hypertensive rats expressing human C-reactive protein.

Dyslipidemia and inflammation play an important role in the pathogenesis of cardiovascular and liver disease. Fenofibrate has a well-known efficacy to reduce cholesterol and triglycerides. Combination with statins can ameliorate hypolipidemic and anti-inflammatory effects of fibrates. In the current study, we tested the anti-inflammatory and metabolic effects of fenofibrate alone and in combination with rosuvastatin in a model of inflammation and metabolic syndrome, using spontaneously hypertensive rats expressing the human C-reactive protein transgene (SHR-CRP transgenic rats). SHR-CRP rats treated with fenofibrate alone (100 mg/kg body weight) or in combination with rosuvastatin (20 mg/kg body weight) vs. SHR-CRP untreated controls showed increased levels of proinflammatory marker IL6, increased concentrations of ALT, AST and ALP, increased oxidative stress in the liver and necrotic changes of the liver. In addition, SHR-CRP rats treated with fenofibrate, or with fenofibrate combined with rosuvastatin vs. untreated controls, exhibited increased serum triglycerides and reduced HDL cholesterol, as well as reduced hepatic triglyceride, cholesterol and glycogen concentrations. These findings suggest that in the presence of high levels of human CRP, fenofibrate can induce liver damage even in combination with rosuvastatin. Accordingly, these results caution against the possible hepatotoxic effects of fenofibrate in patients with high levels of CRP.

Gender-related effects on substrate utilization and metabolic adaptation in hairless spontaneously hypertensive rat.

Cold exposure of rats leads to ameliorated glucose and triglyceride utilization with females displaying better adaptation to a cold environment. In the current study, we used hairless rats as a model of increased thermogenesis and analyzed gender-related effects on parameters of lipid and glucose metabolism in the spontaneously hypertensive (SHR) rats. Specifically, we compared hairless coisogenic SHR-Dsg4 males and females harboring mutant Dsg4 (desmoglein 4) gene versus their SHR wild type controls. Two way ANOVA showed significant Dsg4 genotype (hairless or wild type) x gender interaction effects on palmitate oxidation in brown adipose tissue (BAT), glucose incorporation into BAT determined by microPET, and glucose oxidation in skeletal muscles. In addition, we observed significant interaction effects on sensitivity of muscle tissue to insulin action when Dsg4 genotype affected these metabolic traits in males, but had little or no effects in females. Both wild type and hairless females and hairless males showed increased glucose incorporation and palmitate oxidation in BAT and higher tissue insulin sensitivity when compared to wild type males. These findings provide evidence for gender-related differences in metabolic adaptation required for increased thermogenesis. They are consistent with the hypothesis that increased glucose and palmitate utilization in BAT and muscle is associated with higher sensitivity of adipose and muscle tissues to insulin action.

Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

Autocrine effects of visfatin on hepatocyte sensitivity to insulin action.

Visfatin was originally described as an adipokine with insulin mimetic effects. Recently, it was found that visfatin is identical with the Nampt (nicotinamide phosphoribosyltransferase) gene that codes for an intra- and extracellular NAD biosynthetic enzyme and is predominantly expressed outside the adipose tissue. In the current study, we found strong protein and mRNA expression of visfatin in rat heart, liver, kidney, and muscle, while the expression of visfatin in visceral fat was significantly lower and undetectable in subcutaneous fat. The insulin-mimetic effects of visfatin (extracellular form of Nampt or eNampt) are controversial and even less is known about autocrine effects of visfatin (intracellular form of Nampt or iNampt). Since liver plays a major role in glucose metabolism, we studied visfatin effects on insulin-stimulated cellular glucose uptake in Fao rat hepatocytes using RNA interference (RNAi). RNAi-mediated downregulation of visfatin expression in Fao cells was associated with significantly reduced NAD biosynthesis (0.3+/-0.01 vs. 0.5+/-0.01 mmol/h/g, P<0.05) and with significantly decreased incremental glucose uptake after stimulation with insulin when compared to controls with normal expression of visfatin (0.6+/-0.2 vs. 2.2+/-0.5 nnmol/g/2 h, P=0.02). These results provide evidence that visfatin exhibits important autocrine effects on sensitivity of liver cells to insulin action possibly through its effects on NAD biosynthesis.

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes.

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.