Single nucleotide polymorphism (-468 Gly to A) at the promoter region of SREBP-1c associates with genetic defect of fructose-induced hepatic lipogenesis [corrected

To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity.     

Antibodies to catecholamines and serotonin during alcoholic intoxication in animals with different predispositions to the development of experimental alcoholism

Experiments on C57Bl/6, CBA and DBA/2 mice characterized by different preferences for ethanol have shown that during chronic administration of alcohol to animals with natural ethanol motivation (strain C57Bl/6) the level of antibodies to catecholamines and serotonin was increased on the 3rd month of ethanol intoxication, with the voluntary alcohol consumption in mice decreased by this time. On the contrary in mice rejecting alcohol (strains DBA/2, CBA) no antibodies to catecholamines and serotonin have been found.     

Fructose diet treatment in mice induces fundamental disturbance of cardiomyocyte Ca2+ handling and myofilament responsiveness

High fructose intake has been linked to insulin resistance and cardiac pathology. Dietary fructose-induced myocardial signaling and morphological alterations have been described, but whether cardiomyocyte function is influenced by chronic high fructose intake is yet to be elucidated. The goal of this study was to evaluate the cardiomyocyte excitation-contraction coupling effects of high dietary fructose and determine the capacity for murine cardiomyocyte fructose transport. Male C57Bl/6J mice were fed a high fructose diet for 12 wk. Fructose- and control-fed mouse cardiomyocytes were isolated and loaded with the fura 2 Ca(2+) fluorescent dye for analysis of twitch and Ca(2+) transient characteristics (4 Hz stimulation, 37°C, 2 mM Ca(2+)). Myocardial Ca(2+)-handling protein expression was determined by Western blot. Gene expression of the fructose-specific transporter, GLUT5, in adult mouse cardiomyocytes was detected by real-time and conventional RT-PCR techniques. Diastolic Ca(2+) and Ca(2+) transient amplitude were decreased in isolated cardiomyocytes from fructose-fed mice relative to control (16 and 42%, respectively), coincident with an increase in the time constant of Ca(2+) transient decay (24%). Dietary fructose increased the myofilament response to Ca(2+) (as evidenced by a left shift in the shortening-Ca(2+) phase loop). Protein expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phosphorylated (P) phospholamban (Ser(16)), and P-phospholamban (Thr(17)) was reduced, and protein phosphatase 2A expression increased, in fructose-fed mouse hearts. Hypertension and cardiac hypertrophy were not evident. These findings demonstrate that fructose diet-associated myocardial insulin resistance induces profound disturbance of cardiomyocyte Ca(2+) handling and responsiveness in the absence of altered systemic loading conditions.     

Glucose Transporter 8 (GLUT8) Mediates Fructose-induced de Novo Lipogenesis and Macrosteatosis

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the world, and it is thought to be the hepatic manifestation of the metabolic syndrome. Excess dietary fructose causes both metabolic syndrome and NAFLD in rodents and humans, but the pathogenic mechanisms of fructose-induced metabolic syndrome and NAFLD are poorly understood. GLUT8 (Slc2A8) is a facilitative glucose and fructose transporter that is highly expressed in liver, heart, and other oxidative tissues. We previously demonstrated that female mice lacking GLUT8 exhibit impaired first-pass hepatic fructose metabolism, suggesting that fructose transport into the hepatocyte, the primary site of fructose metabolism, is in part mediated by GLUT8. Here, we tested the hypothesis that GLUT8 is required for hepatocyte fructose uptake and for the development of fructose-induced NAFLD. We demonstrate that GLUT8 is a cell surface-localized transporter and that GLUT8 overexpression or GLUT8 shRNA-mediated gene silencing significantly induces and blocks radiolabeled fructose uptake in cultured hepatocytes. We further show diminished fructose uptake and de novo lipogenesis in fructose-challenged GLUT8-deficient hepatocytes. Finally, livers from long term high-fructose diet-fed GLUT8-deficient mice exhibited attenuated fructose-induced hepatic triglyceride and cholesterol accumulation without changes in hepatocyte insulin-stimulated Akt phosphorylation. GLUT8 is thus essential for hepatocyte fructose transport and fructose-induced macrosteatosis. Fructose delivery across the hepatocyte membrane is thus a proximal, modifiable disease mechanism that may be exploited to prevent NAFLD.     

Protective effect of bile acids on the onset of fructose-induced hepatic steatosis in mice

Fructose intake is being discussed as a key dietary factor in the development of nonalcoholic fatty liver disease (NAFLD). Bile acids have been shown to modulate energy metabolism. We tested the effects of bile acids on fructose-induced hepatic steatosis. In C57BL/6J mice treated with a combination of chenodeoxycholic acid and cholic acid (100 mg/kg body weight each) while drinking water or a 30% fructose solution for eight weeks and appropriate controls, markers of hepatic steatosis, portal endotoxin levels, and markers of hepatic lipogenesis were determined. In mice concomitantly treated with bile acids, the onset of fructose-induced hepatic steatosis was markedly attenuated compared to mice only fed fructose. The protective effects of the bile acid treatment were associated with a downregulation of tumor necrosis factor (TNF)α, sterol regulatory element-binding protein (SREBP)1, FAS mRNA expression, and lipid peroxidation in the liver, whereas hepatic farnesoid X receptor (FXR) or short heterodimer partner (SHP) protein concentration did not differ between groups fed fructose. Rather, bile acid treatment normalized occludin protein concentration in the duodenum, portal endotoxin levels, and markers of Kupffer cell activation to the level of water controls. Taken together, these data suggest that bile acids prevent fructose-induced hepatic steatosis in mice through mechanisms involving protection against the fructose-induced translocation of intestinal bacterial endotoxin.     

Pterostilbene and allopurinol reduce fructose-induced podocyte oxidative stress and inflammation via microRNA-377

High dietary fructose is an important causative factor in the development of metabolic syndrome-associated glomerular podocyte oxidative stress and injury. Here, we identified microRNA-377 (miR-377) as a biomarker of oxidative stress in renal cortex of fructose-fed rats, which correlated with podocyte injury and albuminuria in metabolic syndrome. Fructose feeding increased miR-377 expression, decreased superoxide dismutase (SOD) expression and activity, and caused O₂⁻ and H₂O₂ overproduction in kidney cortex or glomeruli of rats. This reactive oxygen species induction increased p38 MAPK phosphorylation and thioredoxin-interacting protein (TXNIP) expression and activated the NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome to produce interleukin-1β in kidney glomeruli of fructose-fed rats. These pathological processes were further evaluated in cultured differentiated podocytes exposed to 5 mM fructose, or transfected with miR-377 mimic/inhibitor and TXNIP siRNA, or co-incubated with p38 MAPK inhibitor, demonstrating that miR-377 overexpression activates the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway to promote oxidative stress and inflammation in fructose-induced podocyte injury. Antioxidants pterostilbene and allopurinol were found to ameliorate fructose-induced hyperuricemia, podocyte injury, and albuminuria in rats. More importantly, pterostilbene and allopurinol inhibited podocyte miR-377 overexpression to increase SOD1 and SOD2 levels and suppress the O₂⁻/p38 MAPK/TXNIP/NLRP3 inflammasome pathway activation in vivo and in vitro, consistent with the reduction of oxidative stress and inflammation. These findings suggest that miR-377 plays an important role in glomerular podocyte oxidative stress, inflammation, and injury driven by high fructose. Inhibition of miR-377 by antioxidants may be a promising therapeutic strategy for the prevention of metabolic syndrome-associated glomerular podocyte injury.     

Correlation between the steady state and turnover of gamma-aminobutyric acid during brain maturation in mice

In 2 inbred strains of mice (C57Bl/6J, DBA/2J) in 15 areas, the in vivo GABA turnover rates are significantly correlated with the GABA steady-state levels in 21 day-old mice. In 3 month-old mice the correlation stands only in some areas, the same ones in the 2 strains: olfactory bulbs, frontal cortex, septum, amygdala, hypothalamus, hippocampus, cerebellum. Moreover, the turnover rates decrease sharply with age.     

Insufficient peroxiredoxin-2 expression in uterine NK cells obtained from a murine model of abortion

The CBA/J × DBA/2 mouse mating combination is prone to spontaneous embryo loss, in contrast to the MHC-identical CBA/J × BALB/c mating combination, which yields successful pregnancies. The underlying mechanisms for these observations are unclear. In this study, multi-vision immunohistochemical staining (IHC), flow cytometry and Western blot analysis were used to detect peroxiredoxin-2 (PRX-2) expression in the uterine natural killer (uNK) cells from CBA/J × DBA/2 and CBA/J × BALB/c mice. In IHC analysis, co-localization of PRX-2 and lectin from Dolichos biflorus agglutinin (DBA-lectin) was confirmed and the frequency of PRX-2(+) DBA-lectin(+) cells was significantly lower in CBA/J × DBA/2 than CBA/J × BALB/c. In flow cytometry and Western blotting, PRX-2 was found expressed at a significantly lower level in CBA/J × DBA/2 mice. PRX-2 inhibition with a neutralizing antibody significantly decreased PRX-2 expression, increased the cytotoxicity of uNK cells, and increased the percentage of embryo loss in CBA/J × DBA/2J mice. Our data suggest that PRX-2 may be involved in the modulation of maternal-fetal tolerance and that insufficient expression of this protein may correlate with increased embryo loss in CBA/J × DBA/2J mice.     

Strain differences in basal and post-citalopram extracellular 5-HT in the mouse medial prefrontal cortex and dorsal hippocampus: relation with tryptophan hydroxylase-2 activity

We used the microdialysis technique to compare basal extracellular serotonin (5-HT) and the response to citalopram in different strains of mice with functionally different allelic forms of tryptophan hydroxylase-2 (TPH-2), the rate-limiting enzyme in brain 5-HT synthesis. DBA/2J, DBA/2N and BALB/c mice carrying the 1473G allele of TPH-2 had less dialysate 5-HT in the medial prefrontal cortex and dorsal hippocampus (DH) (20-40% reduction) than C57BL/6J and C57BL/6N mice carrying the 1473C allele. Extracellular 5-HT estimated by the zero-net flux method confirmed the result of conventional microdialysis. Citalopram, 1.25, 5 and 20 mg/kg, dose-dependently raised extracellular 5-HT in the medial prefrontal cortex of C57BL/6J mice, with maximum effect at 5 mg/kg, but had significantly less effect in DBA/2J and BALB/c mice and in the DH of DBA/2J mice. A tryptophan (TRP) load enhanced basal extracellular 5-HT in the medial prefrontal cortex of DBA/2J mice but did not affect citalopram's ability to raise cortical and hippocampal extracellular 5-HT. The impairment of 5-HT synthesis quite likely accounts for the reduction of basal 5-HT and the citalopram-induced rise in mice carrying the mutated enzyme. These findings might explain why DBA/2 and BALB/c mice do not respond to citalopram in the forced swimming test. Although TRP could be a useful strategy to improve the antidepressant effect of citalopram (Cervo et al. 2005), particularly in subjects with low 5-HT synthesis, the contribution of serotonergic and non-serotonergic mechanisms to TRP's effect remains to be elucidated.     

Lactobacillus casei Shirota protects from fructose-induced liver steatosis: A mouse model

To test the hypothesis that Lactobacillus casei Shirota (Lcs) protects against the onset of non-alcoholic fatty liver disease (NAFLD) in a mouse model of fructose-induced steatosis, C57BL/6J mice were either fed tap water or 30% fructose solution +/- Lcs for 8 weeks. Chronic consumption of 30% fructose solution led to a significant increase in hepatic steatosis as well as plasma alanine-aminotransferase (ALT) levels, which was attenuated by treatment with Lcs. Protein levels of the tight junction protein occludin were found to be markedly lower in both fructose treated groups in the duodenum, whereas microbiota composition in this part of the intestine was not affected. Lcs treatment markedly attenuated the activation of the Toll-like receptor (TLR) 4 signalling cascade found in the livers of mice only treated with fructose. Moreover, in livers of fructose fed mice treated with Lcs peroxisome proliferator-activated receptor (PPAR)-γ activity was markedly higher than in mice only fed fructose. Taken together, the results of the present study suggest that the dietary intake of Lcs protects against the onset of fructose-induced NAFLD through mechanisms involving an attenuation of the TLR-4-signalling cascade in the liver.     

Identification of two forms of an endogenous murine retroviral env gene linked to the Rmcf locus.

The Rmcf gene restricts the replication of recombinant murine mink cell focus-inducing (MCF) viruses in cell cultures derived from mice carrying the resistance allele (Rmcfr) and may play a role in resistance to retrovirus-induced leukemias in vivo. We have characterized the endogenous gp70 expressed by Rmcfr and Rmcfs mice with a panel of type-specific monoclonal antibodies which discriminate xenotropic and MCF gp70. Embryo and tail skin cultures derived from Rmcfr mice (DBA/2 and CBA/N) expressed gp70 bearing a determinant unique to MCF viruses, whereas cultures from Rmcfs mice expressed either no detectable gp70 (NFS/N and IRW) or a gp70 serologically related to a subgroup of xenotropic viruses (C57BL/6, CBA/J, and A/WySn). Studies of progeny embryos derived from a (C57BL/6 X DBA/2) X C57BL/6 backcross established that the Rmcf resistance allele was linked to the expression of the MCF gp70 and that the gene encoding the xenotropic gp70 expressed by C57BL/6 Rmcfs mice was allelic with the MCF gp70 from Rmcfr mice. These data indicate that the Rmcf locus contains an endogenous gp70 gene having two allelic forms, one of which inhibits exogenous MCF infection in vitro by a mechanism of viral interference.     

Experimental diabetes in mice infected with Coxsackie viruses

The influence of Coxsackie B4 and AI3 viruses on the pancreas of mice (resistant and susceptible to diabetes) was studied. Glucose intolerance and changes in the synthesis of immunoreactive insulin were detected in all the treated groups of animals. Biochemical changes were more prominent in male DBA/2 mice, infected with Coxsackie B4 virus, in FI (CBA X C57Bl/6) hybrids and in female DBA/2 mice infected with Coxsackie AI3 virus and alloxan.     

Differential expression and regulation of myristoylated alanine-rich C kinase substrate (MARCKS) in the hippocampus of C57/BL6J and DBA/2J mice

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in brain that binds the inner surface of the plasma membrane, calmodulin, and cross-links filamentous actin, all in a PKC phosphorylation-reversible manner. MARCKS has been implicated in hippocampal-dependent learning and long-term potentiation (LTP). Previous studies have shown DBA/2 mice to exhibit poor spatial/contextual learning, impaired hippocampal LTP, and hippocampal mossy fiber hypoplasia, as well as reduced hippocampal PKC activity and expression relative to C57BL/6 mice. In the present study, we assessed the expression (mRNA and protein) and subcellular distribution (membrane and cytolsol) of MARCKS in the hippocampus and frontal cortex of C57BL/6 and DBA/2 mice using quantitative western blotting. In the hippocampus, total MARCKS mRNA and protein levels in C57BL/6J mice were significantly lower ( approximately 45%) compared with DBA/2J mice, and MARCKS protein was observed predominantly in the cytosolic fraction. MARCKS expression in frontal cortex did not differ significantly between strains. To examine the dynamic regulation of MARCKS subcellular distribution, mice from each strain were subjected to 60 min restraint stress and MARCKS subcellular distribution was determined 24 h later. Restraint stress resulted in a significant reduction in membrane MARCKS expression in C57BL/6J hippocampus but not in the DBA/2J hippocampus despite similar stress-induced increases in serum corticosterone. Restraint stress did not affect cytosolic or total MARCKS levels in either strain. Similarly, restraint stress (30 min) in rats also induced a significant reduction in membrane MARCKS, but not total or cytosolic MARCKS, in the hippocampus but not in frontal cortex. In rats, chronic lithium treatment prior to stress exposure reduced hippocampal MARCKS expression but did not affect the stress-induced reduction in membrane MARCKS. Collectively these data demonstrate higher resting levels of MARCKS in the hippocampus of DBA/2J mice compared to C57BL/6J mice, and that acute stress leads to a long-term reduction in membrane MARCKS expression in C57BL/6J mice and rats but not in DBA/2J mice. These strain differences in hippocampal MARCKS expression and subcellular translocation following stress may contribute to the differences in behaviors requiring hippocampal plasticity observed between these strains.     

Extinction of passive avoidance response in various mouse strains

Dependence of the passive avoidance extinction dynamics on a mouse strain was shown. Mice C57BL/6J and AKR/J extinguished more quickly relative to DBA/2J, CBA/Lac and BALB/c, and this extinction was stable. Individual instability of extinction was characteristic of C3H/HeJ mice. Extinction of the passive avoidance in mice CBA/Lac and BALB/c was slower: with a delay in the beginning and prolonged retention of memory trace of the shock exposure. In DBA/2J mice, the extinction was impaired. These data suggest that DBA/2J, CBA/Lac and BALB/c mice constitute groups of risk with high predisposition to impairment of extinction of memory of aversive events, which is thought to be a symptom of a depressive-like state.     

Decline in male mouse pheromone with age

An age-related decline in urinary-borne pheromone was found in male C57BL/6J mice aged from 2 to 30 months. Pheromone activity, estimated by bioassay, declined sharply after about 10 months of age. Two other strains of mice tested (DBA/2J and CBA/HT6J) also appeared to show an age-related decline in pheromone activity. Within each strain, however, pheromone activity was consistently similar to or higher than that of the C57BL/6J male mice. The DBA/2J and BALB/cWt strains appeared to be high pheromone producers, and the C57BL/6J and CBA/HT6J strains, low producers. This report is the first demonstration of a decline with age in male mouse pheromone activity. This decline appears to be synchronized with the well-defined loss of reproductive function in female mice.     

The characteristics of the manifestation of hereditarily induced anxiety in male C57Bl/6J and CBA/Lac mice

Behavior of male mice of C57Bl/6J and CBA/Lac strains was tested in the elevated plus-maze and open field in order to estimate state anxiety in novel conditions. The cube and partition tests were used to reveal trait anxiety in the familiar conditions of the home cage. It is concluded that genetically defined state anxiety is more pronounced in CBA/Lac mice and trait anxiety in C57Bl/6J strain.     

Vascular extracellular adenosine metabolism in mice correlates with susceptibility to atherosclerosis

Abstract

Animal models are widely used in atherosclerosis research. The most useful, economic and valid is mouse genetic model of this pathology. Purinergic signaling is an important mechanism regulating processes involved in the vascular inflammation and atherosclerosis. The aim of this study was to measure vascular activities of nucleotide and adenosine-degrading ecto-enzymes in different strains of mice and to compare them to atherosclerotic susceptibility.

The vascular extracellular nucleotide catabolism pathway was analyzed in 6-month-old male genetically unmodified mouse strains: FVB/NJ, DBA/2J, BALB/c, C57Bl/6J and mouse knock-outs on C57Bl/6J background for LDLR (LDLR-/-) and for ApoE and LDLR (ApoE-/-LDLR-/-). LDLR-/- mice were a model of moderate hypercholesterolemia, while ApoE-/-LDLR-/- mice, a model of severe hypercholesterolemia with advanced atherosclerosis.

FVB/NJ, DBA/2J and BALB/c mice showed high rates of vascular extracellular AMP hydrolysis and low activity of adenosine deamination. In turn, all mice with the C57Bl/6J background expressed diminished activity of vascular AMP hydrolysis. Mice with genetically-induced hyperlipidemia and atherosclerosis on the C57Bl/6J background revealed increased ecto-adenosine deaminase activity.

Mouse strains that were resistant to atherosclerosis (FVB/NJ, DBA/2J, BALB/c) exhibited a protective extracellular vascular ecto-enzyme pattern directed toward the production of anti-inflammatory and anti-atherosclerotic adenosine. In turn, mice with genetically induced hypercholesterolemia and atherosclerosis expressed disturbed activities of ecto-5’nucleotidase and ecto-adenosine deaminase related to decreased production and increased degradation of extracellular adenosine.     

Polychlorinated biphenyls (PCBs) inducible monooxygenases in rabbits and mice: species and organ specificities

The effects of the PCBs mixture, Aroclor 1254, as modifiers of monooxygenases were studied in rabbits and mice. From data presented, it is not possible to generalize the biological effects of PCBs observed with rats, namely, that they are potent, nonspecific inducers of monooxygenase activities. This environmental pollutant enhanced microsomal drug-metabolizing enzymes in livers of rabbits and C57Bl/6J and DBA/2J mice. In rabbit lung, it inhibited, and in rabbit kidney, it enhanced the metabolism of ethylmorphine. Further, at dosages used, PCBs were poor inducers of aryl (benzo(a)pyrene) hydroxylase activity in livers of C57BL/6J and DBA/2J mice; they enhanced aryl hydrocarbon hydroxylase activity in rabbit kidney but caused a significant depression of its activity in rabbit lung. These studies demonstrate that the biologic impact of the widely distributed environmental pollutant, PCBs, may differ in different species and emphasize the need to carry out toxicological studies in more than one species of animals. The differential effects observed on various organs may also be important determinants of organ-targeted chemical toxicity.     

H-2 haplotype and sex-related differences in IgG response to ovalbumin in mice.

Specific IgG antibodies were determined by enzyme-linked immunosorbent assay (ELISA) in 7 strains of male and female mice after immunization with ovalbumin. Also, H-2 haplotype and sex-related differences in IgG response to ovalbumin were evaluated using statistical methods, slope ratio assay and parallel line assay. H-2k strain mice (C3H/HeN and CBA/JN) showed higher IgG responsiveness to ovalbumin than H-2d (BALB/cAnN and DBA/2 N) and H-2b (C57BL/6 N) mice. With regard to the sex-related differences in IgG response to ovalbumin, females in some strains showed higher IgG response than males, but some strains showed no sex-related differences, and sex-related differences in IgG response to ovalbumin did not relate to their H-2 haplotypes. These results may be caused by other immune response genes which control the sex-related immune response than H-2 or other unknown factors.     

Interaction of allogeneic lymphocytes and precursor cells during the primary and secondary immune response]

A mixture of lymph node cells from CBA mice and spleen cells from C57Bl/6J mice stimulated by the cheep erythrocytes fro the first or second time was transplanted in the lethally irradiated mice (CBA X C57Bl/6j)Fl. The interaction of allogenic cells during the secondary immune response was accompanied by the complete inactivation of antibody producents. Under the ratio of interacting cell elements 1 : 1-1 : 2, 93-96% of precursor cells and 98% of antibody forming cells were inactivated. Under the ratio 1 : 5, the index of inactivation of precursor cells fell down to 35%. During the primary response, under the ratio 1 : 1, only 20-48% of precursor cells and 68% of antibody forming cells were inactivated. Under the ratio 1 : 2, no inactivation of precursor cells was observed and, under the ratio 1 : 10, the antibody formation was stimulated. Following the delayed by 1-3 days transplantation of CBA lymphocytes, the cooperative effect was registered with respect to the spleen cells from C57Bl/6J mice stimulated by the erythrocytes for the first time. The interaction of allogenic cells resulted in the 3-4-fold increase in the number of antibody forming cells.