Hepatitis C virus acts as a tumor accelerator by blocking apoptosis in a mouse model of hepatocarcinogenesis

We developed hepatitis C virus (HCV) core-E1-E2 and HCV core transgenic mice on a common genetic background to assess the contribution of HCV structural proteins to hepatocarcinogenesis. Eight-week-old core-E1-E2, core, and nontransgenic mice inbred on the FVBxC57Bl/6 background were treated with diethylnitrosamine (DEN) and sacrificed at 32 weeks old. Proliferation and apoptosis were assessed by immunohistochemistry. The effect of viral proteins on apoptosis was evaluated in HepG2 cells in which apoptosis was induced by anti-Fas antibody. HCCs were identified at 32 weeks in the majority of DEN-treated mice from all three groups. The mean size of HCCs was significantly larger in core-E1-E2 transgenic (4.63 +/- 1.48 mm), compared with core transgenic (0.78 +/- 0.26 mm, P = .01), and nontransgenic (1.0 +/- 0.19 mm, P = .002) mice. While there were no differences in proliferation, the apoptotic index in core-E1-E2 transgenic HCCs was significantly lower than those found in core and non-transgenic HCCs. Core-E1-E2 transfected HepG2 cells demonstrated a significantly lower apoptotic index (0.35 +/- 0.11) compared with that of core transfected cells (0.74 +/- 0.07, P = .0103). Analysis of a Fas-induced apoptosis model in HCV transgenic mice confirmed that core-E1-E2 transgenic liver underwent significantly less apoptosis than transgenic tissue expressing core only. In conclusion, HCV core-E1-E2 transgenic mice develop significantly larger tumors than transgenic mice expressing core alone or nontransgenic mice. The accelerated tumor phenotype is attributable to suppression of apoptosis rather than enhanced proliferation. These data implicate HCV E1 and/or E2 in conjunction with core as antiapoptotic, tumor accelerator proteins.     

Extracellular superoxide dismutase, nitric oxide, and central nervous system O2 toxicity.

Although reactive O2 species appear to participate in central nervous system (CNS) O2 toxicity, the exact roles of different reactive O2 species are undetermined. To study the contribution of extracellular superoxide anion (O2-) to CNS O2 toxicity we constructed transgenic mice overexpressing human extracellular superoxide dismutase (ECSOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) in the brain. Remarkably, when exposed to 6 atm (1 atm = 101.3 kPA) of hyperbaric oxygen for 25 min, transgenic mice demonstrated higher mortality (83%) than nontransgenic litter-mates (33%; P < 0.017). Pretreatment with diethyldithiocarbamate, which inhibits both ECSOD and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity, increased resistance to CNS O2 toxicity, in terms of both survival (100% in transgenics and 93% in nontransgenics) and resistance to seizures (4-fold increase in seizure latency in both transgenic and nontransgenic mice; P < 0.05). Thus, O2- apparently protects against CNS O2 toxicity. We hypothesized that O2- decreased toxicity by inactivating nitric oxide (NO.). To test this, we inhibited NO. synthase (EC 1.14.23) with N omega-nitro-L-arginine to determine whether NO. contributes to enhanced CNS O2 toxicity in transgenic mice. N omega-nitro-L-arginine protected both transgenic and nontransgenic mice against CNS O2 toxicity (100% survival and a 4-fold delay in time to first seizure; P < 0.05), as well as abolishing the difference in sensitivity to CNS O2 toxicity between transgenic and nontransgenic mice. These results implicate NO. as an important mediator in CNS O2 toxicity and suggest that ECSOD increases CNS O2 toxicity by inhibiting O2(-)-mediated inactivation of NO.     

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.     

Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome

To determine whether adipocyte storage capacity influences the onset and severity of type 2 diabetes and other components of the metabolic syndrome, we made normal and db/db mice resistant to obesity by overexpressing leptin receptor-b on the aP2-Lepr-b promoter. On a 4% diet, these mice have no phenotype, but on a 60% fat diet, they resist diet-induced obesity because constitutive adipocyte-specific overexpression of Lepr-b prevents obesity via the antilipogenic autocrine/paracrine action of leptin on adipocytes. After 8 months on the same 60% fat diet, body fat of transgenic mice was 70% below WT controls. Cardiac and liver fat was elevated in the transgenics, and their hyperinsulinemia was more marked, suggesting greater insulin resistance. The aP2-Lepr-b transgene also prevented obesity in db/db mice; at 10 weeks of age their body fat was half that of the db/db mice. This lack of obesity was attributable to reduced expression of sterol regulatory element binding protein-1c and its target lipogenic enzymes in adipose tissue and a 6-fold increase in Pref-1 mRNA. Severe diabetes was present in transgenics at 4 weeks of age, 10 weeks before db/db controls. Echocardiographic evidence of cardiomyopathy appeared at 10 weeks, weeks before the db/db mice. Histologically, loss of beta cells and myocardial fibrosis was present in the transgenic group at least 6 weeks before the db/db mice. These results suggest that the expression level of genes that regulate the adipogenic response to overnutrition profoundly influences the age of onset and severity of diet-induced type 2 diabetes and co-morbidities.     

Reduction of diet-induced obesity in transgenic mice overexpressing uncoupling protein 3 in skeletal muscle

Aims/hypothesis It has been suggested that uncoupling protein 3 (UCP3) can increase energy expenditure, thereby regulating body weight. Although studies on UCP3 knock-out mice suggest that lack of UCP3 function does not cause obesity or Type 2 diabetes, it is possible that up-regulation of UCP3 function improves these disorders or their clinical sequelae. A 10- to 20-fold increase of UCP3 gene expression is achievable through physiological or pharmacological stimuli. We examined the phenotype of transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle.Methods We generated transgenic mice with approximately 18-fold overexpression of mouse UCP3 mRNA in skeletal muscle under control of the skeletal muscle-specific muscle creatine kinase gene promoter. The phenotype of these mice was analysed either on a standard diet or on a 4-week high-fat diet.Results In mice on standard chow, there was no difference in body weight, oxygen consumption and mitochondrial protonmotive force between transgenic mice and non-transgenic littermates. However, transgenic mice tended to have lower body weight, increased oxygen consumption and decreased mitochondrial protonmotive force than the control mice. Transgenic mice on a 4-week high-fat diet consumed much more oxygen and had noticeably less weight gain and less epididymal fat, as well as better glucose tolerance than non-transgenic littermates.Conclusions/interpretation Our study shows that 18-fold overexpression of UCP3 mRNA in the skeletal muscle reduced diet-induced obesity. An 18-fold increase of UCP3 mRNA can be attained by physiological or pharmacological stimuli, suggesting that UCP3 has therapeutic potential in the treatment of obesity.Abbreviations UCP3 uncoupling protein 3 - UCP2 uncoupling protein 2 - UCP1 uncoupling protein 1 - PPAR peroxisome-proliferator-activated receptor - MCK muscle creatine kinase - p mitochondrial protonmotive force     

Age-related changes in body composition of bovine growth hormone transgenic mice

GH has a significant impact on body composition due to distinct anabolic and catabolic effects on lean and fat mass, respectively. Several studies have assessed body composition in mice expressing a GH transgene. Whereas all studies report enhanced growth of transgenic mice as compared with littermate controls, there are inconsistencies in terms of the relative proportion of lean mass to fat mass in these animals. The purpose of this study was to characterize the accumulation of adipose and lean mass with age and according to gender in a bovine (b) GH transgenic mouse line. Weight and body composition measurements were assessed in male and female bGH mice with corresponding littermate controls in the C57BL/6J genetic background. Body composition measurements began at 6 wk and continued through 1 yr of age. At the conclusion of the study, tissue weights were determined and triglyceride content was quantified in liver and kidney. Although body weights for bGH mice were significantly greater than their corresponding littermate controls at all time points, body composition measurements revealed an unexpected transition midway through analyses. That is, younger bGH mice had relatively more fat mass than nontransgenic littermates, whereas bGH mice became significantly leaner than controls by 4 months in males and 6 months in females. These results reveal the importance in timing and gender when conducting studies related to body composition or lean and fat tissue in GH transgenic mice or in other genetically manipulated mouse strains in which body composition may be impacted.     

Reproductive abnormalities in human insulin-like growth factor-binding protein-1 transgenic male mice

Adult transgenic mice overexpressing human insulin-like growth factor-binding protein-1 in the liver present reproductive abnormalities in both sexes. In the present work, we have investigated the mechanisms responsible for limiting breeding capacity in these transgenic male mice. Homozygous adult transgenic male mice (3-6 months old) exhibited irregular copulatory behavior and a reduction of the number of pregnancies per female as well as of litter size per pregnancy. Genital tract weight, more specifically epididymal and seminal vesicle weights, were reduced by 45% in homozygous transgenic vs. nontransgenic mice. Homozygous transgenic mice exhibited a 30% reduction of the length of seminiferous tubules (P = 0.007), a 30% decrease in daily sperm production per testis (P = 0.019), and a 50% decrease in the number of spermatozoa in testis (P = 0.037), associated with morphological abnormalities of the sperm heads leading to an approximately 50% reduction of fertilized two-cell eggs (P = 0.002) and of implanted embryos on d 5.5 after mating (P = 0.004). The round spermatids also appeared altered in their morphology. In addition, Leydig cells in homozygous transgenic mice exhibited an altered appearance, with a 1.8-fold increase in lipid droplets in their cytoplasm (P < 0.001). Moreover, the concentration of 3beta-hydroxysteroid dehydrogenase was 66% lower in testis from transgenics compared with those from normal mice (P = 0.01), leading to a tendency toward lower plasma testosterone levels (P = 0.1). Interestingly, LH concentrations were increased by 40% in transgenic pituitary extracts (P = 0.02), and basal LH secretion by pituitary explants in vitro was increased by 60% in homozygous transgenic vs. normal mice (P = 0.04), suggesting an alteration of LH pulsatile secretion in vivo. In conclusion, these data suggest that the breeding impairment of human insulin-like growth factor-binding protein-1 transgenic males is due at least in part to an alteration of the process of spermatogenesis, leading to a diminution of sperm production and of its quality. Minor impairment of steroidogenesis may also contribute to the reduced reproductive capacity of these animals. Our observations are consistent with the idea that normal spermatogenesis and perhaps also steroidogenesis are dependent on the actions of sufficient concentrations of unbound IGF-I.     

Up-regulation of muscle uncoupling protein 3 gene expression in mice following high fat diet, dietary vitamin A supplementation and acute retinoic acid-treatment

OBJECTIVE: To analyse the impact of vitamin A supplementation of both a normal fat (NF) diet and a high fat (HF) diet and of acute retinoic acid (RA)-treatment on the expression of uncoupling protein 3 (UCP3) in mice. DESIGN: C57BL/6J mice were fed for 18 weeks a NF or a HF diet (10 and 45 energy% as fat, respectively), both with the normal vitamin A content or an excess vitamin A (8 mg and 320 mg retinyl palmitate/kg diet, respectively). Body weight and energy intake were recorded periodically. UCP3 mRNA and UCP3 protein levels in skeletal muscle (soleus/gastrocnemius) were analysed, as well as UCP1, UCP2 and UCP3 mRNA levels in interscapular brown adipose tissue (BAT), and UCP2 mRNA, UCP2 protein and leptin mRNA levels in white adipose tissue (WAT) depots. The effect of acute RA-treatment (100 mg/kg/day, 4 days) on UCP3 mRNA levels in skeletal muscle and BAT of NMRI mice was also assessed. RESULTS: Vitamin A supplementation of a NF diet led to increased levels of UCP3 mRNA and UCP3 protein in muscle, UCP1 mRNA in BAT, and UCP2 mRNA in inguinal WAT, but had no impact on body weight or adiposity of B6 mice. HF diet promoted obesity and increased levels of UCP3 mRNA and UCP3 protein in skeletal muscle, and of the mRNAs for all three UCPs in BAT. Supplementing the HF diet with vitamin A had little effect on the final obesity reached and did not lead to further increases of muscle UCP3 mRNA nor BAT UCP1 mRNA over the levels achieved with the non-supplemented HF diet. Adipose leptin mRNA levels were down regulated after vitamin A supplementation, independently of the fat content of the diet. Up-regulation of muscle, but not BAT, UCP3 mRNA levels was also found after acute RA-treatment in NMRI mice. CONCLUSION: The results provide evidence of a stimulatory effect of retinoids on muscle UCP3 expression in vivo, and a differential retinoid-regulation of the UCP3 gene in muscle and BAT.     

Genetic variation in uncoupling protein 3 is associated with dietary intake and body composition in females

The uncoupling proteins (UCPs) are a family of mitochondrial transport proteins that promote proton leakage across the inner mitochondrial membrane, uncoupling oxidative phosphorylation from adenosine triphosphate (ATP) production and releasing energy as heat. Variation in these genes may disrupt biochemical pathways influencing thermogenesis, energy metabolism, and fuel substrate partitioning and oxidation, which may in turn predispose to obesity. We genotyped polymorphisms in UCP2 and UCP3 in a sample of nondiabetic participants (n = 722) of the San Luis Valley Diabetes Study (SLVDS) and found female-specific associations between UCP3 polymorphisms and measures of dietary intake and body composition. The UCP3-5 variant was statistically significantly associated with total caloric intake (P =.012), fat intake (P =.011), fat mass (P =.004), and lean mass (P =.013), with the C allele corresponding to higher dietary intake and lower fat mass and lean mass. The UCP3p-55 and the UCP3-3 polymorphisms, which were in high linkage disequilibrium (D' = 0.9776), showed similar patterns of association with total caloric intake (P =.031 and P =.042, respectively) and lean mass (P =.035 and P =.059, respectively), with the rare alleles corresponding to higher total intake and lean mass. No statistically significant associations were detected between the outcome variables and polymorphisms in UCP2. Two-way analysis of covariance (ANCOVA), used to evaluate the multi-locus effects and interactions between UCP3-5 and UCP3p-55, showed association with the main effect terms, but no evidence for statistically significant interaction between UCP3-5 and UCP3p-55 in regard to dietary intake. The UCP3-5 polymorphism was the only statistically significant genetic predictor of fat mass. The lean mass model showed no statistically significant association with either UCP3 variant. These results support a role for UCP3 in fuel substrate management and energy metabolism, which may influence body weight regulation.     

Nicotine induces uncoupling protein 1 in white adipose tissue of obese mice.

OBJECTIVE: To test the hypothesis that nicotine not only activates uncoupling protein1 (UCP1) in brown adipose tissue (BAT), but also induces UCP1 in white adipose tissue (WAT), which contributes to the mitigation of obesity in obese mice. DESIGN: Weights of the whole body, the gastrocnemius muscle, interscapular BAT and subcutaneous and retroperitoneal WAT, food intake and the mRNA and protein of UCP1 in these tissues were measured and immunohistochemistry using antiserum against UCP1 was also performed in obese yellow KK mice treated with nicotine for 6 months and control mice treated with physiological saline. RESULTS: Obese mice treated with nicotine for 6 months, compared with those injected with saline, weighed significantly less (P < 0.01) and had smaller subcutaneous and retroperitoneal WAT pads (P < 0.01), while obese mice that received nicotine ate less (P < 0.05) than those injected with saline. In mice treated with nicotine, the mRNA and protein of UCP1 was detected not only in BAT, but also in subcutaneous and retroperitoneal WATs. Immunohistochemically, the BAT of obese mice contained large lipid droplets and appeared rather WAT-like, but changed to typical brown adipocytes after nicotine treatment. The fat pads of nicotine-treated mice contained many multilocular cells that were positive for UCP1. CONCLUSION: Nicotine not only activates UCP1 in BAT, but also induces UCP1 in WAT and decreases food intake, which contributes to the mitigation of obesity.     

Fiber type dependent upregulation of human skeletal muscle UCP2 and UCP3 mRNA expression by high-fat diet

OBJECTIVE: To test the hypothesis that consumption of a high-fat diet leads to an increase in UCP mRNA expression in human skeletal muscle. In a group of endurance athletes, with a range in fiber type distribution, we hypothesized that the effect of the high-fat diet on UCP2 and UCP3 mRNA expression is more pronounced in muscle fibers which are known to have a high capacity to shift from carbohydrate to fat oxidation (type IIA fibers). DESIGN: Ten healthy trained athletes (five males, five females) consumed a low-fat diet (17+/-0.9 en% of fat) and high-fat diet (41.4+/-1.4 en% fat) for 4 weeks, separated by a 4 week wash-out period. Muscle biopsies were collected at the end of both dietary periods. MEASUREMENTS: Using RT-PCR, levels of UCP2 and UCP3 mRNA expression were measured and the percentage of type I, IIA and IIB fibers were determined using the myofibrillar ATPase method in all subjects. RESULTS: UCP3L mRNA expression tended to be higher on the high-fat diet, an effect which reached significance when only males were considered (P=0.037). Furthermore, diet-induced change in mRNA expression of UCP3T (r: 0.66, P=0.037), UCP3L (r: 0.61, P=0.06) and UCP2 (r: 0.70, P=0.025), but not UCP3S, correlated significantly with percentage dietary fat on the high-fat diet. Plasma FFA levels were not different during the two diets. Finally, the percentage of type IIA fibers was positively correlated with the diet-induced change in mRNA expression for UCP2 (r: 0.7, P=0.03), UCP3L (r: 0.73, P=0.016) and UCP3T (r: 0.68, P=0.03) but not with UCP3S (r: 0.06, NS). CONCLUSION: UCP2 and UCP3 mRNAs are upregulated by a high-fat diet. This upregulation is more pronounced in humans with high proportions of type IIA fibers, suggesting a role for UCPs in lipid utilization.     

Human uncoupling protein 2 and 3 genes are associated with obesity in Japanese

Human uncoupling proteins (UCPs) are mitochondrial proteins that are involved in the control of energy metabolism and the pathophysiology of obesity. Although there have been several reports on the association between the UCP2/UCP3 locus and the obesity, there have been no haplotype-based case-control studies with gender-specific analysis. The aim of this study was to examine whether there is an association between the UCP2/UCP3 locus and the obesity in the Japanese population when using a single nucleotide polymorphism (SNP)-based and haplotype-based case-control study with gender-specific analysis. We examined a group consisting of 551 subjects, of which 369 were non-obese and 182 were overweight and/or obese. We selected one nonsynonymous SNP (rs660339: Ala55Val) as a genetic marker. Genotyping for all subjects was performed by the TaqMan polymerase chain reaction (PCR) method. Although the overall distributions of genotype and allele were not significantly different between the non-obese and the obese groups, the overall distributions of the genotype were significantly different in men (P = 0.030). In the obese group, male subjects with the Val allele were significantly more frequent in both association studies. There was a significant difference in the overall distribution of the haplotype (UCP3 rs180049, UCP3 rs2075577, UCP2 rs660339) between the weight groups (P = 0.010), and in women, there was a significant difference (P = 0.042) in the overall distribution of the haplotype (UCP3 rs2075577, UCP2 rs660339). Nonsynonymous rs660339 in the human UCP2 gene in men, and the haplotype (UCP3 rs2075577-UCP2 rs660339) in women might be good obesity markers.     

Activation of the hexosamine signaling pathway in adipose tissue results in decreased serum adiponectin and skeletal muscle insulin resistance

Overexpression of the rate-limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase) in muscle and adipose tissue of transgenic mice was previously shown to result in insulin resistance and hyperleptinemia. Explanted muscle from transgenic mice was not insulin resistant in vitro, suggesting that muscle insulin resistance could be mediated by soluble factors from fat tissue. To dissect the relative contributions of muscle and fat to hexosamine-induced insulin resistance, we overexpressed glutamine:fructose-6-phosphate amidotransferase 2.5-fold, specifically in fat under control of the aP2 promoter. Fasting glucose, insulin, and triglycerides were unchanged in the transgenic mice; leptin and beta-hydroxybutyrate levels were 91% and 29% higher, respectively. Fasted transgenic mice have mild glucose intolerance and skeletal muscle insulin resistance in vivo. In fasting transgenic mice, glucose disposal rates with hyperinsulinemia were decreased 27% in females and 10% in males. Uptake of 2-deoxy-D-glucose into muscle was diminished by 45% in female and 21% in male transgenics. Serum adiponectin was also lower in the fasted transgenics, by 37% in females and 22% in males. TNF alpha and resistin mRNA levels in adipose tissue were not altered in the fasted transgenics; levels of mRNA for leptin were increased and peroxisome proliferator-activated receptor gamma decreased. To further explore the relationship between adiponectin and insulin sensitivity, we examined mice that have been refed for 6 h after a 24-h fast. Refeeding wild-type mice resulted in decreased serum adiponectin and increased leptin. In transgenic mice, however, the regulation of these hormones by refeeding was lost for adiponectin and diminished for leptin. Refed transgenic female and male mice no longer exhibited decreased serum adiponectin in the refed state, and they were no longer insulin resistant as by lower or unchanged insulin and glucose levels. We conclude that increased hexosamine levels in fat, mimicking excess nutrient delivery, are sufficient to cause insulin resistance in skeletal muscle. Changes in serum adiponectin correlate with the insulin resistance of the transgenic animals.     

Expression of HLA-DR4 and human CD4 transgenes in mice determines the variable region beta-chain T-cell repertoire and mediates an HLA-DR-restricted immune response.

Inherited susceptibility to rheumatoid arthritis is associated with genes encoding the human major histocompatibility complex class II molecule HLA-DR4. To study the immune function of HLA-DR4 and attempt to generate a murine model of rheumatoid arthritis we have produced triple transgenic mice expressing HLA-DRA*0101, -DRB1*0401, and human CD4. The expression of the HLA transgenes is driven by the promoter of the murine major histocompatibility complex class II I-E alpha gene and was found on murine cells that normally display major histocompatibility complex class II molecules. The expression of the human CD4 transgene is driven by the murine CD3 delta-promoter, and therefore its gene product was found on cells that express murine CD3. In contrast to other HLA-DR and HLA-DQ transgenic mouse lines, the transgenes are functional in our mice. In H-2 I-E-negative transgenic mice, T cells expressing variable region beta chain (V beta) 3, 5, 6, 7, 9, 11, 12, or 13 were either absent or significantly reduced, in contrast to H-2 I-E-negative nontransgenic littermates. In addition, the peptide antigen influenza A virus hemagglutinin 307-319, which binds to the HLA-DRA*0101/-DRB1*0401 heterodimer with high affinity and induces an HLA-DR-restricted and CD4+ T-cell response in humans, also induced a T-cell response in the triple transgenic mice but not in nontransgenic littermates. Thus, these transgenic mice should permit extensive testing of the antigen-presentation capabilities of the HLA-DRA*0101/-DRB1*0401 molecule.     

Body composition, bone mass and microstructural analysis in GH-transgenic mice reveals that skeletal changes are specific to bone compartment and gender.

Experimental and clinical studies suggest that high serum levels of growth hormone (GH) increase cortical but not trabecular bone. We studied body composition and bone structure in transgenic mice (MT-bGH) with systemic overexpression of GH. Body composition was examined with dual-energy X-ray absorptiometry (DXA), ashing, and chemical analysis, and the femora with DXA and micro computerized tomography. The absolute fat and bone tissue contents were significantly higher in GH transgenic mice vs controls (P < or = 0.05), but no significant difference was noted when normalizing the values to body weight. Male transgenics displayed no change in apparent (volumetric) femoral bone density, relative cortical area and trabecular bone volume fraction. Female transgenic mice demonstrated an increase in apparent femoral density and in trabecular bone volume fraction (+130%; P < or = 0.01). The mineralized tissue matrix density was decreased in male and female transgenic mice (P < or = 0.05). The results show that chronic GH excess affects trabecular bone in a gender-specific manner and that bone changes depend on the compartment investigated.     

Growth response and androgen receptor expression in seminal vesicles from aging transgenic mice expressing human or bovine growth hormone genes.

Previous work has shown that expression of human (h) GH in transgenic mice is associated with significant age-related enlargement of seminal vesicles. To further explore this aberrant growth activity, we have characterized seminal vesicles from various GH transgenic lines and examined their androgen receptor (AR) content and distribution. Six groups of animals were initially studied: young adult (3-5 months) control mice, old (greater than 12 months) control mice, young adult hGH transgenic mice, old hGH transgenics, young adult bovine (b) GH transgenics, and old bGH transgenic mice. Young transgenic mice (hGH and bGH) possessed seminal vesicles with similar relative weights, DNA and protein contents, and AR levels as nontransgenic littermates. Histologically, the glands appeared similar. With aging, the hGH transgenic seminal vesicles exhibited massive stromal hyperplasia, whereas the glands from controls and bGH transgenic mice did not show this response. Seminal vesicles from old hGH mice presented with a marked increase in cell number (DNA content) and a marked decrease in cell size and/or glandular secretions (protein/DNA ratio) compared to those from old controls and young hGH transgenic mice. Tissue AR content was markedly reduced in old hyperplastic hGH seminal vesicles compared to that in seminal vesicles from young hGH transgenics, old controls, and old bGH transgenic mice. Immunohistochemistry indicated the absence of AR in the proliferating stromal cells, whereas acinar epithelial cells showed similar or moderately reduced AR staining intensity compared to control seminal vesicles. To examine whether the above results may be due to insertional mutagenesis rather than hGH itself, two additional GH transgenic lines were examined. Aged transgenic mice expressing bGH with an alternate promoter possessed seminal vesicle weights that were not different from those of old controls, whereas aged transgenic mice expressing an hGH. V gene (variant gene, placental origin) possessed significantly larger vesicles than the controls, which further suggests that vesicular hyperplasia is specifically related to hGH. To assess androgen responsiveness, aged control and hGH transgenic mice were castrated and examined after 15 days. While control seminal vesicles significantly decreased in size, glands from transgenic mice did not. Regressive changes were observed in the remaining epithelium of hGH transgenic mice; however, stromal tissue exhibited no response to androgen withdrawal. The present results suggest that the aging-associated seminal vesicle hyperplasia in hGH transgenic mice is a result of a massive increase in stromal tissue that is low or devoid of AR, suggesting a loss of direct androgen regulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage

Hepatic metabolism of ethanol by cytochrome P450 2E1 (CYP2E1) is believed to contribute to alcoholic liver damage. To further evaluate CYP2E1 in alcoholic liver disease, we created a transgenic mouse containing human CYP2E1 complementary DNA (cDNA) under the control of mouse albumin enhancer-promoter. Two experiments were performed. In the first experiment, transgenic and nontransgenic mice were fed normal chow. In the second experiment, transgenic and nontransgenic mice were pair fed a nutritionally complete liquid diet for 16 weeks. The liquid diet contained 30% of calories as ethanol (or dextrose) and 25% of calories as corn oil. Liver damage was assessed by measuring serum alanine aminotransferase (ALT) levels and examining liver histology. Transgenic animals reproduced and were phenotypically normal. Hepatic levels of CYP2E1 messenger RNA (mRNA), protein, and enzyme activity did not differ between chow-fed transgenic and nontransgenic mice. Livers from transgenic mice fed the alcohol diet contained significantly more CYP2E1 protein and enzyme activity than livers from nontransgenic mice fed the same diet. Transgenic mice receiving the alcohol diet had significantly higher serum ALT levels than nontransgenic mice. Histologic examination of the livers showed higher histologic scores in transgenic mice fed ethanol compared with nontransgenic mice fed ethanol. Ballooning hepatocytes were seen in livers from transgenic mice fed ethanol. Apoptosis, as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay, did not differ between groups. In conclusion, we have produced a transgenic mouse that expresses human CYP2E1 in the liver. When fed a nutritionally complete alcohol diet, transgenic mice develop more liver damage than nontransgenic mice.     

Dietary calcium regulates ROS production in aP2-agouti transgenic mice on high-fat/high-sucrose diets

OBJECTIVE: We have previously demonstrated that 1alpha, 25(OH)2D3 promotes adipocyte reactive oxygen species (ROS) production. We have now evaluated whether decreasing 1alpha, 25(OH)2D3 levels by increasing dietary calcium will decrease oxidative stress in vivo. METHODS: We fed low-calcium (0.4% Ca) and high-calcium (1.2% Ca from CaCO3) obesity-promoting (high sucrose/high fat) diets to aP2-agouti transgenic mice and assessed regulation of ROS production in adipose tissue and skeletal muscle. RESULTS: Mice on the high-calcium diet gained 50% of the body weight (P=0.04) and fat (P<0.001) as mice on the low-calcium diet (0.4% Ca). The high-calcium diet significantly reduced adipose intracellular ROS production by 64 and 18% (P<0.001) and inhibited adipose tissue nicotinamide adenine dinucleotide phosphate oxidase expression by 49% (P=0.012) and 63% (P=0.05) in visceral and subcutaneous adipose tissue, respectively. Adipocyte intracellular calcium ([Ca2+]i) levels were suppressed in mice on the high-calcium diet by 73-80% (P<0.001). The high-calcium diet also induced 367 and 191% increases in adipose mitochondrial uncoupling protein 2 (UCP2) expression (P<0.001) in visceral and subcutaneous adipose tissue, respectively. The pattern of UCP3 expression and indices of ROS production in skeletal muscle were consistent with those in adipose tissue. The high-calcium diet also suppressed 11beta-hydroxysteroid dehydrogenase (11beta-HSD) expression in visceral adipose tissue by 39% (P=0.034). 11beta-HSD expression was markedly higher in visceral vs subcutaneous adipose tissue in mice on the low-calcium diet (P=0.034), whereas no difference was observed between the fat depots in mice on the high-calcium diet. CONCLUSION: These data support a potential role for dietary calcium in the regulation of obesity-induced oxidative stress.     

乙型肝炎病毒转基因小鼠免疫耐受状态及血液生物化学指标的研究

目的 了解HBV转基因鼠HBV基因的复制表达和免疫耐受状态,为探讨乙型肝炎发病机制和抗HBV新药评价提供可靠的参考依据.方法 选取遗传背景相同的SPE级HBsAg阴性非转基因鼠和转基因鼠.化学发光法检测HBsAg、HBeAg、HBV DNA,ELISA检测前S1、HBcAg,肝组织行病理学检查,免疫组织化学染色检测不同时期转基因鼠肝HBsAg表达,流式细胞仪检测小鼠淋巴细胞增殖情况,酶联免疫斑点检测(ELISPOT)分泌IFNγ的T淋巴细胞斑点数,双色免疫荧光法检测脾细胞悬液和脾树突状细胞(DC)中Toll样受体(TLR)2和TLR9的表达.数据行t检验和F检验.结果 HBV转基因鼠可复制表达HBsAg、前S1、HBeAg、HBcAg和HBVDNA,而抗-HBs、抗-HBc、抗-HBe均阴性;肝组织无明显病理改变,肝细胞中HBsAg在胞质表达,HBcAg在胞核表达.HBsAg刺激后,HBV转基因鼠T淋巴细胞增殖能力为(697.6±67.3)cpm,显著低于非转基因鼠的(1315.5±191.6)cpm.经HBsAg刺激后,HBV转基因鼠脾细胞分泌IFNγ的T淋巴细胞斑点数为8.25±1.10,低于非转基因鼠的28.50±4.21(F=155.967,P=0.000).HBV转基因DC表达CD11c+、TLR2和TLR9与非转基因鼠比较,差异无统计学意义(均P＞0.05).在18日龄胎鼠和1日龄仔鼠肝组织观察到HBsAg表达.结论 HBV转基因鼠有HBV相关抗原表达,并对HBV相关抗原存在免疫耐受,其先天和获得性免疫功能均正常,类似于人类慢性HBV无症状携带者.HBV转基因鼠是比较理想的动物模型.