Altered gene expression in the liver of gamma-glutamyl transpeptidase-deficient mice

We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.     

Oxygen-Induced Pulmonary Injury in g-Glutamyl Transpeptidase-Deficient Mice

We used mice with a targeted disruption in g-glutamyl transpeptidase (GGT-deficient mice) to study the role of glutathione (GSH) in protection against oxygen-induced lung injury. These mice had reduced levels of lung GSH and restricted ability to synthesize GSH because of low levels of cysteine. When GGT-deficient mice were exposed to 80% oxygen, they developed diffuse pulmonary injury and died within eight days. Ten of 12 wild-type mice were alive after 18 days. Administration of N-acetylcysteine (NAC) to GGT-deficient mice corrected GSH values and prevented the development of severe pulmonary injury and death. Oxygen exposure induced an increase in lung GSH levels in both wild-type and GGT-deficient mice, but induced levels in the mutant mice were <50% of those in wild-type mice. Cysteine levels were approximately 50-fold lower than GSH levels the lungs of both wild-type and GGT-deficient mice. Levels of lung RNA coding for the heavy subunit of g-glutamyl cysteine synthetase rose three- to fourfold after oxygen exposure in both wild-type and GGT-deficient mice. In contrast, oxygen exposure failed to provoke increases in glutathione synthetase, glutathione peroxidase, glutaredoxin, or thioredoxin.     

Reproductive defects in gamma-glutamyl transpeptidase-deficient mice

Mice deficient in gamma-glutamyl transpeptidase (GGT) are growth retarded as a result of cysteine deficiency secondary to excessive glutathione excretion in urine and display coat color defects and cataracts. Although GGT is widely expressed throughout the mouse reproductive axis, little is known about its role in reproduction. Here, we present an analysis of the reproductive phenotypes of GGT-deficient mice. Mutant male mice have reduced testis and seminal vesicle size and suppressed serum insulin-like growth factor I and FSH levels and are infertile. Although these mice are severely oligospermic, histological analysis of testes reveals grossly normal stages of spermatogenesis, including late stage spermatids, but the tubule diameter is reduced. GGT-deficient female mice are also hypogonadal and infertile. At 6 weeks of age, the ovaries of mutant mice are histologically indistinguishable from those of its wild-type counterpart. However, the absence of antral follicles and corpora lutea and follicular degeneration are apparent by 11-13 weeks. In addition, immature female mutant mice (at 21-23 days) are insensitive to exogenous gonadotropin administration and fail to superovulate, suggesting an intraovarian defect. Consistent with these mutant phenotypes, HPLC analysis of adult mutant testes and ovaries showed a reduction in intracellular cysteine levels. Administration of N-acetylcysteine in the drinking water beginning on day 21 to mutant mice for 2 weeks restored testis, seminal vesicle, and ovary sizes to values comparable to those in wild-type mice. Furthermore, N-acetylcysteine-fed (continuously) mutant male and female mice were fertile and produced normal numbers of offspring when mated to wild-type control mice. These results demonstrate that GGT itself is not necessary for reproductive function. However, GGT plays an important role in cysteine homeostasis within the mouse reproductive axis.     

gamma-Glutamyl transpeptidase overexpression increases metastatic growth of B16 melanoma cells in the mouse liver.

B16 melanoma (B16M) cells with high glutathione (GSH) content show rapid proliferation in vitro and high metastatic activity in the liver in vivo. gamma-Glutamyl transpeptidase (GGT)-mediated extracellular GSH cleavage and intracellular GSH synthesis were studied in vitro in B16M cells with high (F10) and low (F1) metastatic potential. GGT activity was modified by transfection with the human GGT gene (B16MF1/Tet-GGT cells) or by acivicin-induced inhibition. B16MF1/Tet-GGT and B16MF10 cells exhibited higher GSH content (35 +/- 6 and 40 +/- 5 nmol/10(6) cells, respectively) and GGT activity (89 +/- 9 and 37 +/- 7 mU/10(6) cells, respectively) as compared (P <.05) with B16MF1 cells (10 +/- 3 nmol GSH and 4 mU GGT/10(6) cells). Metastasis (number of foci/100 mm(3) of liver) increased in B16MF1 cells pretreated with GSH ester ( approximately 3-fold, P <.01), and decreased in B16MF1/Tet-GGT and B16MF10 cells pretreated with the GSH synthesis inhibitor L-buthionine (S,R)-sulphoximine ( approximately 5-fold and 2-fold, respectively, P <.01). Liver, kidney, brain, lung, and erythrocyte GSH content in B16MF1/Tet-GGT- or B16MF10-bearing mice decreased as compared with B16MF1- and non-tumor-bearing mice. Organic anion transporting polypeptide 1-independent sinusoidal GSH efflux from hepatocytes increased in B16MF1/Tet-GGT- or B16MF10-bearing mice ( approximately 2-fold, P <.01) as compared with non-tumor-bearing mice. Our results indicate that tumor GGT activity and an intertissue flow of GSH can regulate GSH content of melanoma cells and their metastatic growth in the liver.     

Reversible skeletal abnormalities in gamma-glutamyl transpeptidase-deficient mice

Gamma-glutamyl transpeptidase (GGT) is a widely distributed ectopeptidase responsible for the degradation of glutathione in the gamma-glutamyl cycle. This cycle is implicated in the metabolism of cysteine, and absence of GGT causes a severe intracellular decrease in this amino acid. GGT-deficient (GGT-/-) mice have multiple metabolic abnormalities and are dwarf. We show here that this latter phenotype is due to a decreased of the growth plate cartilage total height resulting from a proliferative defect of chondrocytes. In addition, analysis of vertebrae and tibiae of GGT-/- mice revealed a severe osteopenia. Histomorphometric studies showed that this low bone mass phenotype results from an increased osteoclast number and activity as well as from a marked decrease in osteoblast activity. Interestingly, neither osteoblasts, osteoclasts, nor chondrocytes express GGT, suggesting that the observed defects are secondary to other abnormalities. N-acetylcysteine supplementation has been shown to reverse the metabolic abnormalities of the GGT-/- mice and in particular to restore the level of IGF-1 and sex steroids in these mice. Consistent with these previous observations, N-acetylcysteine treatment of GGT-/- mice ameliorates their skeletal abnormalities by normalizing chondrocytes proliferation and osteoblastic function. In contrast, resorbtion parameters are only partially normalized in GGT-/- N-acetylcysteine-treated mice, suggesting that GGT regulates osteoclast biology at least partly independently of these hormones. These results establish the importance of cysteine metabolism for the regulation of bone remodeling and longitudinal growth.     

Abnormal hepatic methionine and glutathione metabolism in patients with alcoholic hepatitis

BACKGROUND: Abnormal methionine metabolism occurs in animals fed ethanol and in end-stage cirrhotic patients. Expected consequences of these abnormalities include reduced hepatic S-adenosylmethionine and glutathione (GSH) levels, impaired transmethylation, and reduced homocysteine catabolism, resulting in the often-observed hyperhomocystinemia in cirrhotic patients. These parameters have not been examined simultaneously in patients with less advanced alcoholic liver disease. METHODS: Six patients hospitalized for alcoholic hepatitis were studied. Plasma was analyzed for homocysteine, methionine, and GSH levels. Liver biopsies diagnosed acute alcoholic hepatitis and underlying fibrosis. Liver specimens were processed for messenger RNA (mRNA) levels and various metabolites and were compared with those of six normal controls. RESULTS: Three patients had cirrhosis, and three had only portal fibrosis. Plasma levels of homocysteine and methionine were increased in two of the three patients with cirrhosis but not in the patients with fibrosis. All patients had markedly lower plasma GSH levels (mean +/- SD: 0.27 +/- 0.19 microM, which is at least 10-fold lower than the normal range). Hepatic S-adenosylmethionine levels were reduced by 50%, whereas methionine, GSH, and cysteine levels were reduced by 70-80%. The mRNA levels of most enzymes involved in methionine metabolism and GSH synthesis were decreased, whereas albumin expression was unchanged. Despite the well known induction of cytochrome P450 2E1 in chronic alcoholics, its mRNA levels were nearly 70% lower in these patients. CONCLUSIONS: In alcoholic hepatitis, abnormal hepatic gene expression in methionine and GSH metabolism occurs and often contributes to decreased hepatic methionine, S-adenosylmethionine, cysteine, and GSH levels. It may be important to replenish these thiols in patients hospitalized with alcoholic hepatitis.     

Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase

Because alcoholic liver disease has been linked to oxidative stress, we investigated the effect of a compromised antioxidant defense system, Cu, Zn-superoxide dismutase (Sod1) deficiency, on alcohol-induced liver injury. C57BL/129SV wild-type (Sod1(+/+)) and Sod1 knockout (Sod1(-/-)) mice were fed dextrose or ethanol (10% of total calories) liquid diets for 3 weeks. Histologic evaluation of liver specimens of Sod1(-/-) mice fed ethanol showed the development of liver injury ranging from mild to extensive centrilobular necrosis and inflammation. Sod1(+/+) mice fed ethanol showed mild steatosis; both Sod1(+/+) and Sod1(-/-) mice fed the dextrose diet had normal histology. Alanine transaminase levels were significantly elevated only in Sod1(-/-) mice fed ethanol. Cytochrome P450 2E1 (CYP2e1) activity was elevated about 2-fold by ethanol in Sod1(+/+) and Sod1(-/-) mice. Ethanol consumption increased levels of protein carbonyls and lipid peroxidation aldehydic products in the liver of Sod1(-/-) mice. Hepatic adenosine triphosphate (ATP) content was reduced dramatically in Sod1(-/-) mice fed ethanol in association with a decrease in the mitochondrial reduced glutathione (GSH) level and activity of MnSOD. Immunohistochemical determination of 3-nitrotyrosine (3NT) residues in liver sections of the Sod1 knockout mice treated with ethanol showed a significant increase of 3NT staining in the centrilobular areas. In conclusion, a rather moderate ethanol consumption promoted oxidative stress in Sod1(-/-) mice, with increased formation of peroxynitrite, protein carbonyls, and lipid peroxidation and decreased mitochondrial GSH and MnSOD. We speculate that the increased oxidative stress causes mitochondrial damage and reduction of ATP content, leading to alcoholic liver injury. This model may be useful in further mechanistic studies on alcohol-induced liver injury.     

Bleomycin-induced pulmonary fibrosis is attenuated in gamma-glutamyl transpeptidase-deficient mice

To investigate repair mechanisms in bleomycin-induced pulmonary fibrosis, we used mice deficient in gamma-glutamyl transpeptidase (GGT-/-), a key enzyme in glutathione (GSH) and cysteine metabolism. Seventy-two hours after bleomycin (0.03 U/g), GGT-/- mice displayed a different inflammatory response to wild-type mice as judged by a near absence of neutrophils in lung tissue and bronchoalveolar lavage and a less pronounced rise in matrix metalloproteinase-9. Inflammation in GGT-/- mice consisted mainly of lymphocytes and macrophages. At 1 month, lungs from bleomycin-treated GGT-/- mice exhibited minimal areas of fibrosis compared with wild-type mice(light microscopy fibrosis index: 510 +/- 756 versus 1975 +/- 817, p < 0.01). Lung collagen content revealed a significant increase in bleomycin-treated wild-type (15.1 +/- 3.8 versus 8.5 +/- 0.7 microg hydroxy(OH)-proline/mg dry weight, p < 0.01) but not in GGT-/- (10.4 +/- 1.7 versus 8.8 +/- 0.8). Control lungs from GGT-/- showed a significant reduction of cysteine (0.03 +/- 0.005 versus 0.055 +/- 0.001, p < 0.02) and GSH levels (1.24 +/- 0.055 versus 1.79 +/- 0.065, p < 0.002). These values decreased after 72 hours of bleomycin in both GGT-/- and wild-type but reached their respective control values after 1 month. Supplementation with N-acetyl cysteine partially ameliorated the effects of GGT deficiency. These findings suggest that increased neutrophils and matrix metalloproteinase-9 during the early inflammatory response and adequate thiol reserves are key elements in the fibrotic response after bleomycin-induced pulmonary injury.     

Progressive development of insulin resistance phenotype in male mice with complete aromatase (CYP19) deficiency

Aromatase (CYP19) is a cytochrome P450 enzyme that catalyzes the formation of aromatic C18 estrogens from C19 androgens. It is expressed in various tissues and contributes to sex-specific differences in cellular metabolism. We have generated aromatase-knockout (ArKO) mice in order to study the role of estrogen in the regulation of glucose metabolism. The mean body weights of male ArKO (-/-) mice (n=7) and wild-type littermates (+/+) (n=7) at 10 and 12 weeks of age were 26.7+/-1.9 g vs 26.1+/-0.8 g and 28.8+/-1.4 g vs 26.9+/-1.0 g respectively. The body weights of the ArKO and wild-type mice diverged between 10 and 12 weeks of age with the ArKO males weighing significantly more than their wild-type littermates (P<0.05). The ArKO males showed significantly higher blood glucose levels during an intraperitoneal glucose tolerance test compared with wild-type littermates beginning at 18 weeks of age. By 24 weeks of age, they had higher fasting blood glucose levels compared with wild-type littermates (133.8+/-22.8 mg/dl vs 87.8+/-20.3 mg/dl respectively; P<0.01). An intraperitoneal injection of insulin (0.75 mU insulin/g) caused a continuous decline in blood glucose levels in wild-type mice whereas ArKO males at 18 weeks and older exhibited a rebound increase in glucose levels 30 min after insulin injection. Thus, ArKO male mice appear to develop glucose intolerance and insulin resistance in an age-dependent manner. There was no difference in fasting serum triglyceride and total cholesterol levels between ArKO male mice and wild-type littermates at 13 and 25 weeks of age. However, serum triglyceride and cholesterol levels were significantly elevated following a meal in ArKO mice at 36 weeks of age. Serum testosterone levels in ArKO male mice were continuously higher compared with wild-type littermates. Treatment of ArKO males with 17beta-estradiol improved the glucose response as measured by intraperitoneal glucose and insulin tolerance tests. Treatment with fibrates and thiazolidinediones also led to an improvement in insulin resistance and reduced androgen levels. As complete aromatase deficiency in man is associated with insulin resistance, obesity and hyperlipidemia, the ArKO mouse may be a useful animal model for examining the role of estrogens in the control of glucose and lipid homeostasis.     

Role of macrophage-derived lipoprotein lipase in lipoprotein metabolism and atherosclerosis

Lipoprotein lipase (LPL) synthesis by macrophages is upregulated in early atherogenesis, implicating the possible involvement of LPL in plaque formation. However, it is still unclear whether macrophage-derived LPL displays a proatherosclerotic or an antiatherosclerotic role in atherosclerotic lesion development. In this study, the role of macrophage-derived LPL on lipid metabolism and atherosclerosis was assessed in vivo by transplantation of LPL-deficient (LPL-/-) and wild-type (LPL+/+) bone marrow into C57BL/6 mice. Eight weeks after bone marrow transplantation (BMT), serum cholesterol levels in LPL-/--->C57BL/6 mice were reduced by 8% compared with those in LPL+/+-->C57BL/6 mice (P:<0.05, n=16), whereas triglycerides were increased by 33% (P:<0.05, n=16). Feeding the mice a high-cholesterol diet increased serum cholesterol levels in LPL-/--->C57BL/6 and LPL+/+-->C57BL/6 mice 5-fold and 9-fold, respectively, resulting in a difference of approximately 50% (P:<0. 01) after 3 months on the diet. No effects on triglyceride levels were observed under these conditions. Furthermore, serum apolipoprotein E levels were reduced by 50% in the LPL-/--->C57BL/6 mice compared with controls under both dietary conditions. After 3 months on a high-cholesterol diet, the atherosclerotic lesion area in LPL-/--->C57BL/6 mice was reduced by 52% compared with controls. It can be concluded that macrophage-derived LPL plays a significant role in the regulation of serum cholesterol, apolipoprotein E, and atherogenesis, suggesting that specific blockade of macrophage LPL production may be beneficial for decreasing atherosclerotic lesion development.     

Interleukin 10-deficient mice exhibit defective colonic Muc2 synthesis before and after induction of colitis by commensal bacteria

Germ-free (GF) interleukin 10-deficient (IL-10) mice develop chronic colitis after colonization by normal enteric bacteria. Muc2 is the major structural component of the protective colonic mucus. Our aim was to determine whether primary or induced aberrations in Muc2 synthesis occur in GF IL-10 mice that develop colitis after bacterial colonization. GF IL-10 and wild-type mice were colonized with commensal bacteria for various intervals up to 6 weeks. Colitis was quantified by histologic score and IL-12 secretion. Muc2 synthesis, total level of Muc2, and Muc2 sulfation were measured quantitatively. GF IL-10 mice showed 10-fold lower Muc2 synthesis and Muc2 levels compared with GF wild-type mice, but Muc2 sulfation was not different. When bacteria were introduced, IL-10 mice developed colitis, whereas wild-type mice remained healthy. Muc2 synthesis was unchanged in wild-type mice, but IL-10 mice showed a peak increase in Muc2 synthesis 1 week after bacterial introduction, returning to baseline levels after 2 weeks. Total Muc2 levels decreased 2-fold in wild-type mice but remained at stable low levels in IL-10 mice. Upon introducing bacteria, Muc2 sulfation increased 2-fold in wild-type mice, whereas in IL-10 mice Muc2 sulfation decreased 10-fold. In conclusion, a primary defect in colonic Muc2 synthesis is present in IL-10 mice, whereas bacterial colonization and colitis in these mice led to reduced Muc2 sulfation. These quantitative and structural aberrations in Muc2 in IL-10 mice likely reduce the ability of their mucosa to cope with nonpathogenic commensal bacteria and may contribute to their susceptibility to develop colitis.     

Obesity and diabetes in transgenic mice expressing proSAAS

ProSAAS is a neuroendocrine peptide precursor that potently inhibits prohormone convertase 1 in vitro. To explore the function of proSAAS and its derived peptides, transgenic mice were created which express proSAAS using the beta-actin promoter. The body weight of transgenic mice was normal until approximately 10-12 weeks, and then increased 30-50% over wild-type littermates. Adult transgenic mice had a fat mass approximately twice that of wild-type mice, and fasting blood glucose levels were slightly elevated. In the pituitary, the levels of several fully processed peptides in transgenic mice were not reduced compared with wild-type mice, indicating that the proSAAS transgene did not affect prohormone convertase 1 activity in this tissue. Because the inhibitory potency of proSAAS-derived peptides towards prohormone convertase 1 is much greater in the absence of carboxypeptidase E activity, the proSAAS transgene was also expressed in carboxypeptidase E-deficient Cpe (fat/fat) mice. Although the transgenic mice were born in the expected frequency, 21 of 22 proSAAS transgenic Cpe (fat/fat) mice died between 11 and 26 weeks of age, presumably due to greatly elevated blood glucose. The levels of several pituitary peptides were significantly reduced in the proSAAS transgenic Cpe (fat/fat) mice relative to non-transgenic Cpe (fat/fat) mice, suggesting that the transgene inhibited prohormone convertase 1 in these mice. Taken together, these results are consistent with a role for proSAAS-derived peptides as neuropeptides that influence body weight independently of their function as inhibitors of prohormone convertase 1.     

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2((-/-))). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2((+/+))) mice and GstP1/P2((-/-)) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2((+/+)) and GstP1/P2((-/-)) mice, GSH levels only recovered in the GstP1/P2((-/-)) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity.     

Effect of age on alteration of glutathione metabolism following chronic cigarette smoke inhalation in mice

Cigarette smoke is the most common oxidant stress in daily life and may affect the antioxidant capacity in humans and animals. The antioxidant functions may play an important role in preventing age-related disorders. However, influences of chronic cigarette smoke on the antioxidant capacity of visceral organs have not been investigated in the age. Senescene-accelerated mice (SAM) are good models for studying physiologic and/or pathologic aging. A senescene-prone strain, SAMP2, shows characteristics of premature aging. The senescence-resistant strain, SAMR1, exhibits relatively normal aging. In this study we examined the effects of chronic cigarette smoke exposure on the glutathione (GSH) metabolism of visceral organs in the two strains of mice that were 6 and 18 months old. After a 4-week cigarette or air exposure, total GSH and oxidized GSH (GSSG) in the organs were examined. In the young (6-month-old) mice, exposure to cigarette smoke caused a significant decrease of GSH in liver, blood, and lung of SAMP2 but not in those of SAMR1. In the aged (18-month-old) mice reduced GSH with a marked increase of GSSG were found in liver of both strains of SAM following cigarette smoke exposure. The baseline values of GSH and the GSSG/GSH ratio after air exposure were slightly changed with age, and the values after exposure to cigarette smoke were changed markedly with advancing age. These results indicate that GSH metabolism may be impaired by chronic cigarette smoke exposure in mice and that aged mice are more susceptible to cigarette smoke than young mice.     

β-catenin is essential for ethanol metabolism and protection against alcohol-mediated liver steatosis in mice

The liver plays a central role in ethanol metabolism, and oxidative stress is implicated in alcohol-mediated liver injury. β-Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β-catenin regulates the hepatic response to ethanol ingestion. Female liver-specific β-catenin knockout (KO) mice and wild-type (WT) littermates were fed the Lieber-Decarli liquid diet (5% ethanol) in a pairwise fashion. Liver histology, biochemistry, and gene-expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol-fed (EtOH) KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and 5 to 6-fold higher serum alanine aminotransferase and aspartate aminotransferase levels. KO mice had a modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N-Acetylcysteine did not prevent ethanol-induced mortality in KO mice. In WT livers, β-catenin was found to coprecipitate with forkhead box O3, the upstream regulator of SOD2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were up-regulated in EtOH WT mice, but were nearly undetectable in KO mice. These changes in ethanol-metabolizing enzymes were associated with 30-fold higher blood alcohol levels in KO mice. CONCLUSION: β-Catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol-mediated liver steatosis. Our results strongly suggest that integration of these functions by β-catenin is critical for adaptation to ethanol ingestion in vivo.     

Hypoglycemia with enhanced hepatic glycogen synthesis in recombinant mice lacking hexose-6-phosphate dehydrogenase

Hexose-6-phosphate dehydrogenase (H6PDH) knockout (KO) mice have reduced generation of nicotinamide adenine dinucleotide phosphate (reduced) within the endoplasmic reticulum. As a consequence, 11beta-hydroxysteroid dehydrogenase type 1 enzyme activity switches from a reductase to a dehydrogenase leading to glucocorticoid inactivation. 11beta-Hydroxysteroid dehydrogenase type 1 has emerged as an important factor in regulating hepatic glucose output; therefore, we examined aspects of glucose homeostasis in KO mice. Compared with wild-type mice, KO mice reduced weight gain, displayed peripheral fasting hypoglycemia, improved glucose tolerance, and elevated plasma corticosterone concentrations. Plasma insulin levels in fed and fasted KO mice are normal; however, insulin and plasma glucose levels are reduced 4 h after fasted animals are refed, indicating improved insulin sensitivity. There is preserved induction and activity of the glucocorticoid-responsive gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in fasted KO mice. Glycogen storage is elevated in fed KO liver, with fed glycogenesis rates increased in KO mice. There is normal flux of lactate through gluconeogenesis recovered as plasma glucose, coupled with increased glycogen derived from lactate. These data suggest partial retention of glucocorticoid sensitivity at the level of the liver. We therefore postulate that increased glycogen synthesis may reflect increased flux of glucose-6-phosphate (H6PDH substrate) through to glycogen in the absence of H6PDH mediated metabolism.     

Angiotensin-converting enzyme C-terminal catalytic domain is the main site of angiotensin I cleavage in vivo

Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polypeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.     

Hepatic fatty acid synthesis is suppressed in mice with fatty livers due to targeted apolipoprotein B38.9 mutation

Humans and genetically engineered mice with hypobetalipoproteinemia due to truncation-producing mutations of the apolipoprotein B (apoB) gene frequently have fatty livers, because the apoB defect impairs the capacity of livers to export triglycerides (TGs). We assessed the adaptation of hepatic lipid metabolism in our apoB-38.9-bearing mice. Hepatic TG contents were 2- and 4-fold higher in heterozygous and homozygous mice, respectively, compared with wild-type mice. Respective in vivo hepatic fatty acid synthetic rates were reduced to 40% and 15% of the wild-type rate. Hepatic mRNAs for sterol regulatory element-binding protein (SREBP)-1c, fatty acid synthase (FAS), and stearoyl coenzyme A desaturase-1 were coordinately decreased. FAS and SREBP-1c mRNA levels were strongly and positively correlated with each other and inversely correlated with hepatic TGs, suggesting that impaired TG export is a potent inhibitor of fatty acid synthesis. In contrast, levels of plasma beta-hydroxybutyrate and of hepatic carnitine palmitoyl transferase and peroxisome proliferator-activated receptor-alpha mRNAs were not altered, implying that beta-oxidation was not affected. Fasting followed by refeeding increased hepatic fatty acid synthesis 56-fold over fasting in normal and heterozygous mice but only 24-fold in homozygous mice. Parallel changes occurred in FAS and SREBP-1c mRNAs. Thus, impairment of very low density lipoprotein export downregulates hepatic fatty acid synthesis, but the adaptation is incomplete, resulting in fatty livers. The signals mediating suppression of FAS and SREBP-1c levels remain to be identified.     

Effects of hypercholesterolemia on myocardial ischemia-reperfusion injury in LDL receptor-deficient mice

Hypercholesterolemia is a primary risk factor for atherosclerosis, coronary artery disease, and myocardial infarction. We subjected low density lipoprotein receptor-deficient (LDLr -/-) and control (wild-type) mice to 30 minutes of myocardial ischemia and 120 minutes of reperfusion. Myocardial infarction per area at risk (AAR) was noted under baseline conditions to be significantly (P<0.05) smaller in the LDLr -/- mice compared with wild-type mice (24.7+/-3. 2% and 38.8+/-4.3% of AAR, respectively). Subsequently, mice were fed a high-cholesterol diet (HCD) for 2 or 12 weeks, which resulted in significant increases in serum cholesterol levels in both LDLr -/- and wild-type groups. After 2 weeks of the HCD, the LDLr -/- mice demonstrated a significant elevation (P<0.01) in myocardial necrosis per AAR (50.2+/-5.36% of AAR) compared with the normal-diet LDLr -/- group, whereas the short-term HCD-fed wild-type mice demonstrated no significant difference from baseline. In contrast, wild-type mice fed the HCD for 12 weeks revealed a significant (P<0. 05) decrease in necrosis per AAR, which was 22.5+/-3.2% of the AAR in comparison with that in the normal-diet wild-type mice (38.8+/-4. 3% of AAR). LDLr -/- mice on the same long-term HCD showed a similar significantly (P<0.05) decreased infarct size, which was 13.2+/-4.0% of the AAR. In additional experiments, we determined that myocardial tissue total glutathione (GSH) levels were reduced after 2 weeks of the HCD and were significantly increased after 12 weeks of the HCD in the LDLr -/- mouse heart. These data suggest that short-term cholesterol feeding renders the myocardium of LDLr -/- mice more susceptible to ischemia-reperfusion injury, whereas more long-term hypercholesterolemia confers cardioprotection in the LDLr -/- mouse heart.     

Effect of rotavirus infection on small gut pathophysiology in a mouse model

AIM: To study the effect of rotavirus infection on amino acid uptake in the small intestine in an experimental mouse model. METHODS: Infant mice (7-8 days of age) were orally infected with the EB serotype 3 strain of rotavirus and animals (both infected and control) were killed on days 0, 1, 3, 5, 7 and 10 post inoculation (PI) after recording their bodyweights. Gamma-glutamyltranspeptidase (GGT), leucine amino peptidase (LAP), glutathione (GSH) and leucine amino acid uptake in the jejunum and ileum were assessed. RESULTS: Infected animals showed significant reduction in their bodyweights and intestinal lengths on the third day PI compared with controls. Leucine amino peptidase, GGT, GSH and leucine uptake were significantly reduced in the ileum on the third and fifth day PI compared with controls while uptake in the jejunum was only reduced on third day PI. The LAP, GGTP, GSH and leucine uptake attained normal levels on the seventh day when diarrhoea stopped. Histology showed damage to the villi in the jejunum and prominent cytoplasmic vacuolation in the ileum of infected animals on the third and fifth day PI. CONCLUSION: Rotavirus affects amino acid uptake in the small intestine at the time of peak infection.