América Giménez

Effects of S-adenosylmethionine on lipid peroxidation and liver fibrogenesis in carbon tetrachloride-induced cirrhosis.

BACKGROUND/AIM The aim of this study was to investigate the effects of S-adenosylmethionine on liver peroxidation and liver fibrogenesis in carbon tetrachloride-induced cirrhosis. METHODS Cirrhosis was induced in three groups of six rats by repeated injections of carbon tetrachloride over 9 weeks. One group of animals was treated only with carbon tetrachloride, and the other two received carbon tetrachloride plus S-adenosylmethionine (10 mg/kg intramuscularly daily) from week 3 to week 9, and from week 6 to week 9 of the study, respectively. Two additional groups of six rats, a control group and a group treated only with S-adenosylmethionine, were also studied. Glutathione concentration, thiobarbituric acid-reactive substances, collagen content, prolyl hydroxylase activity, and procollagen type I mRNA expression were determined in liver samples. RESULTS All carbon tetrachloride-treated rats had cirrhosis at the end of the study. Cirrhosis was also present in five of the six carbon tetrachloride-treated rats receiving S-adenosylmethionine for 3 weeks, but in only one of the six rats that received S-adenosylmethionine for 6 weeks. Hepatic glutathione was significantly diminished in carbon tetrachloride-treated rats (2.7 +/- 0.3 mumol/g tissue) and returned to normal in rats receiving S-adenosylmethionine for 3 or 6 weeks (3.7 +/- 0.13 and 3.9 +/- 0.11 mumol/g tissue, respectively). The hepatic thiobarbituric acid-reactive substances were significantly lower in rats treated with carbon tetrachloride and S-adenosylmethionine for 6 weeks (98 +/- 5 nmol/g) than in rats treated with carbon tetrachloride (134 +/- 12 nmol/g) and in those treated with carbon tetrachloride and S-adenosylmethionine for 3 weeks (127 +/- 13 nmol/g). There were no differences in either hepatic collagen and prolyl hydroxylase activity between rats that received only carbon tetrachloride and those treated with S-adenosylmethionine for 3 weeks. In contrast, carbon tetrachloride-treated rats receiving S-adenosylmethionine for 6 weeks had significantly lower collagen and prolyl hydroxylase activity than the other two groups. A much greater increase in procollagen type I mRNA was found in carbon tetrachloride-treated rats than in rats treated with carbon tetrachloride and S-adenosylmethionine for 6 weeks. Furthermore, there was a significant correlation between the hepatic thiobarbituric acid-reactive substances and prolyl hydroxylase activity and hepatic collagen. CONCLUSIONS We conclude that the early administration of S-adenosylmethionine in a model of carbon tetrachloride-induced liver injury restores glutathione levels and reduces lipid peroxidation, resulting in less advanced liver fibrosis.

Fibrogenic and collagenolytic activity in carbon-tetrachloride-injured rats: beneficial effects of zinc administration

Collagen synthesis and degradation in normal and carbon-tetrachloride-injured male Wistar rats at early and late stages of liver fibrosis, and the potential beneficial effects of zinc supplementation on liver fibrogenesis and collagenolysis have been assessed by measuring hepatic collagen content and prolyl hydroxylase and collagenase activities. No significant changes in hepatic collagen and prolyl hydroxylase activities were observed between control rats (82 +/- 25 cpm/mg protein) and rats with induced cirrhosis (107 +/- 23 cpm/mg protein) after 4 weeks of carbon tetrachloride injury. By this time, hepatic collagenase activity was significantly lower in rats with induced cirrhosis (61 +/- 9 micro units/mg protein) than in control rats (133 +/- 31 micro units/mg protein) (p < 0.05). This result was prevented by zinc administration, since hepatic collagenase activity was similar in zinc-supplemented, carbon-tetrachloride-injured rats and normal rats (148 +/- 19 micromicrons/mg protein). After 16 weeks, all carbon-tetrachloride-injured rats had cirrhosis. Hepatic collagen content and prolyl hydroxylase activity were significantly higher in carbon-tetrachloride-injured rats than in controls. These effects were partially prevented by zinc administration, since only two of the seven zinc-supplemented, carbon-tetrachloride-injured rats had cirrhosis. Moreover, prolyl hydroxylase activity was significantly lower in zinc-supplemented injured rats (263 +/- 27 cpm/mg protein) than in the non-supplemented respective controls (389 +/- 52 cpm/mg protein) (p < 0.05). No significant changes in hepatic collagenase activity were observed at this stage of liver injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Pattern of methionine adenosyltransferase isoenzyme expression during rat liver regeneration after partial hepatectomy

The present report investigates the pattern of expression of liver‐specific and extrahepatic methionine adenosyltransferase (MAT) isoenzymes in regenerating rat liver after partial hepatectomy. The results show that there is a switch in the expression of these isoenzymes that is coincident with maximal cell proliferation in the remaining liver lobes. Extrahepatic MAT levels increase about three times 4 h after hepatectomy, reaching a maximum 36 h later. This is accompanied by a rapid and transient increase in total MAT activity and levels of related metabolites S‐adenosyl‐l‐methionine and S‐adenosyl‐l‐homocysteine. Liver‐specific MAT levels are reversibly down‐regulated within 24–48 h post surgery. Our results indicate that MAT isoenzyme expression is tightly regulated during liver regeneration after two‐third hepatectomy. The implications of these observations for evaluation of the degree of liver regeneration are briefly discussed.

Role of zinc in the process of pancreatic fibrosis in chronic alcoholic pancreatitis.

Zinc acts as a cofactor in many enzymatic processes, including collagen synthesis. The observation of increased activity of prolylhydroxylase (PHase), an enzyme that takes part in the synthesis of collagen, in zinc-depleted liver tissue suggests that zinc deficiency is associated with hepatic collagen deposition. The pancreatic zinc content in chronic alcoholic pancreatitis (CAP) is still unknown. The objectives of this study were (a) To assess zinc concentrations in the pancreatic tissue in CAP; (b) to establish a possible relation between pancreatic zinc content, fibrosis, and PHase activity; and (c) to evaluate the relation between serum and pancreatic zinc levels. Sixteen surgical specimens of pancreatic tissue from patients with CAP were analyzed; control pancreatic samples from II organ donors were also studied. Zinc concentration, Phase activity, and the amount of fibrous tissue were assessed in the pancreatic tissue of each individual. The amount of fiber deposited in the pancreas in CAP was 68.4 ± 19.8%, and that of the control group, 5 ± 2% (p < 0.001). PHase activity in CAP was 754 ± 230 cpm/mg of protein and that of the control group was 405 ± 151 cpm/mg of protein (p < 0.001). The amount of pancreatic zinc in the former was 15.0 ± 9.7 pg/g of tissue and that of the latter was 28.1 ± 18.1 pg/g of tissue (p = 0.023). Serum zinc levels were similar in both groups. There was a significant inverse correlation between pancreatic zinc content and the quantity of fiber deposited in the tissue (r = −0.437; p = 0.026), as well as between pancreatic zinc and PHase activity (r = −0.466; p = 0.029). The reduction of zinc in the pancreatic tissue in CAP may enhance collagen synthesis and increase fibrous tissue deposition in this organ. It could also influence the unfavorable evolution of the disease.

Synthesis and degradation of collagen in pancreatic fibrogenesis.

The mechanism of fibrogenesis in the pancreas is not well known. We analyzed the role of prolylhydroxylase and collagenase activities in the development of fibrosis in chronic alcoholic pancreatitis (CAP). Nineteen patients with CAP and 11 controls (organ donors) with normal pancreatic histology were included in the study. Pancreatic tissue was obtained from all subjects to measure (a) area of fibrosis (histomorphometric method); (b) prolylhydroxylase activity (PHase), which reflects the intracellular synthesis of collagen (Huttons method); and (c) collagenase activity, which reflects the degradation of collagen (collagenase assay system, 3H). The percentage of the fibrosis area in relation to the total area of pancreatic tissue was significantly higher in CAP than in the control group (70.6+/-20.2% vs. 4.6+/-1.8%; p<0.001). Mean pancreatic PHase activity was also significantly higher in CAP than in the control group (775+/-258 cpm/mg protein/h vs. 405+/-151 cpm/mg protein/h; p<0.001). The collagenase activity was significantly lower in CAP than in the control group (8.7+/-3.5 cpm/cpm added/mg protein vs. 18.0+/-3.9 cpm/cpm added/mg protein; p<0.001). A significant correlation was observed between percentage fibrosis evaluated histomorphometrically and PHase activity in all patients (r = 0.72; p<0.001), and between PHase and collagenase activities in controls (r = 0.70; p = 0.024), but not in CAP. Pancreatic tissue in CAP has an increased fibrogenic activity and an impaired collagen-degradation capacity. These findings might explain the excessive development of fibrosis in CAP.

Cyclin-Dependent Kinase 4 Hyperactivity Promotes Autoreactivity in the Immune System but Protects Pancreatic β Cell Mass from Autoimmune Destruction in the Nonobese Diabetic Mouse Model

Cyclin-dependent kinase 4 (Cdk4) plays a central role in perinatal pancreatic β cell replication, thus becoming a potential target for therapeutics in autoimmune diabetes. Its hyperactive form, Cdk4R24C, causes β cell hyperplasia without promoting hypoglycemia in a nonautoimmune-prone mouse strain. In this study, we explore whether β cell hyperproliferation induced by the Cdk4R24C mutation balances the autoimmune attack against β cells inherent to the NOD genetic background. To this end, we backcrossed the Cdk4R24C knockin mice, which have the Cdk4 gene replaced by the Cdk4R24C mutated form, onto the NOD genetic background. In this study, we show that NOD/Cdk4R24C knockin mice exhibit exacerbated diabetes and insulitis, and that this exacerbated diabetic phenotype is solely due to the hyperactivity of the NOD/Cdk4R24C immune repertoire. Thus, NOD/Cdk4R24C splenocytes confer exacerbated diabetes when adoptively transferred into NOD/SCID recipients, compared with NOD/wild-type (WT) donor splenocytes. Accordingly, NOD/Cdk4R24C splenocytes show increased basal proliferation and higher activation markers expression compared with NOD/WT splenocytes. However, to eliminate the effect of the Cdk4R24C mutation specifically in the lymphocyte compartment, we introduced this mutation into NOD/SCID mice. NOD/SCID/Cdk4R24C knockin mice develop β cell hyperplasia spontaneously. Furthermore, NOD/SCID/Cdk4R24C knockin females that have been adoptively transferred with NOD/WT splenocytes are more resistant to autoimmunity than NOD/SCID WT female. Thus, the Cdk4R24C mutation opens two avenues in the NOD model: when expressed specifically in β cells, it provides a new potential strategy for β cell regeneration in autoimmune diabetes, but its expression in the immune repertoire exacerbates autoimmunity.

Hepatic fibrogenic activity in chronic alcoholic pancreatitis

The prevalence and the mechanisms of hepatic fibrosis in chronic alcoholic pancreatitis remain uncertain. The aim of this study was to investigate the fibrogenic activity of the liver in patients with chronic pancreatitis and its relation with either the alcohol or cholestasis. Liver biopsies were obtained from 16 patients with chronic pancreatitis at the time of surgery and from 10 organ donors. Samples were processed for histologic examination to assess the presence and extent of fibrosis, inflammatory reactions, and cholestasis- and alcohol-related lesions. In other samples, the collagen content was measured by morphometry, and prolylhydroxylase activity was determined. Liver-function tests, ultrasonography, and endoscopic retrograde cholangiopancreatography were performed before surgery in all the patients. Of patients with chronic pancreatitis, 75% had significantly greater hepatic fibrosis and prolylhydroxylase activity than the control group. Moreover, prolylhydroxylase activity in patients with chronic pancreatitis was higher in those with cholestasis or partial obstruction of the common bile duct than in those without cholestasis or partial obstruction of the common bile duct. Both the fibrogenic activity and the collagen content in the livers of patients with chronic alcoholic pancreatitis are significantly increased, even in those without histologic lesions, and these alterations may be secondary to a partial occlusion of the common bile duct.

Serum amino-terminal propeptide of type III procollagen levels in chronic pancreatitis.

The severity of pancreatic fibrosis, a characteristic feature of patients with chronic pancreatitis (CP), can be assessed only by direct histologic analysis of pancreatic tissue. Since serum levels of the amino-terminal type III procollagen propeptide (PIIIP) can reflect the degree of fibrogenic activity in several diseases associated with fibrosis, the current study was aimed at investigating whether PIIIP are increased in chronic pancreatitis, the relationship between PIIIP and pancreatic fibrogenic activity, and the influence of pancreatectomy, pancreatic exocrine function, and duration of disease on PIIIP levels. Serum PIIIP was measured in 18 patients with CP (15 without liver disease and three with cholestasis) and in 21 healthy controls. The effect of pancreatectomy on PIIIP was evaluated in seven patients, in whom PIIIP was measured immediately before and 2 months after surgery. Prolylhydroxylase (PHase) activity as an index of pancreatic fibrogenesis was evaluated in pancreatic tissue from 11 patients who had undergone subtotal pancreatectomy and from 11 organ donors. The bentiromide (BT)-PABA test as an index of exocrine pancreatic function was measured in all patients. PIIIP was significantly higher in patients who had or had not undergone pancreatectomy (17.3 ± 4.0 and 25 ± 11.4 ng/ml, respectively) than in controls (12.3 ± 3.1 ng/ml) (p < 0.001). PIIIP decreased significantly after pancreatectomy (before, 32.0 ± 9.3 ng/ml; after, 18.4 ± 4.8 ng/ml; p = 0.00s). PHase was significantly higher in patients (773 ± 250 cpm/mg protein) than in controls (405 ± 121 cpm/mg protein) (p < 0.001). PIIIP was correlated with pancreatic PHase (r = 0.7, p = 0.001) but not with BTPABA or with the duration of the disease. In conclusion, serum PIIIP levels are increased in patients with CP and reflect the severity of pancreatic fibrogenic activity. No relationship between the serum PIIIP levels and the pancreatic exocrine function and duration of disease was found.

Effects of ethanol feeding and malnutrition on collagen synthesizing and degrading enzymes in rat pancreas

This study was performed to delineate the combined effects of a low-fat diet and chronic ethanol ingestion on collagen metabolism in rat pancreas. Rats fed a very low-fat diet (5% of total calories as lipid) for 12 weeks developed malnutrition as judged by weight loss (-33% of the initial body weight) and low serum albumin and amylase levels. The pancreas of malnourished rats showed increased collagenase activity with respect to animals fed a 35% lipid diet (p < 0.05). Hydroxyproline content was higher in the pancreas of malnourished rats and collagenase activity correlated well with hydroxyproline content (r = 0.57, p = 0.0013). Ethanol feeding for 12 weeks, regardless of the nutritional state of the rats, did not change the synthesis and degradation rates of collagen in the pancreas. The present study suggests that malnutrition may have profound effects on collagen metabolism.

Influence of dietary zinc on hepatic collagen and prolyl-hydroxylase activity in alcoholic rats

The effects of dietary zinc on hepatic collagen and prolyl hydroxylase activity in normal and alcoholic rats has been investigated in four groups of pair-fed male Wistar rats given either liquid ethanol or a control diet for 12 wk. Each group of pair-fed animals received a diet with a different zinc concentration (standard diet, 7.6 mg/L; low-zinc diet, 3.4 mg/L; zinc-supplemented diet, 76 mg/L; and zinc-extrasupplemented, 300 mg/L. There were no significant differences in hepatic collagen concentration and prolyl hydroxylase activity between alcoholic and normal rats receiving a standard diet (collagen, 77 +/- 5 and 73 +/- 6 micrograms/mg protein; and prolyl hydroxylase; 37 +/- 26 and 36 +/- 22 cpm/mg protein). Alcoholic rats fed a low-zinc diet showed increased prolyl hydroxylase activity (75 +/- 10 cpm/mg protein, p less than 0.05), although no changes in hepatic collagen (77 +/- 10 micrograms/mg protein) were observed in comparison with rats fed a standard alcoholic diet. By contrast, hepatic collagen was significantly lower in alcoholic rats fed a zinc-supplemented diet (66 +/- 4 and 63 +/- 3 micrograms/mg protein, p less than 0.05 and p less than 0.01, respectively), and hepatic prolyl hydroxylase activity was particularly lower in rats receiving zinc 300 mg/L (18 +/- 20 cpm/mg protein). Similar effects were observed in normal rats. We conclude that dietary zinc influences hepatic prolyl hydroxylase activity and collagen deposition in alcoholic rats, and in consequence, the control of dietary zinc is necessary to assess the effects of alcohol on collagen metabolism in rats.