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Objective: The present pilot trial was carried out to evaluate the effects of an acute treatment with a mixture containing 500 million of Lactobacillus bulgaricus and Streptococcus thermophilus per day in patients with non alcoholic fatty liver disease (NAFLD)., Research Methods: A sample of 30 patients with NAFLD (diagnosed by liver biopsy) was enrolled and 28 patients were analyzed in a double blind randomized clinical trial. Patients were randomized to one of the following treatments during 3 months: group I, treated with one tablet per day with 500 million of Lactobacillus bulgaricus and Streptococcus thermophilus and group II, treated with one placebo tablet (120 mg of starch)., Results: In group I, alanine amino transferase (ALT: 67.7 +/- 25.1 vs. 60.4 +/- 30.4 UI/L; p < 0.05), aspartate aminotransferase activity (AST: 41.3 +/- 15.5 vs. 35.6 +/- 10.4 UI/L; p < 0.05) and gammaglutamine transferase levels (gammaGT: 118.2 +/- 63.1 vs. 107.7 +/- 60.8 UI/L; p < 0.05) decreased. In group II, all liver function parameters remained unchanged (ALT: 60.7 +/- 32.1 vs. 64.8 +/- 35.5 UI/L; p < 0.05), aspartate aminotransferase activity (AST: 31.7 +/- 13.1 vs. 36.4 +/- 13.8 UI/L; ns) and gammaglutamine transferase levels (gammaGT: 82.1 +/- 55.1 vs. 83.6 +/- 65.3 UI/L; ns). Anthropometric parameters and cardiovascular risk factors remained unchanged after treatment in both groups., Conclusion: A tablet of 500 million of Lactobacillus bulgaricus and Streptococcus thermophilus, with a randomized clinical design, improved liver aminotransferases levels in patients with NAFLD.

Background: Osteocalcin is a hormone with a complex cross-talk between adipose tissue and the skeleton. The aim of the present study was to explore the relation of osteocalcin with histopathological changes of NALFD patients., Subjects: A population of 69 NAFLD patients was analyzed. A liver biopsy was realized. Weight, fat mass, body mass index, basal glucose, insulin, insulin resistance (HOMA), total cholesterol, LDLcholesterol, HDL-cholesterol, triglycerides and osteocalcin levels were measured., Results: Patients were divided in two groups by median osteocalcin value (11.34 ng/mL), group I (patients with the low values) and group II (patients with the high values). Only liver fibrosis frequencies were different between groups (group I: 22.9% vs group II: 9.4%; p < 0.05). Patients in group I had higher levels of glucose (115.6 ± 28.1 mg/dL vs. 103.7 ± 24.3 mg/dL; p < 0.04), HOMA (4.6 ± 3.1 units vs. 3.6 ± 1.8 units; p < 0.04), weight (102.9 ± 32.4 kg vs. 85.9 ± 16.8 kg; p = 0.002) and body mass index (38.3 ± 11.4 kg/m(2) vs. 30.1 ± 5.7 kg/m(2); p = 0.001)) than patients in group II. Osteocalcin was inverse correlated with glucose (r =-0.4; p = 0.002) and HOMA (r = -0.3:p = 0.01)., Conclusion: Osteocalcin is associated with liver fibrosis. However, this association disappeared in a multivariate analysis, and HOMA remained as an independent factor.

Background: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease. The aim of the present study was to explore the relation of visfatin with underlying histopathological changes of NAFLD patients., Subjects: A population of 55 NAFLD patients was analyzed in a cross-sectional study. A liver biopsy was realized. Weight, basal glucose, insulin, insulin resistance (HOMA), total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, and visfatin levels were measured. A bioimpedance was performed., Results and Conclusions: The mean age was 42.8 +/- 11.2 years, the mean BMI was 33.1 +/- 10.2 with 37 males (67.3%) and 18 females (32.7%). Probabilities to have; portal inflammation increased 1.11 (CI95%:1.03-1.50) with each increment of 1 ng/ml of visfatin concentration, high grade of steatosis increased 1.25 (CI 95%:1.06-1.61) with each unit of insulin concentrations, fibrosis increased 1.12 (CI 95%:1.02-1.43) with each unit of fat mass and lobulillar inflammation increased 13.4 (CI 95%:1.3-147) with each unit of HOMA-IR. Portal inflammation frequencies were different between groups (low visfatin group 13.07 < ng/ml: 37.5% versus high visfatin group 13.07 > ng/ml: 62.5%; P < 0.05). In conclusion, several histopathological changes in liver biopsies could be explained by insulin concentrations, HOMA-IR, and fat mass amount. Moreover, visfatin plasma concentrations could predict the presence of portal inflammation in NAFLD patients.

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents.

The in vivo radioactivity decay of glutathione S-transferase (GST) was observed in livers of normal-fed (N), protein-depleted (D), and re-fed mice (R), labelled with [35S]methionine. Half-lives in days at N, D and R, respectively, were: total GST, 1.7, 1.4, 4.3; Yb1-subunit, 2.0, 1.8, 3.3; Yc-subunit, 2.4, 1.0, 4.2; Yf-subunit, 1. 1, 2.1, 5.0. These findings, together with the fact that the proportions of GST-subunits depend on dietary protein, show that breakdown contributes differentially to control the content of each GST subunit. Data also indicate that GSTs are long-life proteins.

The effect of protein depletion followed by refeeding with a normal diet on the content of mouse liver cytosolic proteins was studied. By peptide-mass fingerprinting and N-terminal sequencing, three polypeptides whose contents changed with dietary protein level were identified as glutathione S-transferases (GST) Yb1, Yc and Yf subunits. Five days of depletion caused the increase of Yb1 and Yf (21.6% and 78.5%, respectively) and the decrease of Yc (31.2%). After two days of refeeding, Yb1 and Yc were practically restored, while the neoplastic marker Yf remained higher (63.4%). None of the nutritional conditions tested induced new GSTs. While protein depletion-refeeding altered the ratios between the constitutive GST subunits, total liver GST content and activity were unaffected by depletion and slightly increased by refeeding. The increased amounts of Yb1 and Yf, and the maintenance of total GST content, indicate that during protein depletion, the GST subunits levels are controlled by mechanisms different from the majority of cytosolic proteins.

The effect of protein depletion and refeeding with a normal diet on mouse liver soluble homogenate calpain activity was studied. It was unchanged when expressed in terms of whole liver (units/liver). However, when expressed in terms of degradable protein (units/mg protein) it increased with depletion and decreased with refeeding. DEAE Sephacel chromatography of soluble homogenate yielded three calpain activities which were eluted at 0.04, 0.16 and 0.23 M NaCl, respectively. On the basis of whole liver, they decreased with depletion and increased with refeeding. Immunochemical analysis revealed similar changes in the mass of the calpain eluted with 0.16 M NaCl. The sum of these three activities (total liver calpain activity) was higher than the activity displayed by the soluble homogenate, indicating that they were separated from calpastatin. Furthermore, the percentage of total calpain activity displayed by soluble homogenate increased with depletion and decreased with refeeding, suggesting that depleted liver had the lowest calpastatin content. This was confirmed by direct measurements which indicated that depleted homogenate had in average 5.5 and 3.1 times less calpastatin compared to normal and 16 hours refed liver, respectively. It is concluded that a remarkable decrease in calpastatin content maintained unchanged whole liver soluble homogenate calpain activity during protein depletion and refeeding and contributes to an increased calpain activity related to degradable protein in depleted livers. This increase is in accordance with the high in vivo rate of protein breakdown depicted by these livers.

The proteolytic activity in isolated liver nuclei from mice subjected to different conditions of protein nutrition and its relationship with histones metabolism was studied. After five days of protein depletion, the nuclear azocaseinolytic activity increases concomitantly with a decrease in the concentration of histones. This activity resembles, in localization, optimum pH and inhibition behavior to rat liver chromatin neutral proteinase that degrades histones. Moreover, these proteins were identified as its main endogenous substrates. Refeeding of the protein depleted mice for 16h with a normal diet was unable to either diminish proteolytic activity or recover the normal histone level. Activity of the multicatalytic proteinase complex (proteasome) was not detected in nuclei. Furthermore, treatments known to activate this enzyme were ineffective. Taken together, these results suggest that nuclear proteases are mainly involved in the regulation of histone levels.

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a M(r) 36,000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The M(r) 36,000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.

The effect of protein depletion and refeeding on the metabolism of mouse liver nuclear proteins was studied. Five days protein depletion caused a 35% decrease in total nuclear protein. A fast recovery of the lost proteins, except histones, was induced when depleted mice were refed with a normal diet. Depletion caused a decrease in total nuclear protein synthesis, whereas refeeding quickly restored its normal value. The rates of total nuclear protein breakdown were estimated either as the difference between synthesis and protein gain or from the decay of radioactivity in protein labeled by the administration of both sodium [14C]bicarbonate and [35S]methionine. By these procedures, it was found that refeeding caused a slowdown in total nuclear protein breakdown. Hence, the recovery of the protein content observed during refeeding is due to both a restoration of synthesis and a decrease of breakdown. The [14C]bicarbonate procedure did not permit to obtain a high efficiency of label and, therefore, it was unsatisfactory for the measurement of the breakdown of fractionated nuclear proteins. A labeling procedure using [35S]methionine was designed for adequate measures of the decay of radioactivity in these proteins. This allows us to find that a slow down in breakdown affects similarly during refeeding to histones, to non histones, and to a fraction which contains ribonucleoproteins and soluble proteins.

Proteolytic activities at pH 5.0 and pH 7.4 in both kidney and liver during refeeding of protein-depleted mice were determined. Under this nutritional condition, the in vivo rate of protein breakdown is inhibited. Protein depletion caused in both kidney and liver a loss of pH 5.0 proteolytic activity. It was restored to normal values after 12 hours of refeeding with a complete diet. The pH 7.4 proteolytic activity also decreased as a consequence of protein depletion, but remained low during refeeding. Both re-fed kidney and liver lysosomes showed an increased stability to osmotic shock and, therefore, a decreased autophagic activity. The results indicate that both kidney and liver exhibit a similar behavior. The decreased activity of the lysosomal-vacuolar system, and the low pH 7.4 proteolytic activity may account for the observed in vivo protein breakdown inhibition during refeeding of protein-depleted mice.

The rapid restoration of liver protein mass observed in protein-depleted mice when they are fed with an adequate diet is quantitatively explained by a large decrease in the average rate of breakdown of total liver proteins. This study was performed in order to know whether this inhibition of breakdown affects in the same way all the protein constituents of the tissue, or only affects a group of these proteins belonging to a particular subcellular fraction. Subcellular fractions were obtained by differential centrifugation. The relative rates of breakdown of their proteins were estimated by the conservation of radioactivity in these proteins previously labelled by the administration of NaH14CO3 to mice. The results obtained indicated: 1) a general decrease in the rate of breakdown of proteins of subcellular fractions from re-fed livers compared with livers of protein-depleted mice; 2) a decrease of breakdown of proteins from cytosol in re-fed mice which is higher as lower is the molecular weight of the proteins subunits.

In the livers of 5-days-protein-depleted mice there is a decrease of 47% of the ribosome mass. When these animals are fed with an adequate diet, ribosome content is restored to the normal value after 1 day of re-feeding. The mechanisms underlying this phenomenon were studied. It was found that: (1) the activity of RNA polymerase I in the nuclei of livers from re-fed animals showed an enhancement of about 2-fold compared with the activity in normal and protein-depleted liver nuclei; (2) ribosome degradation, measured by the disappearance of radioactivity from ribosomal proteins previously labelled by the administration of NaH14CO3 to the mice, stopped during the first day after re-feeding.

A study is presented of the liver protein gain during the early stages of postnatal development. Fractional rates of protein synthesis and degradation were determined in vivo in livers of 4-day-old mice. At this age, liver protein accumulated at a rate of 18% per day. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was extimated as the balance between synthesis and accumulation of stable liver proteins, or from the disappearance of radioactivity from liver protein previously labelled by the administration of NaH14CO3. We found that the neonatal livers: (1) synthesize 139% as much protein per unit time and unit mass as adult tissue, which is accounted for by a higher ribosome concentration (synthesis per mg of RNA was the same); (2) retain 39% of the newly synthesized protein as stable liver components (compared with 48% in adult mice); (3) degrade protein at 56% of the rate in the adult liver. This lower rate of degradation is quantitatively the most significant difference between the growing and non-growing liver.

Shortly after feeding protein-depleted mice with a meal containing protein, the RNA polymerase I activity in isolated liver nuclei shows a 2-fold increase over the values in the nuclei of either normal or protein-depleted mice. The activity of the RNA polymerase I solubilized from nuclei of re-fed mice was slightly enhanced, probably reflecting an increase in enzyme amount. However, this increase only accounts for about 30% of the stimulation of transcription in the intact nuclei. Administration of pactamycin, an inhibitor of protein synthesis, to normal or protein-depleted mice has almost no inhibitory effect on the RNA polymerase I activity in the isolated nuclei. On the contrary, within 15 min after treatment with the drug, the stimulated activity in nuclei from re-fed mice declines towards the values in normal or protein-depleted mice and then remains constant. The activity of the solubilized enzyme remains slightly elevated for at least 2 1/2 h after re-fed mice are treated with pactamycin. These observations indicate that the stimulation of the RNA polymerase I activity in the intact nuclei after re-feeding is controlled by mechanisms other than an increase in the enzyme amount and suggest the presence of short-lived proteins required for inducing an activated state of transcription.

The effect of hypophysectomy on the protein metabolism of the liver in vivo was studied. Fractional rates of protein synthesis and degradation were determined in the livers of normal and hypophysectomized rats. Synthesis was measured after the injection of massive amounts of radioactive leucine. Degradation was estimated either as the balance between synthesis and accumulation of stable liver proteins or from the disappearance of radioactivity from the proteins previously labelled by the injection of NaH14CO3. The results indicate that: (1) hypophysectomy diminishes the capacity of the liver to synthesize proteins in vivo, mainly of those that are exported as plasma proteins; (2) livers of both normal and hypophysectomized rats show identical protein-degradation rates, whereas plasma proteins are degraded slowly after hypophysectomy.

Fractional rates of synthesis and degradation of liver porteins were estimated during the rapid restoration of liver mass observed in protein-depleted mice when they are fed with an adequate diet. 1. Net protein gain was fastest 12h after the nutritional shift, when it reached a rate of 48% per day. 2. The RNA/protein ratio in livers of protein-depleted animals was essentially the same as in normal livers; it increased by a maximum of 13% 12h after the nutritional shift. 3. Rates of protein synthesis in vivo were measured by the incorporation into liver protein of massive amounts of L-[1-14C]leucine. In protein-depleted animals, the rate of synthesis per mg of RNA was 72% of that in normal livers. Normal rates were recovered within 12h of the nutritional shift. 4. The fraction of newly synthesized protein retained by the liver was studied after they were pulse-labelled by the intravenous injection of radioactive leucine, and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis); 3h later the livers in both experimental situations retained 58% of the newly synthesized protein. 5. Fractional rates of protein degradation were estimated either from the difference between the synthesis of stable liver proteins and the net protein increase, or by the disappearance of radioactivity from the liver protein previously labelled by the administration to the mice of NaH14CO3. Both procedures demonstrated a large decrease in the rate of protein degradation during liver growth.

1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins.