Decreased immunoexpression of survivin could be a potential marker in human non-alcoholic fatty liver disease progression?

BACKGROUND/AIM: Regulation of apoptosis in non-alcoholic fatty liver disease (NAFLD) has been a theme of growing debate. Although no other study assessed the role of survivin in NAFLD, its expression has been reported in hepatic carcinogenesis because of other aetiological factors with relevant discrepancies. The aim of this study was to assess the pattern of survivin immunoexpression by tissue microarray along the whole spectrum of NAFLD, including non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC). METHODS: Liver biopsies from 56 patients with NAFLD were evaluated: 18 with steatosis, 21 non-cirrhotic NASH, 10 NASH-related cirrhosis, seven NASH-related HCC, as compared with 71 HCC related to other causes and with 12 normal livers. RESULTS: Survivin immunoexpression in NAFLD was restricted to cytoplasm and was found to be progressively lower in advanced stages, including cirrhosis and HCC: steatosis vs NASH-related cirrhosis (P=0.0243); steatosis vs NASH-related HCC (P=0.0010); NASH vs NASH-related cirrhosis (P=0.0318); and NASH vs NASH-related HCC (P=0.0007), thus suggesting a deregulation of apoptosis from NAFLD towards HCC. Interestingly, survivin immunoreactivity in NASH-related HCC was also found to be significantly lower than in HCC related to other causes (P<0.05). Remarkably, nuclear staining for survivin was not detected in any case of NAFLD, contrasting to its presence in all other cases of HCC. CONCLUSIONS: Survivin immunoexpression in NASH-related HCC is herein originally found substantially different than in HCC related to other causes, thus requiring further studies to elucidate the role of survivin in human NAFLD progression.

Intake of trans fatty acids causes nonalcoholic steatohepatitis and reduces adipose tissue fat content.

We investigated the effects of dietary trans fatty acids, PUFA, and SFA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SFA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPARalpha, PPARgamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SFA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1c and PPARgamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPARalpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome.

Prevention and reversion of nonalcoholic steatohepatitis in OB/OB mice by S-nitroso-N-acetylcysteine treatment.

OBJECTIVE: To evaluate the role oral administration of S-nitroso-N-acetylcysteine (SNAC), a NO donor drug, in the prevention and reversion of NASH in two different animal models. METHODS: NASH was induced in male ob/ob mice by methionine-choline deficient (MCD) and high-fat (H) diets. Two animal groups received or not SNAC orally for four weeks since the beginning of the treatment. Two other groups were submitted to MCD and H diets for 60 days receiving SNAC only from the 31(st) to the 60(th) day. RESULTS: SNAC administration inhibited the development of NASH in all groups, leading to a marked decrease in macro and microvacuolar steatosis and in hepatic lipid peroxidation in the MCD group. SNAC treatment reversed the development of NASH in animals treated for 60 days with MCD or H diets, which received SNAC only from the 31(st) to the 60(th) day. CONCLUSIONS: Oral administration of SNAC markedly inhibited and reversed NASH induced by MCD and H diets in ob/ob mice.

Modulation of hepatic microsomal triglyceride transfer protein (MTP) induced by S-nitroso-N-acetylcysteine in ob/ob mice.

We evaluated the effects of a potent NO donor, S-nitroso-N-acetylcysteine (SNAC), on microsomal triglyceride transfer protein (MTP) expression in ob/ob mice. NAFLD was induced in male ob/ob mice using a methionine-choline deficient diet (MCD) concomitantly with oral SNAC fed solution (n=5) or vehicle (control; n=5) by gavage daily for 4 weeks. Livers were collected for histology and for assessing MTP by RT-qPCR, Western blot, immunohistochemistry and immunogold electron microscopy analyses. Histological analysis showed diffuse macro and microvesicular steatosis, moderate hepatocellular ballooning and moderate inflammatory infiltrate in ob/ob mice fed the MCD diet. With SNAC, mice showed a marked reduction in liver steatosis (p<0.01), in parenchymal inflammation (p=0.02) and in MTP protein immunoexpression in zone III (p=0.05). Moreover, SNAC caused reduction of MTP protein in Western blot analysis (p<0.05). In contrast, MTP mRNA content was significantly higher (p<0.05) in mice receiving SNAC. Immuno-electron microscopy showed MTP localized in the rough endoplasmic reticulum of hepatocytes in both treated and untreated groups. However with SNAC treatment, MTP was also observed surrounding fat globules. Histological improvement mediated by a nitric oxide donor is associated with significantly altered expression and distribution of MTP in this animal model of fatty liver disease. Further studies are in progress to examine possible mechanisms and to develop SNAC as a possible therapy for human fatty liver disease.

Oral administration of S-nitroso-N-acetylcysteine prevents the onset of non alcoholic fatty liver disease in rats.

AIM: To evaluate the potential of S-nitroso-N-acetylcysteine (SNAC) in inhibition of lipid peroxidation and the effect of oral SNAC administration in the prevention of nonalcoholic fatty liver disease (NAFLD) in an animal model. METHODS: NAFLD was induced in Wistar male rats by choline-deficient diet for 4 wk. SNAC-treated animals (n=6) (1.4 mg/kg per day of SNAC, orally) were compared to 2 control groups: one (n=6) received PBS solution and the other (n=6) received NAC solution (7 mg/kg per day). Histological variables were semiquantitated with respect to macro and microvacuolar fat changes, its zonal distribution, foci of necrosis, portal and perivenular fibrosis, and inflammatory infiltrate with zonal distribution. LOOHs from samples of liver homogenates were quantified by HPLC. Nitrate levels in plasma of portal vein were assessed by chemiluminescence. Aqueous low-density lipoprotein (LDL) suspensions (200 microg protein/mL) were incubated with CuCl(2) (300 micromol/L) in the absence and presence of SNAC (300 micromol/L) for 15 h at 37 degree Celsius. Extent of LDL oxidation was assessed by fluorimetry. Linoleic acid (LA) (18.8 micromol/L) oxidation was induced by soybean lipoxygenase (SLO) (0.056 micromol/L) at 37 degree Celsius in the presence and absence of N-acetylcysteine (NAC) and SNAC (56 and 560 micromol/L) and monitored at 234 nm. RESULTS: Animals in the control group developed moderate macro and microvesicular fatty changes in periportal area. SNAC-treated animals displayed only discrete histological alterations with absence of fatty changes and did not develop liver steatosis. The absence of NAFLD in the SNAC-treated group was positively correlated with a decrease in the concentration of LOOH in liver homogenate, compared to the control group (0.7+/-0.2 nmol/mg vs 3.2+/-0.4 nmol/mg protein, respectively, P<0.05), while serum levels of aminotransferases were unaltered. The ability of SNAC in preventing lipid peroxidation was confirmed in in vitro experiments using LA and LDL as model substrates. CONCLUSION: Oral administration of SNAC prevents the onset of NAFLD in Wistar rats fed with choline-deficient diet. This effect is correlated with the ability of SNAC to block the propagation of lipid peroxidation in vitro and in vitro.

Long-term nutritional outcome after gastric bypass.

BACKGROUND: Weight loss and nutritional status 5 or more years after Roux-en-Y gastric bypass was prospectively documented. The hypothesis was that even after clinical adaptation, imbalances might still occur. METHODS: Seventy-five consecutive patients (age 49.3 +/- 10.6 years, 89.3% females) were recruited 83.4 +/- 14.3 months after the intervention. Weight loss and nutritional abnormalities were registered. RESULTS: Body mass index (BMI) was 56.5 +/- 10.0 preoperatively, 29.4 +/- 6. 2 by 24 months and 34.4 +/- 14.6 when last seen. Major current deficit occurred for magnesium (32.1% of the patients), hemoglobin (50.8%), iron (29.8%), ferritin (36.0%), zinc (40.5%), vitamin B(12) (61.8%), vitamin D(3) (60.5%), and beta-carotene (56.8%). Low preoperative measurements had already been unveiled for iron, transferrin, zinc, and vitamin B(12). Total drug consumption tended to decrease after operation, and present findings correlated with excess weight loss (EWL). Also presence of diabetes and BMI value were predictors of long-term EWL, along with biochemical profile by 2 years. Multivitamin supplementation and gastrointestinal complaints partially correlated with nutritional results. CONCLUSIONS: (1) Good initial weight loss with moderate late regain, anemia, and multiple nutrient deficits was the common pattern. (2) Massive weight loss, frequent vomiting, dumping syndrome, and women in reproductive age were risk factors for hemoglobin or vitamin deficits, whereas superobesity, diabetes, and use of multiple drugs were associated with EWL result. (3) Most laboratory tests became stable by 2 years and along with BMI correlated with late EWL. (4) Two-year nutritional investigation is especially recommended because of its long-term predictive value.

Pro- and anti-inflammatory cytokines in steatosis and steatohepatitis.

BACKGROUND: Fatty liver disease is a problem in both bariatric patients and in patients with moderate obesity. Tumor necrosis factor (TNF)-alpha has been frequently measured in nonalcoholic steatohepatitis (NASH) with or without diabetes, but less is known about interleukin (IL)-6 and IL-10. METHODS: Moderately obese patients (n = 80) with histologically proven steatosis (n = 29) and NASH (n = 51) were recruited. Serum levels of cytokines were documented along with clinical information. The aim was to identify the correlates of such biomolecules in a stable population. RESULTS: Diabetes tended to be more associated with NASH (52.5% instead of 41.4%, P = 0.015), with no difference of age, gender, or body mass index regarding steatosis. For the entire population, cytokine changes were not significant, including TNF-alpha and IL-6. In diabetics only, all markers tended to diminish with NASH, especially IL-10 (P = 0.000). IL-10 correlated with homeostatic model assessment index (P = 0.000) and other variables of glucose homeostasis in diabetes, thus representing a major marker of the disease. CONCLUSIONS: (1) Generally inconsistent changes in pro- and anti-inflammatory cytokines occurred when NASH was globally compared to steatosis. (2) In contrast, downregulation of IL-6 and IL-10 was perceived in diabetics with NASH. (3) Arterial hypertension did not play a role in these circumstances. (4) IL-10 maintained strong correlations with glucose metabolism indices. (5) TNF-alpha could not be incriminated for progressive liver damage, as values failed to increase in NASH. (6) Investigations of IL-10 and other counterregulatory cytokines are lacking in this context and deserve further studies.

A rodent model of NASH with cirrhosis, oval cell proliferation and hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a well recognized complication of advanced NASH (non-alcoholic steatohepatitis). We sought to produce a rat model of NASH, cirrhosis and HCC. METHODS: Adult Sprague-Dawley rats, weighing 250-300g, were fed a choline-deficient, high trans-fat diet and exposed to DEN in drinking water. After 16 weeks, the animals underwent liver ultrasound (US), sacrifice and assessment by microscopy, immunohistochemistry and transmission electron microscopy (TEM). RESULTS: US revealed steatosis and focal lesions in 6 of 7. All had steatohepatitis defined as inflammation, advanced fibrosis and ballooning with Mallory-Denk bodies (MDB) with frank cirrhosis in 6. Areas of more severe injury were associated with anti-CK19 positive ductular reaction. HCC, present in all, were macro-trabecullar or solid with polyhedral cells with foci of steatosis and ballooned cells. CK19 was positive in single or solid nests of oval cells and in neoplastic hepatocytes. TEM showed ballooning with small droplet fat, dilated endoplasmic reticulum and MDB in non-neoplastic hepatocytes and small droplet steatosis in some cancer cells. CONCLUSIONS: This model replicated many features of NASH including steatohepatitis with ballooning, fibrosis, cirrhosis and hepatocellular carcinoma. Oval cell proliferation was evident and the presence anti-CK 19 positivity in the cancer suggests oval cell origin of the malignancy.