Niemann-Pick type C2 protein supplementation in experimental non-alcoholic fatty liver disease

Background and aims Hepatic cholesterol deposition drives inflammation and fibrosis in non-alcoholic steatohepatitis (NASH). The Niemann-Pick type C2 (NPC2) protein plays an important role in regulating intracellular cholesterol trafficking and homeostasis. We hypothesized that intravenous NPC2 supplementation reduces cholesterol accumulation, hepatic inflammation and fibrogenesis in a nutritional NASH rat model. Methods: Rats were fed a high-fat, high-cholesterol (HFHC) diet for four weeks resulting in moderately severe NASH. Animals were treated with intravenous NPC2 or placebo twice weekly for either the last two weeks or the entire four weeks. End-points were liver/body- and spleen/body weight ratios, histopathological NASH scores, fibrosis, serum liver enzymes, cholesterol, lipoproteins, cytokines, and quantitative polymerase chain reaction derived hepatic gene expression related to cholesterol metabolism, inflammation, and fibrosis. Results: HFHC rats developed hepatomegaly, non-fibrotic NASH histopathology, elevated liver enzymes, serum cholesterol, and pro-inflammatory cytokines. Their sterol regulatory element binding factor 2 (SREBF2) and low-density lipoprotein receptor (LDL-R) mRNAs were down-regulated compared with rats on standard chow. NPC2 did not improve liver weight, histopathology, levels of serum liver enzymes or pro-inflammatory tumor necrosis factor-α (TNFα), Interleukin (IL)-6, or IL-1β in HFHC rats. Two weeks of NPC2 treatment lowered hepatic TNFα and COL1A1 mRNA expression. However, this effect was ultimately reversed following additional two weeks of treatment. Four weeks NPC2 treatment of rats raised ATP-binding cassette A1 (ABCA1) and low-density lipoprotein receptor (LDLR) mRNAs in the liver, concurrent with a strong tendency towards higher serum high-density lipoprotein (HDL). Furthermore, the peroxisome proliferator activated receptor-ɣ (PPARG) gene expression was reduced. Conclusions: NPC2 proved inefficient at modifying robust hepatic NASH end-points in a HFHC NASH model. Nonetheless, our data suggest that hepatic ABCA1 expression and reverse cholesterol transport were upregulated by NPC2 treatment, thus presenting putative therapeutic effects in diseases associated with deregulated lipid metabolism

Non-alcoholic steatohepatitis weakens the acute phase response to endotoxin in rats

Background & Aims\ud \ud Patients with non-alcoholic steatohepatitis (NASH) have increased mortality, including from infections. We, therefore, tested in a rodent model of steatohepatitis whether the hepatic acute phase response is intact.\ud \ud Methods\ud \ud Steatohepatitis was induced in rats by feeding a high-fat, high-cholesterol diet for 4 (early) and 16 weeks (advanced NASH). 2 h after low-dose LPS (0.5 mg/kg i.p.), we measured the serum concentrations of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We also measured liver mRNA's and the serum concentrations of acute phase proteins 24 h after LPS.\ud \ud Results\ud \ud Non-alcoholic steatohepatitis in itself increased the liver mRNA levels of TNF-α and IL-6 and also the liver mRNA and serum levels of the acute phase proteins. The exposure to LPS increased serum TNF-α in both early and advanced NASH and more so than in the control rats. However, the increases in acute phase protein genes in liver tissue and proteins in the blood were lower than in the control rats.\ud \ud Conclusion\ud \ud In rats with early or advanced experimental NASH, LPS despite an increased interleukin release resulted in a blunted acute phase protein response. This tachyphylaxis may be part of the mechanism for the increased infection susceptibility of patients with NASH. We speculate that the steatosis-related interleukin release desensitises the signalling pathway leading to acute phase protein synthesis

Experimental nonalcoholic steatohepatitis compromises ureagenesis, an essential hepatic metabolic function

Nonalcoholic steatohepatitis (NASH) is increasing in prevalence, yet its consequences for liver function are unknown. We studied ureagenesis, an essential metabolic liver function of importance for whole body nitrogen homeostasis, in a rodent model of diet-induced NASH. Rats were fed a high-fat, high-cholesterol diet for 4 and 16 wk, resulting in early and advanced experimental NASH, respectively. We examined the urea cycle enzyme mRNAs in liver tissue, the hepatocyte urea cycle enzyme proteins, and the in vivo capacity of urea-nitrogen synthesis (CUNS). Early NASH decreased all of the urea cycle mRNAs to an average of 60% and the ornithine transcarbamylase protein to 10%, whereas the CUNS remained unchanged. Advanced NASH further decreased the carbamoyl phosphate synthetase protein to 63% and, in addition, decreased the CUNS by 20% [from 5.65 ± 0.23 to 4.58 ± 0.30 μmol × (min × 100 g)−1; P = 0.01]. Early NASH compromised the genes and enzyme proteins involved in ureagenesis, whereas advanced NASH resulted in a functional reduction in the capacity for ureagenesis. The pattern of urea cycle perturbations suggests a prevailing mitochondrial impairment by NASH. The decrease in CUNS has consequences for the ability of the body to adjust to changes in the requirements for nitrogen homeostasis e.g., at stressful events. NASH, thus, in terms of metabolic consequences, is not an innocuous lesion, and the manifestations of the damage seem to be a continuum with increasing disease severity

Alcoholic Hepatitis Markedly Decreases the Capacity for Urea Synthesis.

Data on quantitative metabolic liver functions in the life-threatening disease alcoholic hepatitis are scarce. Urea synthesis is an essential metabolic liver function that plays a key regulatory role in nitrogen homeostasis. The urea synthesis capacity decreases in patients with compromised liver function, whereas it increases in patients with inflammation. Alcoholic hepatitis involves both mechanisms, but how these opposite effects are balanced remains unclear. Our aim was to investigate how alcoholic hepatitis affects the capacity for urea synthesis. We related these findings to another measure of metabolic liver function, the galactose elimination capacity (GEC), as well as to clinical disease severity.We included 20 patients with alcoholic hepatitis and 7 healthy controls. The urea synthesis capacity was quantified by the functional hepatic nitrogen clearance (FHNC), i.e., the slope of the linear relationship between the blood α-amino nitrogen concentration and urea nitrogen synthesis rate during alanine infusion. The GEC was determined using blood concentration decay curves after intravenous bolus injection of galactose. Clinical disease severity was assessed by the Glasgow Alcoholic Hepatitis Score and Model for End-Stage Liver Disease (MELD) score.The FHNC was markedly decreased in the alcoholic hepatitis patients compared with the healthy controls (7.2±4.9 L/h vs. 37.4±6.8 L/h, P<0.01), and the largest decrease was observed in those with severe alcoholic hepatitis (4.9±3.6 L/h vs. 9.9±4.9 L/h, P<0.05). The GEC was less markedly reduced than the FHNC. A negative correlation was detected between the FHNC and MELD score (rho = -0.49, P<0.05).Alcoholic hepatitis markedly decreases the urea synthesis capacity. This decrease is associated with an increase in clinical disease severity. Thus, the metabolic failure in alcoholic hepatitis prevails such that the liver cannot adequately perform the metabolic up-regulation observed in other stressful states, including extrahepatic inflammation, which may contribute to the patients' poor prognosis

Time course of compromised urea synthesis in patients with alcoholic hepatitis

<p><b>Objectives</b>: Alcoholic hepatitis (AH) markedly decreases the urea synthesis capacity. We aimed to investigate the time course of this compromised essential liver function in patients with AH and its relation to treatment and survival.</p> <p><b>Materials and methods</b>: Thirty patients with AH were included in a prospective cohort study. We measured the substrate-independent urea synthesis capacity, i.e., the functional hepatic nitrogen clearance (FHNC), in the patients at study entry and again at three months (survivors/available: <i>n</i> = 17). Patients with severe disease (Glasgow Alcoholic Hepatitis Score ≥9, <i>n</i> = 17) were randomized to receive either prednisolone or pentoxifylline and were in addition examined after 14 days (<i>n</i> = 9).</p> <p><b>Results</b>: FHNC (normal range = 25–45 L/h) was markedly decreased at study entry (median = 5.6 (IQR = 3.0–9.6) L/h) and increased by three-fold in survivors at three months (15.1 (12.0–22.9) L/h; <i>p</i> < .001). In patients with severe AH, FHNC was also increased after 14 days of pharmacologic treatment and showed the greatest increase in the patients taking prednisolone (prednisolone 25.4 (20.6–26.2) L/h vs. pentoxifylline 12.3 (8.0–15.3) L/h; <i>p</i> = .05). FHNC at study entry was lower in 90-day non-survivors than in survivors (<i>p</i> = .04).</p> <p><b>Conclusions</b>: The decrease in the urea synthesis capacity in patients with AH was the most marked in short-term non-survivors and partly recovered in survivors at three months. In patients on pharmacologic treatment, recovery was observed already after 14 days, and it was nearly complete in those on prednisolone. Thus, metabolic liver failure in AH seems to be prognostically important, is potentially reversible, and may recover more rapidly following treatment with prednisolone.</p

Relationship between the functional hepatic nitrogen clearance and galactose elimination capacity.

<p>Relationship between the functional hepatic nitrogen clearance (FHNC) and galactose elimination capacity (GEC) in patients with non-severe (hollow circles) (GAHS<9, N = 8) and severe (circles) (GAHS≥9, N = 10) alcoholic hepatitis. No significant correlation was observed between the FHNC and GEC.</p

Relationship between the functional hepatic nitrogen clearance and Model for End-Stage Liver Disease score.

<p>Relationship between the functional hepatic nitrogen clearance (FHNC) and Model for End-Stage Liver Disease (MELD) score in patients with non-severe (hollow circles) (GAHS<9, N = 9) and severe (circles) (GAHS≥9, N = 11) alcoholic hepatitis. The linear regression line shows the correlation (rho = -0.49; P<0.05).</p

Baseline characteristics of the patients with alcoholic hepatitis and healthy controls.

<p>Baseline characteristics of the patients with alcoholic hepatitis and healthy controls.</p

Functional hepatic nitrogen clearance in patients with non-severe and severe alcoholic hepatitis and healthy controls.

<p>Functional hepatic nitrogen clearance (FHNC) in patients with non-severe (GAHS<9, N = 9) and severe (GAHS≥9, N = 11) alcoholic hepatitis and healthy controls (N = 7). The solid horizontal lines indicate the mean values. The FHNC was decreased in the AH patients compared with the healthy controls (P<0.01) and the largest decrease was observed in those with severe AH (P<0.05).</p

Hepatic gene expression related to inflammation, fibrogenesis and cholesterol metabolism.

<p>Relative mRNA expressions compared with isocaloric diet fed controls of <i>TNF</i> and <i>COL1A1</i> (Panel A), <i>TGFB1</i> and <i>PPARG</i> (Panel B), <i>SREBF2</i>, <i>LDLR</i>, <i>HMGCR</i> and <i>ABCA1</i> (Panel C) and <i>INSIG1</i>, <i>SRB1</i>, <i>GNMT</i> and <i>NPC2</i> (Panel D) in isocaloric controls, high-fat-high-cholesterol (HFHC) controls, two-week NPC2 (treatment) and four-week NPC2 (prevention) animals. Bars represent median ± interquartile range. *: P < 0.05 compared with Isocaloric Controls. **: P < 0.005 compared with Isocaloric controls. ***: P < 0.0005 compared with Isocaloric controls. #: P< 0.05 compared with HFHC Controls. ¤: P<0.05 compared with two-week NPC2.</p