Similar insulin regulation of splanchnic FFA and VLDL-TG in men with nonalcoholic hepatic steatosis and steatohepatitis.

This study aimed to determine whether obese men with nonalcoholic fatty liver disease (NAFLD) display differences between those with simple steatosis versus steatohepatitis (NASH) in splanchnic and hepatic FFA and VLDL-triglycerides (VLDL-TG) balances. The study involved 17 obese men with biopsy-proven NAFLD (9 with NASH and 8 with simple steatosis). We used hepatic vein catheterization in combination with [3H]palmitate and [14C]VLDL-TG tracers to measure splanchnic palmitate and VLDL-TG uptake and release rates during basal and hyperinsulinemic conditions. Indocyanine green was used to measure splanchnic plasma flow. Splanchnic palmitate uptake was similar in the two groups and significantly reduced during hyperinsulinemia (NASH: 62 (48-77) versus 38 (18-58) µmol/min; simple steatosis: 62 (46-78) versus 45 (25-65) µmol/min, mean (95% CI), basal versus clamp periods, respectively, P = 0.02 time-effect). Splanchnic palmitate release was also comparable between groups and nonsignificantly diminished during hyperinsulinemia. The percent palmitate delivered to the liver originating from visceral adipose tissue lipolysis was similar and unchanged by hyperinsulinemia. Splanchnic uptake and release of VLDL-TG were similar between groups. Hyperinsulinemia suppressed VLDL-TG release (P <0.05 time-effect) in both groups. Insulin-mediated glucose disposal was similar in the two groups (P = 0.54). Obese men with NASH and simple steatosis have similar splanchnic uptake and release of FFA and VLDL-TG and a similar proportion of FFA from visceral adipose tissue lipolysis delivered to the liver. These results demonstrate that the splanchnic balances of FFA and VLDL-TG do not differ between obese men with NASH and those with simple steatosis.

Glucagon augments the secretion of FGF21 and GDF15 in MASLD by indirect mechanisms.

INTRODUCTION: Glucagon receptor agonism is currently explored for the treatment of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The metabolic effects of glucagon receptor agonism may in part be mediated by increases in circulating levels of Fibroblast Growth Factor 21 (FGF21) and Growth Differentiation Factor 15 (GDF15). The effect of glucagon agonism on FGF21 and GDF15 levels remains uncertain, especially in the context of elevated insulin levels commonly observed in metabolic diseases. METHODS: We investigated the effect of a single bolus of glucagon and a continuous infusion of glucagon on plasma concentrations of FGF21 and GDF15 in conditions of endogenous low or high insulin levels. The studies included individuals with overweight with and without MASLD, healthy controls (CON) and individuals with type 1 diabetes (T1D). The direct effect of glucagon on FGF21 and GDF15 was evaluated using our in-house developed isolated perfused mouse liver model. RESULTS: FGF21 and GDF15 correlated with plasma levels of insulin, but not glucagon, and their secretion was highly increased in MASLD compared with CON and T1D. Furthermore, FGF21 levels in individuals with overweight with or without MASLD did not increase after glucagon stimulation when insulin levels were kept constant. FGF21 and GDF15 levels were unaffected by direct stimulation with glucagon in the isolated perfused mouse liver. CONCLUSION: The glucagon-induced secretion of FGF21 and GDF15 is augmented in MASLD and may depend on insulin. Thus, glucagon receptor agonism may augment its metabolic benefits in patients with MASLD through enhanced secretion of FGF21 and GDF15.

Exercise increases myocardial free fatty acid oxidation in subjects with metabolic dysfunction-associated fatty liver disease.

BACKGROUND AND AIMS: Metabolic dysfunction-associated fatty liver disease (MAFLD) is associated with dyslipidemia and may promote cardiac lipotoxicity. Myocardial free fatty acids (FFA) oxidation (MOFFA) is normal in pre-diabetes, but reduced in heart failure. We hypothesized that during exercise MOFFA, very low-density lipoprotein triglycerides (VLDL-TG) secretion, hepatic FFA utilization, and lactate production differ among obese subjects with and without MAFLD. METHODS: Nine obese subjects with MAFLD and 8 matched subjects without MAFLD (Control) without a history of heart failure and cardiovascular disease were compared before and after 90-min exercise at 50% Peak oxygen consumption. Basal and exercise induced cardiac and hepatic FFA oxidation, uptake and re-esterification and VLDL-TG secretion were measured using [11C]palmitate positron-emission tomography and [1-14C]VLDL-TG. RESULTS: In the heart, increased MOFFA was observed after exercise in MAFLD, whereas MOFFA decreased in Control (basal vs exercise, MAFLD: 4.1 (0.8) vs 4.8 (0.8) µmol·100 ml-1 min-1; Control: 4.9 (1.8) vs 4.0 (1.1); µmol·100 ml-1 min-1, mean (SD), p < 0.048). Hepatic FFA fluxes were significantly lower in MAFLD than Control and increased ≈ two-fold in both groups. VLDL-TG secretion was 50% greater in MAFLD at rest and similarly suppressed during exercise. Plasma lactate increased significantly less in MAFLD than Control during exercise. CONCLUSIONS: Using robust tracer-techniques we found that obese subjects with MAFLD do not downregulate MOFFA during exercise compared to Control, possibly due to diminished lactate supply. Hepatic FFA fluxes are significantly lower in MAFLD than Control, but increase similarly with exercise. VLDL-TG export remains greater in MAFLD compared to Control. Basal and post-exercise myocardial and hepatic FFA, VLDL-TG and lactate metabolism is abnormal in subjects with MAFLD compared to Control.

Impaired Glucagon-Mediated Suppression of VLDL-Triglyceride Secretion in Individuals With Metabolic Dysfunction-Associated Fatty Liver Disease (MAFLD).

Individuals with metabolic dysfunction-associated fatty liver disease (MAFLD) have elevated plasma lipids as well as glucagon, although glucagon suppresses hepatic VLDL-triglyceride (TG) secretion. We hypothesize that the sensitivity to glucagon in hepatic lipid metabolism is impaired in MAFLD. We recruited 11 subjects with severe MAFLD (MAFLD+), 10 with mild MAFLD (MAFLD-), and 7 overweight control (CON) subjects. We performed a pancreatic clamp with a somatostatin analog (octreotide) to suppress endogenous hormone production, combined with infusion of low-dose glucagon (0.65 ng/kg/min, t = 0-270 min, LowGlucagon), followed by high-dose glucagon (1.5 ng/kg/min, t = 270-450 min, HighGlucagon). VLDL-TG and glucose tracers were used to evaluate VLDL-TG kinetics and endogenous glucose production (EGP). HighGlucagon suppressed VLDL-TG secretion compared with LowGlucagon. This suppression was markedly attenuated in MAFLD subjects compared with CON subjects (MAFLD+: 13% ± [SEM] 5%; MAFLD-: 10% ± 3%; CON: 36% ± 7%, P < 0.01), with no difference between MAFLD groups. VLDL-TG concentration and VLDL-TG oxidation rate increased between LowGlucagon and HighGlucagon in MAFLD+ subjects compared with CON subjects. EGP transiently increased during HighGlucagon without any difference between the three groups. Individuals with MAFLD have a reduced sensitivity to glucagon in the hepatic TG metabolism, which could contribute to the dyslipidemia seen in MAFLD patients. ClinicalTrials.gov: NCT04042142.

Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis.

BACKGROUND: Animal models of non-alcoholic steatohepatitis (NASH) are important tools in preclinical research and drug discovery. Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mice represent a model of fibrosing NASH. The present study directly assessed the clinical translatability of the model by head-to-head comparison of liver biopsy histological and transcriptome changes in GAN DIO-NASH mouse and human NASH patients. METHODS: C57Bl/6 J mice were fed chow or the GAN diet rich in saturated fat (40%), fructose (22%) and cholesterol (2%) for ≥38 weeks. Metabolic parameters as well as plasma and liver biomarkers were assessed. Liver biopsy histology and transcriptome signatures were compared to samples from human lean individuals and patients diagnosed with NASH. RESULTS: Liver lesions in GAN DIO-NASH mice showed similar morphological characteristics compared to the NASH patient validation set, including macrosteatosis, lobular inflammation, hepatocyte ballooning degeneration and periportal/perisinusoidal fibrosis. Histomorphometric analysis indicated comparable increases in markers of hepatic lipid accumulation, inflammation and collagen deposition in GAN DIO-NASH mice and NASH patient samples. Liver biopsies from GAN DIO-NASH mice and NASH patients showed comparable dynamics in several gene expression pathways involved in NASH pathogenesis. Consistent with the clinical features of NASH, GAN DIO-NASH mice demonstrated key components of the metabolic syndrome, including obesity and impaired glucose tolerance. CONCLUSIONS: The GAN DIO-NASH mouse model demonstrates good clinical translatability with respect to the histopathological, transcriptional and metabolic aspects of the human disease, highlighting the suitability of the GAN DIO-NASH mouse model for identifying therapeutic targets and characterizing novel drug therapies for NASH.

Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion.

BACKGROUND & AIMS: We recently showed that the functional capacity for ureagenesis is deficient in non-alcoholic fatty liver disease (NAFLD) patients. The aim of this study was to assess expression of urea cycle-related genes to elucidate a possible gene regulatory basis to the functional problem. METHODS: Liver mRNA expression analyses within the gene pathway governing hepatic nitrogen conversion were performed in 20 non-diabetic, biopsy-proven NAFLD patients (8 simple steatosis; 12 non-alcoholic steatohepatitis [NASH]) and 12 obese and 14 lean healthy individuals. Sixteen NAFLD patients were included for gene expression validation. Relationship between gene expressions and functional capacity for ureagenesis was described. RESULTS: Gene expression of most urea cycle-related enzymes were downregulated in NAFLD vs both control groups; markedly so for the urea cycle flux-generating carbamoyl phosphate synthetase (CPS1) (~3.5-fold, P < .0001). In NASH, CPS1 downregulation paralleled the deficit in ureagenesis (P = .03). Additionally, expression of several genes involved in amino acid uptake and degradation, and the glucagon receptor gene, were downregulated in NAFLD. Conversely, glutamine synthetase (GS) expression increased >1.5-fold (P ≤ .03), inversely related to CPS1 expression (P = .004). CONCLUSIONS: NAFLD downregulated the expression of urea cycle-related genes. Downregulation of urea cycle flux-generating CPS1 correlated with the loss of functional capacity for ureagenesis in NASH. On gene level, these changes coincided with an increase in the major ammonia scavenging enzyme GS. The effects seemed related to a fatty liver as such rather than NASH or obesity. The findings support gene regulatory mechanisms involved in the deficient ureagenesis of NAFLD, but it remains unexplained how hepatocyte fat accumulation exerts these effects.

Hepatic exposure of metformin in patients with non-alcoholic fatty liver disease.

AIMS: Metformin is first-line treatment of type 2 diabetes mellitus and reduces cardiovascular events in patients with insulin resistance and type 2 diabetes. Target tissue for metformin action is thought to be the liver, where metformin distribution depends on facilitated transport by polyspecific transmembrane organic cation transporters (OCTs). Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in the western world with strong associations to insulin resistance and the metabolic syndrome, but whether NAFLD affects metformin biodistribution to the liver is not known. In this study, the primary aim was to investigate in vivo hepatic uptake of metformin dynamically in humans with variable degrees of liver affection. As a secondary aim, we wished to correlate hepatic metformin distribution with OCT gene transcription determined in diagnostic liver biopsies. METHODS: Eighteen patients with biopsy-proven NAFLD were investigated using 11C-metformin PET/CT technique. Gene transcripts of OCTs were determined by real-time polymerase chain reaction (PCR). RESULTS: We observed similar hepatic volume of distribution of metformin between patients with simple steatosis and non-alcoholic steatohepatitis (NASH) (Vd 2.38 ± 0.56 vs. 2.10 ± 0.39, P = 0.3). There was no association between hepatic exposure to metformin and the degree of inflammation or fibrosis, and no clear correlation between metformin distribution and OCT gene transcription. CONCLUSION: Metformin is distributed to the liver in patients with NAFLD and the distribution is not impaired by inflammation or fibrosis. The findings imply that metformin action in liver in patients with NAFLD may be preserved.

Hepatic transcriptome signatures in patients with varying degrees of nonalcoholic fatty liver disease compared with healthy normal-weight individuals.

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of conditions ranging from simple steatosis (NAFL), over nonalcoholic steatohepatitis (NASH) with or without fibrosis, to cirrhosis with end-stage disease. The hepatic molecular events underlying the development of NAFLD and transition to NASH are poorly understood. The present study aimed to determine hepatic transcriptome dynamics in patients with NAFL or NASH compared with healthy normal-weight and obese individuals. RNA sequencing and quantitative histomorphometry of liver fat, inflammation and fibrosis were performed on liver biopsies obtained from healthy normal-weight ( n = 14) and obese ( n = 12) individuals, NAFL ( n = 15) and NASH ( n = 16) patients. Normal-weight and obese subjects showed normal liver histology and comparable gene expression profiles. Liver transcriptome signatures were largely overlapping in NAFL and NASH patients, however, clearly separated from healthy normal-weight and obese controls. Most marked pathway perturbations identified in both NAFL and NASH were associated with markers of lipid metabolism, immunomodulation, extracellular matrix remodeling, and cell cycle control. Interestingly, NASH patients with positive Sonic hedgehog hepatocyte staining showed distinct transcriptome and histomorphometric changes compared with NAFL. In conclusion, application of immunohistochemical markers of hepatocyte injury may serve as a more objective tool for distinguishing NASH from NAFL, facilitating improved resolution of hepatic molecular changes associated with progression of NAFLD. NEW & NOTEWORTHY Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in Western countries. NAFLD is associated with the metabolic syndrome and can progress to the more serious form, nonalcoholic steatohepatitis (NASH), and ultimately lead to irreversible liver damage. Using gold standard molecular and histological techniques, this study demonstrates that the currently used diagnostic tools are problematic for differentiating mild NAFLD from NASH and emphasizes the marked need for developing improved histological markers of NAFLD progression.

[Treatment of non-alcoholic fatty liver disease].

Non-alcoholic fatty liver disease and especially non-alcoholic steatohepatitis (NASH) and fibrosis are associated with severe liver disease and increased cardiovascular risk. It is therefore important to identify patients with NASH fibrosis. Therapeutical options include life style intervention and pharmacological treatment with vitamin E and pioglitazone; however, evidence of effect is scarce for all options. New treatments are under investigation and include glucagon-like peptide-1, farnesoid receptor as well as peroxisome proliferator-activated receptor-α/δ agonists. Bariatric surgery may be an option in selected patients.

Urea cycle dysregulation in non-alcoholic fatty liver disease.

BACKGROUND & AIMS: In non-alcoholic steatohepatitis (NASH), the function of urea cycle enzymes (UCEs) may be affected, resulting in hyperammonemia and the risk of disease progression. We aimed to determine whether the expression and function of UCEs are altered in an animal model of NASH and in patients with non-alcoholic fatty liver disease (NAFLD), and whether this process is reversible. METHODS: Rats were first fed a high-fat, high-cholesterol diet for 10 months to induce NASH, before being switched onto a normal chow diet to recover. In humans, we obtained liver biopsies from 20 patients with steatosis and 15 with NASH. Primary rat hepatocytes were isolated and cultured with free fatty acids. We measured the gene and protein expression of ornithine transcarbamylase (OTC) and carbamoylphosphate synthetase (CPS1), as well as OTC activity, and ammonia concentrations. Moreover, we assessed the promoter methylation status of OTC and CPS1 in rats, humans and steatotic hepatocytes. RESULTS: In NASH animals, gene and protein expression of OTC and CPS1, and the activity of OTC, were reversibly reduced. Hypermethylation of Otc promoter genes was also observed. Additionally, in patients with NAFLD, OTC enzyme concentration and activity were reduced and ammonia concentrations were increased, which was further exacerbated in those with NASH. Furthermore, OTC and CPS1 promoter regions were hypermethylated. In primary hepatocytes, induction of steatosis was associated with Otc promoter hypermethylation, a reduction in the gene expression of Otc and Cps1, and an increase in ammonia concentration in the supernatant. CONCLUSION: NASH is associated with a reduction in the gene and protein expression, and activity, of UCEs. This results in hyperammonemia, possibly through hypermethylation of UCE genes and impairment of urea synthesis. Our investigations are the first to describe a link between NASH, the function of UCEs, and hyperammonemia, providing a novel therapeutic target. LAY SUMMARY: In patients with fatty liver disease, the enzymes that convert nitrogen waste into urea may be affected, leading to the accumulation of ammonia, which is toxic. This accumulation of ammonia can lead to scar tissue development, increasing the risk of disease progression. In this study, we show that fat accumulation in the liver produces a reversible reduction in the function of the enzymes that are involved in detoxification of ammonia. These data provide potential new targets for the treatment of fatty liver disease.

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-1ß 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-É£ (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.

Preserved liver regeneration capacity after partial hepatectomy in rats with non-alcoholic steatohepatitis.

AIM: To evaluate the liver regeneration capacity (LRC) after partial hepatectomy (PH) in experimental non-alcoholic steatohepatitis (NASH). METHODS: Fifty-four female rats were fed a high-fat, high-cholesterol diet (HFCD, 65% fat, 1% cholesterol) or standard diet (STD) for 16 wk. A 70% PH was performed and the animals were euthanised before PH or 2 or 5 d post-PH. LRC was evaluated using: The total number of Ki-67 positive hepatocytes in the caudate lobe, N(Ki-67, lobe) evaluated in a stereology-based design, the regenerated protein ratio (RPR), prothrombin-proconvertin ratio (PP), and mRNA expression of genes related to regeneration. RESULTS: The HFCD NASH model showed significant steatosis with ballooning and inflammation, while no fibrosis was present. Mortality was similar in HFCD and STD animals following PH. HFCD groups were compared to respective STD groups and HFCD animals had a significantly elevated alanine transaminase at baseline (P < 0.001), as well as a significantly elevated bilirubin at day 2 after PH (P < 0.05). HFCD animals had a higher N(Ki-67, lobe) at baseline, (P < 0.0001), day 2 after PH (P = 0.06) and day 5 after PH (P < 0.025). We found no significant difference in RPR or PP neither 2 or 5 d post-PH. Expression of liver regeneration genes (e.g., hepatic growth factor) was higher at both day 2 and 5 post-PH in HFCD groups (P < 0.05). CONCLUSION: NASH rats had a preserved LRC after hepatectomy when compared to STD rats. The methods and models of NASH are essential in understanding and evaluating LRC.

Placebo-controlled, randomised clinical trial: high-dose resveratrol treatment for non-alcoholic fatty liver disease.

OBJECTIVE: "The obesity epidemic" has led to an increase in obesity-related conditions including non-alcoholic fatty liver disease (NAFLD), for which effective treatments are in demand. The polyphenol resveratrol prevents the development of experimental NAFLD through modulation of cellular pathways involved in calorie restriction. We aimed to test the hypothesis that resveratrol alleviates NAFLD in a randomised, clinical trial. MATERIALS AND METHODS: A total of 28 overweight patients with transaminasemia and histological NAFLD were randomised 1:1 to placebo or resveratrol 1.5 g daily for 6 months. Twenty-six participants completed the trial and underwent repeated clinical investigation, blood work, MR spectroscopy; and 19 participants agreed to a repeat liver biopsy. RESULTS: Resveratrol treatment was generally not superior to placebo in improving plasma markers of liver injury (primary outcome: alanine transaminase, p = 0.51). Resveratrol-treated patients showed a 3.8% decrease in liver lipid content (p = 0.03), with no difference between the two treatment arms (p = 0.38) and no improvement of histological features. Resveratrol treatment was not associated with improvements in insulin sensitivity or markers of the metabolic syndrome, except for a transient decrease in systolic BP. Microarray analysis and qRT-PCR revealed no major changes in expression profile. Also, we report a serious adverse event in a patient who developed fever and bicytopenia. CONCLUSIONS: In this placebo-controlled, high-dose and long-term study, resveratrol treatment had no consistent therapeutic effect in alleviating clinical or histological NAFLD, though there may be a small ameliorating effect on liver function tests and liver fat accumulation.

Effect of resveratrol on experimental non-alcoholic fatty liver disease depends on severity of pathology and timing of treatment.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease with few therapeutic options. Resveratrol (RSV) prevents the development of steatosis in a number of experimental fatty liver (non-alcoholic fatty liver [NAFL]) models, but the preventive or therapeutic effects on experimental NASH are not yet clarified, and clinical results on non-alcoholic fatty liver disease are ambiguous. Thus, we aimed to compare the RSV-mediated preventive and therapeutic effects on experimental NAFL and NASH. METHODS: We used a high-fat (HF) diet to generate a rat NAFL model and a high-fat, high-cholesterol (HFC) diet to generate a rat NASH model. The preventive and therapeutic potential of RSV was tested by adding RSV to the HF and HFC diet from study start or after 1 week of the diets. Animals were sacrificed after 8 weeks with appropriate controls. Blood and liver were harvested for analysis, including measurement of RSV metabolites. RESULTS: Resveratrol reduced the development of histological steatosis (P = 0.03) and partly triglyceride accumulation (fold change reduced from 3.6 to 2.4, P = 0.08) in the male NAFL model, although effects were moderate. In NASH prevention, RSV reduced the accumulation of triglyceride in hepatic tissue (P < 0.01), while there was no effect on biochemical, histopathological, or transcriptional NASH changes. Further, RSV had no therapeutic effect on established NASH. We found RSV metabolites but no parent RSV in serum or liver tissue, confirming low bioavailability. CONCLUSIONS: These experimental findings suggest that a weak hepatic benefit of RSV treatment is seen in prevention of steatosis only.

Plasma levels of OLFM4 in normals and patients with gastrointestinal cancer.

Olfactomedin 4 (OLFM4) is a secreted glycoprotein predominantly expressed in bone marrow and gastrointestinal tissues. Aberrant expression of OLFM4 has been shown in several cancers. However, the clinical significance hereof is currently controversial. OLFM4 has been proposed as a candidate biomarker of gastrointestinal cancers. To address this, we developed monoclonal antibodies against synthetic peptides representing various segments of OLFM4. We examined expression of OLFM4 in epithelial cells by immunohistochemistry and found that OLFM4 is highly expressed in proliferating benign epithelial cells and in some carcinoma cells. We developed an Enzyme Linked Immunosorbent Assay for OLFM4 and investigated whether plasma levels of OLFM4 reflect colorectal malignancies, but were unable to see any such association. Instead, we observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 µg/ml, mean 0.028 µg/ml while 10% of both normals and patients with cancers had OLFM4 between 4 and 60 µg/ml, mean 15 µg/ml. The levels were constant over time. The background for this high plasma level is not known, but must be taken into account if OLFM4 is used as biomarker for GI cancers.

Effect of resveratrol on experimental non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis (NASH) are increasing clinical problems for which effective treatments are required. The polyphenol resveratrol prevents the development of fatty liver disease in a number of experimental studies. We hypothesized that it could revert steatohepatitis, including hepatic inflammation and fibrosis, in an experimental NASH model. To induce hepatic steatohepatitis, a 65% fat, 2% cholesterol and 0.5% cholate (HFC) diet was fed to rats for 1 or 16 weeks, prior to treatment. Subsequently, the diet was supplemented with resveratrol (approx. 100mg/rat/day) to three intervention groups; week 2-4, 2-7 or 17-22. Treated animals were sacrificed at the end of each intervention period with appropriate control and HFC diet controls. Blood and liver were harvested for analysis. When commenced early, resveratrol treatment partially mitigated transaminase elevations, hepatic enlargement and TNFα induced protein-3 protein expression, but generally resveratrol treatment had no effect on elevated hepatic triglyceride levels, histological steatohepatitis or fibrosis. We observed a slight reduction in Collagen1α1 mRNA expression and no reduction in the mRNA expression of other markers of fibrosis, inflammation or steatosis (TGFß, TNFα, α2-MG, or SREBP-1c). Resveratrol metabolites were detected in serum, including trans-resveratrol-3-O-sulphate/trans-resveratrol-4'-O-sulphate (mean concentration 7.9 µg/ml). Contrary to the findings in experimental steatosis, resveratrol treatment had no consistent therapeutic effect in alleviating manifest experimental steatohepatitis.

Effects of resveratrol in experimental and clinical non-alcoholic fatty liver disease.

The prevalence of obesity and related conditions like non-alcoholic fatty liver disease (NAFLD) is increasing worldwide and therapeutic options are limited. Alternative treatment options are therefore intensively sought after. An interesting candidate is the natural polyphenol resveratrol (RSV) that activates adenosinmonophosphate-activated protein kinase (AMPK) and silent information regulation-2 homolog 1 (SIRT1). In addition, RSV has known anti-oxidant and anti-inflammatory effects. Here, we review the current evidence for RSV-mediated effects on NAFLD and address the different aspects of NAFLD and non-alcoholic steatohepatitis (NASH) pathogenesis with respect to free fatty acid (FFA) flux from adipose tissue, hepatic de novo lipogenesis, inadequate FFA ß-oxidation and additional intra- and extrahepatic inflammatory and oxidant hits. We review the in vivo evidence from animal studies and clinical trials. The abundance of animal studies reports a decrease in hepatic triglyceride accumulation, liver weight and a general improvement in histological fatty liver changes, along with a reduction in circulating insulin, glucose and lipid levels. Some studies document AMPK or SIRT1 activation, and modulation of relevant markers of hepatic lipogenesis, inflammation and oxidation status. However, AMPK/SIRT1-independent actions are also likely. Clinical trials are scarce and have primarily been performed with a focus on overweight/obese participants without a focus on NAFLD/NASH and histological liver changes. Future clinical studies with appropriate design are needed to clarify the true impact of RSV treatment in NAFLD/NASH patients.

Propranolol treatment of portal hypertension in cirrhosis patients is better the higher the untreated pressure: a single-centre prospective experience.

OBJECTIVE: To assess the effect of propranolol treatment on the hepatic venous pressure gradient (HVPG) and the relationship between native HVPG and the effect of propranolol in patients with cirrhosis and portal hypertension in a prospective, observational, single-center study. MATERIAL AND METHODS: The HVPG was registered prospectively in 124 consecutive cirrhosis patients with and without treatment with propranolol 80 mg daily. Results. 41% of the patients responded to the treatment with the intended reduction of HVPG to <12 mm Hg and/or by >20%. The HVPG reduction was larger for higher native HVPG values (p < 0.001). There was no significant relation between changes in heart rate and changes in HVPG (p = 0.8). CONCLUSIONS: The high fraction of hemodynamic non-responders supports the rationale of measuring the HVPG with and without propranolol treatment to assist the clinical assessment and avoid meaningless and potentially harmful treatment. The positive association between a high native HVPG and propranolol-induced HVPG reduction indicates that pharmacological treatment also benefits patients with advanced portal hypertension.

[Prognosis and treatment of non-alcoholic fatty liver disease and steatohepatitis]. / Prognose og behandling af nonalkoholisk fedtleversygdom og steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) are among the most common liver diseases. NAFLD with pure steatosis is considered benign. NASH, however, may progress to liver cirrhosis with risk of liver cancer. Therefore, it is important to search for treatment modalities for NASH. Because obesity and insulin resistance are risk factors for NASH, weight loss and exercise are considered mandatory, although limited data are in support. Presently, we focus on the prognosis of NAFLD and NASH and on pharmaceutical treatment, especially in the form of vitamin E and pioglitazone.

Smarcc1 expression: a significant predictor of disease-specific survival in patients with clinically localized prostate cancer treated with no intention to cure.

OBJECTIVE: The clinical outcome of prostate cancer (PC) is extremely variable and therefore difficult to predict at the early stage of the disease. Since curative-intended therapies are bound up with the risk of severe adverse events, identification of new prognostic markers in PC is essential in individualized clinical treatment. The Smarcc1 protein, a part of the intranuclear SWI/SNF complex, is up-regulated in PC, and has been suggested to be implicated in tumour dedifferentiation, progression and biochemical recurrence. This makes Smarcc1 a possible candidate marker for PC survival. MATERIAL AND METHODS: Immunohistochemistry was used to measure protein expression levels of Smarcc1in on a tissue microarray containing specimens from 100 patients suffering from clinically localized PC treated with no intention to cure and followed to death. RESULTS: The median age at diagnosis was 75.5 years (55-95 years) and the median survival time was 5 years (0.01-15 years). In total, 41 patients (41%) died of PC. Statistically, there was no significant association between Smarcc1 immunostaining (negative/positive) and Gleason score (p = 0.7/0.8) or the clinical T stage (p = 0.9). Positive staining for Smarcc1 in patients with clinically localized PC correlated with a prolonged disease-free survival as opposed to negative staining (p = 0.025). CONCLUSION: In patients with clinically localized PC treated without intention of cure, Smarcc1 expression was a statistically significant and independent predictor of disease-specific survival.