Reduced Lysosomal Acid Lipase Activity in Adult Patients With Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is characterized by intra-hepatic fat accumulation and mechanisms involved in its pathogenesis are not fully explained. Lysosomal Acid Lipase (LAL) is a key enzyme in lipid metabolism. We investigated its activity in patients with fatty liver. LAL activity (nmol/spot/h) was measured in 100 adult healthy subjects (HS) and in 240 NAFLD patients. A sub-analysis on 35 patients with biopsy-proven non-alcoholic steatohepatitis (NASH) was performed. Median LAL activity was 1.15 (0.95-1.72) in HS. It was significantly reduced in NAFLD [0.78 (0.61-1.01), p < 0.001 vs. HS]. A further reduction was observed in the subgroup of NASH [0.67 (0.51-0.77), p < 0.001 vs. HS]. Patients with LAL activity below median had higher values of serum total cholesterol (p < 0.05) and LDL-c (p < 0.05), and increased serum liver enzymes (ALT, p < 0.001; AST, p < 0.01; GGT, p < 0.01). At multivariable logistic regression analysis, factors associated with LAL activity below median were ALT (OR: 1.018, 95% CI 1.004-1.032, p = 0.011) and metabolic syndrome (OR: 2.551, 95% CI 1.241-5.245, p = 0.011), whilst statin use predicted a better LAL function (OR: 0.464, 95% CI 0.248-0.866, p = 0.016). Our findings suggest a strong association between impaired LAL activity and NAFLD. A better knowledge of the role of LAL may provide new insights in NAFLD pathogenesis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is characterized by intra-hepatic fatty acid (lipid) accumulation, affecting a growing number of people worldwide (Vernon et al., 2011, Bellentani et al., 2000). NAFLD includes a wide spectrum of diseases extending from simple fatty liver to non-alcoholic steatohepatitis (NASH) and cirrhosis (Farrell and Larter, 2006), and, before long, will become the main cause of liver failure in the next future (Kemmer et al., 2013).

Many pathophysiological mechanisms have been associated to NAFLD, including insulin resistance (Angelico et al., 2005), dyslipidaemia (Corey et al., 2015), oxidative imbalance (Del Ben et al., 2014a), gut microbiota (Compare et al., 2012) alteration and genetic factors (Del Ben et al., 2014b). Several therapeutic intervention have been proposed (Pastori et al., 2015a, Del Ben et al., 2014c, Angelico et al., 2007, Chalasani et al., 2012).

In the liver, hyperinsulinemia is responsible for impaired mitochondrial oxidation of fatty acids, which consequently accumulate and are then partly metabolised by peroxisomes and microsomes with the production of reactive oxygen species and lipid peroxidation (Del Ben et al., 2014a).

Only a minority of patients with simple steatosis will develop NASH, and no reliable biomarkers of disease progression are available (Sanyal et al., 2015).

Lysosomal Acid Lipase (LAL) is a hydrolase that plays a key role in intra-cellular cholesterol trafficking. Inside the lysosomes, LAL hydrolyses triglycerides and cholesterol esters derived from plasma lipoproteins via LDL-receptor pathway (Fasano et al., 2012). A reduced LAL activity promotes an increased cholesterol ester storage in lysosomes, as observed in two genetic diseases, namely Wolman and Cholesterol Ester Storage Disease (CESD) (Fasano et al., 2012), which are characterized by total or sub-total LAL deficiency (Thelwall et al., 2013, Pisciotta et al., 2009, Fouchier and Defesche, 2013). These conditions are associated with severe liver steatosis and rapid liver failure (Bernstein et al., 2013, Reiner et al., 2014, Reynolds, 2013). Furthermore, LAL supplementation in young adults with CESD was associated with an improvement of liver steatosis (Valayannopoulos et al., 2014).

Our hypothesis was that a reduction of LAL activity may contribute to intracellular fatty acid accumulation in adult NAFLD. Thus, we measured the activity of LAL in a cohort of adult patients affected by NAFLD, and we investigated factors associated with reduced LAL activity.

Methods

Study Design

The study was performed in 240 consecutive patients with ultrasonography (US) evidence of fatty liver, referring to the Day Service of Internal Medicine of the Policlinico Umberto I University Hospital in Rome.

Inclusion criteria were: no history of excessive alcohol consumption defined as a mean daily intake of alcohol > 20 g; no history of Hepatitis C–B viruses infection with negative tests for the presence of hepatitis B surface antigen and antibody to hepatitis C virus; no history for other chronic liver diseases; and no therapy with drugs known to promote liver steatosis (e.g., amiodarone). Subjects underwent routine biochemical evaluation including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), fasting total and HDL-cholesterol, triglycerides, glucose and insulin. Waist circumference, height and weight were recorded and body mass index (BMI) was calculated.

The homeostasis model of insulin resistance (HOMA-IR) was used as a measure of IR (Matthews et al., 1985). Metabolic syndrome was diagnosed according to the ATP III modified criteria (Anon., 2001). The presence of diabetes and arterial hypertension was defined according to international guidelines (Authors et al., 2013, Mancia et al., 2013).

All patients provided signed informed consent before the study. The study was approved by the local ethical board of Sapienza University of Rome (ref. n° 2277/2011), and conducted according to the ethical principles embodied in the Declaration of Helsinki. Funding source: none.

Ultrasonography Evaluation of Fatty Liver

Liver US scanning was performed to assess the degree of steatosis. All US were performed by the same operator who was blinded to laboratory values using a GE Vivid S6 apparatus equipped with a convex 3.5 MHz probe. Liver steatosis was defined according to Hamaguchi criteria based on the presence of abnormally intense, high level echoes arising from the hepatic parenchyma, liver–kidney difference in echo amplitude, echo penetration into deep portion of the liver and clarity of liver blood vessel structure (Saverymuttu et al., 1986, Hamaguchi et al., 2007).

Liver Biopsy

Percutaneous liver biopsy was performed under US guide in 35 of the above fatty liver patients with clinical suspicion of NASH by their treating hepatologists. The decision to perform the biopsy was individualized and based on a persistent elevation of serum alanine aminotransferase levels (> 1.5 upper normal values) for more than 6 months and the presence of bright liver at US scan. A single operator performed ultrasound-guided liver biopsies. Pathologist who examined biopsies specimen was blinded to patients' identity or clinical information. NASH diagnosis was defined using standard criteria (Sanyal et al., 2011). All patients who underwent liver biopsy satisfied histological criteria for NASH.

Lysosomal Acid Lipase Activity Assay

All blood samples were taken after a 12-hour fast. LAL-activity was dosed with dried blood spot (DBS) technique using the inhibitors Lalistat 2. Ethylene-diamine-tetra acetic acid (EDTA) blood, obtained by venepuncture, was spotted on to filter paper (Whatman grade 903 Schleicher & Schuell) and allowed to dry overnight at room temperature. Samples were stored double-bagged with desiccant at − 20 °C and analysed within 2 weeks of storage. Uninhibited and inhibited with Lalistat 2 activities were dosed. LAL activity was determined by subtracting activity in the inhibited reaction from uninhibited reaction (total lipase) and expressed as nmol/spot/h of 4 MU (methylumbelliferone) (Hamilton et al., 2012). DBS tests were performed in Bambino Gesù Hospital in Rome. Physicians analysing LAL activity were unaware of clinical and biochemical characteristics of any enrolled patient. Inter and intra-assay variations were 2.4% and 2.3%, respectively.

To establish a normal value of LAL activity in adults, we performed DBS tests in 100 normal weight healthy subjects (HS), matched for age and sex with NAFLD patients. HS were not taking any drug or supplement before the blood sample collection, had no ultrasound evidence of fatty liver disease and did not suffer for any acute or chronic disease.

Statistical Analysis

Distribution of continuous variables was tested using a Kolmogorov–Smirnov test. Data are expressed as the mean ± standard deviation for normally distributed variables and as median followed by the 25th and 75th percentiles in parenthesis for non-normally distributed data. Group comparisons were performed by unpaired Student's t-test and by Mann–Whitney or Kruskal–Wallis test for non-normally distributed variables.

Proportions and categorical variables were tested by the χ2 test or by the 2-tailed Fisher's exact.

For the analyses, we divided the cohort according to the median value of LAL activity. We performed a multivariable logistic regression analysis with LAL activity below median as dependent variable. After testing for collinearity, the following covariates were used for the model: female gender, body mass index, alanine aminotransferase (ALT), statin therapy, anti-hypertensive drug, triglycerides, gamma-glutamyl transpeptidase (γ-gt), and metabolic syndrome. Moreover, a multivariable logistic regression analysis was performed to evaluate the independent predictors of the presence of NASH after controlling for gender, age, platelets, metabolic syndrome, homeostasis model assessment-insulin resistance (HOMA-IR), serum total cholesterol, triglycerides, γ-GT, ALT and LAL activity below median. All tests are two-tailed, and a p < 0.05 was considered as cut-off for statistical significance. Statistical analysis was performed by using the SPSS statistical software version 20.0 for Windows (SPSS, Inc., Chicago. Illinois).

Results

LAL Activity in Healthy Subjects

Median blood LAL activity in 100 HS was 1.15 (IQR 0.95–1.72) nmol/spot/h; no difference between males (n = 55) and females (n = 45) [1.08 (0.94–1.70) vs. 1.17 (0.96–1.74) p = 0.486] was found. HS group had mean age of 53.0 ± 11.3 years; LAL activity was not correlated with age (rs = − 0.53, p = 0.590).

Analysis of LAL Activity in NAFLD Patients

Clinical and biochemical characteristics of 240 NAFLD patients are listed in Table 1. Mean age was 55.4 ± 11.0 years; 60.4% were men.

Table 1

Characteristics of population.

	NAFLD patients (N = 240)
Age (years)	55.4 ± 11.0
Male gender (%)	60.4
Body mass index (kg/m2)	30.5 ± 4.7
Waist circumference (cm)a	106.0 (101.0–113.8)
Diabetes mellitus (%)	31.9
Coronary heart disease (%)	6.8
Metabolic syndrome (%)	70.5
Statin users (%)	35.7
Total cholesterol (mg/dl)	198.3 ± 38.8
LDL cholesterol (mg/dl)	117.5 ± 33.0
HDL cholesterol (mg/dl)	48.4 ± 15.6
Triglycerides (mg/dl)a	146.5 (105.5–187.5)
ALT (U/l)a	30.0 (21.0–44.0)
AST (U/l)a	22.0 (18.0–32.0)
GGT (U/l)a	28.5 (18.0–43.5)
Glycaemia (mg/dl)a	90.0 (100.0–109.0)
Insulin (mU/l)a	14.3 (10.6–20.2)
HOMA-IRa	3.7 (2.6–5.6)
Creatinine (mg/dl)a	0.9 (0.8–1.0)

ALT: alanine aminotransferase; AST: aspartate aminotransferase; GGT: gamma-glutamyl transpeptidase; HDL: high-density lipoprotein; HOMA IR: homeostasis model assessment-insulin resistance; LDL: low-density lipoprotein.

Data expressed as median and interquartile range.

LAL activity was significantly reduced in patients with NAFLD, as compared to those without [1.15 (0.94–1.72) vs. 0.78 (0.61–1.01) nmol/spot/h, p < 0.001].

To investigate factors associated with reduced LAL activity, we divided NAFLD patients in two groups according to the median value of LAL (0.78 nmol/spot/h, Table 2).

Table 2

Clinical and biochemical characteristics of patients with NAFLD above or below the median of Lysosomal Acid Lipase (LAL) activity.

	LAL activity< 0.78 nmol/spot/h (N = 120)	LAL activity≥ 0.78 nmol/spot/h (N = 120)	p
Age (years)	55.0 ± 11.0	56.0 ± 11.0	ns
Male gender (%)	63.4	59.5	ns
Body mass index (kg/m2)	30.6 ± 4.7	30.4 ± 4.7	ns
Waist circumference (cm)a	106.0 (101.0–114.0)	105.0 (100.0–113.0)	ns
Diabetes mellitus (%)	31.5	33.3	ns
Coronary heart disease (%)	5.9	7.6	ns
Metabolic syndrome (%)	76.1	65.0	0.07
Statin users (%)	28.0	43.3	< 0.05
Total cholesterol (mg/dl)	203.9 ± 40.8	192.8 ± 36.1	< 0.05
LDL cholesterol (mg/dl)	122.4 ± 32.1	112.7 ± 33.2	< 0.05
HDL cholesterol (mg/dl)	47.6 ± 17.7	49.2 ± 13.2	ns
Triglycerides (mg/dl)a	147.0 (112.0–177.0)	146.0 (103.0–195.0)	ns
ALT (U/l)a	35.0 (24.0–53.0)	26.0 (20.0–37.0)	< 0.01
AST (U/l)a	23.0 (19.0–35.0)	21.0 (18.0–27.0)	< 0.01
γ-GT (U/l)a	32.0 (21.0–51.0)	25.0 (17.0–42.0)	< 0.01
Glycaemia (mg/dl)	103.0 (90.0–115.0)	97.0 (88.0–122.0)	ns
Insulin (mU/l)a	15.2 (10.5–21.6)	13.7 (10.6–19.5)	ns
HOMA-IRa	3.9 (2.5–5.8)	3.3 (2.6–5.6)	ns
Creatinine (mg/dl)a	0.9 (0.8–1.0)	0.9 (0.8–1.0)	ns

ALT: alanine aminotransferase; AST: aspartate aminotransferase; γ-GT: gamma-glutamyl transpeptidase; HDL: high-density lipoprotein; HOMA IR: homeostasis model assessment-insulin resistance; LDL: low-density lipoprotein.

Data expressed as median and interquartile range.

We found that NAFLD patients with LAL activity below median had higher values of serum total cholesterol (p < 0.05) and LDL-c (p < 0.05), and increased serum liver enzymes (ALT, p < 0.001; AST, p < 0.01; GGT, p < 0.01). Moreover, in the same group of patients a significant lower prevalence of statin therapy (28% vs. 43.3%, p < 0.05) was present. In addition, values of LAL activity were higher in patients treated with statins compared to those without (Fig. 2).

Fig. 2

Lysosomal Acid Lipase activity in patients treated or not with statins.

At multivariable logistic regression analysis (Table 3), factors associated with impaired LAL activity (below median) were ALT (OR: 1.018, 95% CI 1.004–1.032, p = 0.011) and metabolic syndrome (OR: 2.551, 95% CI 1.241–5.245, p = 0.011), whilst statin use predicted a better LAL function (OR: 0.464, 95% CI 0.248–0.866, p = 0.016).

Table 3

Multivariate logistic regression analysis of factors associated with reduced Lysosomal Acid Lipase activity (below median).

	B	SE	Beta	p value	95.0% C.I. for B
Age	0.005	0.014	1.005	0.744	0.977	1.034
Female gender	− 0.023	0.298	0.977	0.938	0.545	1.752
Body mass index	− 0.020	0.033	0.980	0.539	0.919	1.045
ALT	0.018	0.007	1.018	0.011	1.004	1.032
Statin therapy	− 0.768	0.318	0.464	0.016	0.248	0.866
Anti-hypertensive drugs	− 0.160	0.330	0.852	0.628	0.447	1.626
Triglycerides	0.000	0.001	1.000	0.630	0.998	1.001
γ-GT	0.003	0.003	1.003	0.293	0.998	1.008
Metabolic syndrome	0.937	0.368	2.551	0.011	1.241	5.245

ALT: alanine aminotransferase; γ-GT: gamma-glutamyl transpeptidase.

LAL Activity and NASH

We also performed a sub-analysis in 35 patients with biopsy-proven NASH. We observed a progressive decrease of LAL activity from patients with simple steatosis [0.84 (0.62–1.08) nmol/spot/h, p < 0.001 vs. HS] to those with NASH [0.67 (0.51–0.77) nmol/spot/h, p < 0.001 vs. HS; p < 0.001, among groups, Fig. 1].

Fig. 1

Lysosomal Acid Lipase activity in healthy subjects (HS), non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

To further investigate the association between NASH and low LAL activity, a multiple logistic regression analysis showed a positive association of LAL activity below median (OR: 5.34, 95% CI 1.14–25.1, p < 0.05) and ALT values (OR: 1.11, 95% CI 1.07–1.16, p < 0.001) with the presence of NASH (R2 = 0.43), after adjustment for potential confounders.

Discussion

This is the first study reporting normal values of blood LAL activity in healthy adult subjects; we found a median value of LAL activity of 1.15 nmol/spot/h, with no age and sex differences. We also investigated LAL activity in a cohort of adult patients with NAFLD, and we found a significant reduction of LAL activity in NAFLD patients, compared to healthy subjects. In particular, patients with NAFLD had a 27.0% reduction of LAL activity, which increased to 41.8% in the subgroup of patients with biopsy proven NASH.

Patients with LAL activity below median had a significant elevation of serum liver enzymes and a worse lipid profile (higher total and LDL cholesterol). This last finding is in keeping with data by Muntoni S et al. showing a similar lipid profile with a polygenic hypercholesterolemia phenotype in E8SJM LAL mutation carriers (Muntoni et al., 2013).

Of note, there are no data in the literature on the correlation between circulating and hepatic LAL activity, and there is no general agreement on whether a reduced blood LAL activity directly reflects hepatic content of triglycerides and cholesterol esters (Puri et al., 2007). In this context, Singh et al. (Singh et al., 2009) described a direct involvement of autophagy and lysosomal pathway in the degradation of intracellular lipid droplets in the liver. This may suggest that a reduction of LAL activity might reduce droplets clearance and promote accumulation of both esterified and unesterified cholesterol in hepatocytes.

LAL activity was inversely associated with ALT values and with the presence of metabolic syndrome, whilst statin use was associated to higher LAL values, at multivariable analysis. The inverse association between LAL and ALT values may suggest that impaired LAL activity is associated with liver damage. This finding is partially supported by the further reduction of LAL activity observed in patients with biopsy-proven NASH. However, whether low LAL activity contributes to liver damage progression, or is itself a consequence of liver failure is still unknown.

Of particular interest is the inverse association between metabolic syndrome and LAL activity. This finding may indicate a dual relationship between the activity of LAL and the presence of metabolic risk factors. Thus, we do not know if metabolic factors modulate LAL activity or if, in turn, LAL activity may contribute to the onset of dyslipidaemia typical of metabolic syndrome. Nevertheless, measurement of LAL activity may help to differentiate patients with a major LAL deficiency-related NAFLD, from those with NAFLD due to metabolic syndrome.

The association between statin use and improved LAL function is another interesting finding of the study. The use of statins is controversial in patients with genetic deficiency of LAL, as in some CESD patients dyslipidaemia persisted despite the lipid-lowering treatment, and liver histology did not improve with statins (Reiner et al., 2014). Interestingly, Fouchier and Defesche (Fouchier and Defesche, 2013) proposed a combined approach with statins and LAL replacement therapy in patients with mutations of Lysosomal Acid Lipase gene.

Our data suggest that in adult non-genetic LAL deficiency, statin could modulate epigenetic expression of this enzyme or potentiate its activity. However, it is unclear whether statins directly influence LAL activity, perhaps as the result of a pleiotropic effect or, alternatively, modulate it through their cholesterol-lowering activity. Nevertheless, the causal relationship cannot be explored in the present study, and an ad hoc interventional study is needed.

Finally, it has been recently reported that LAL released by macrophages in the extracellular space contributes to modification of LDL within the artery wall (Dubland and Francis, 2015). The Authors speculated that early stage atherosclerosis may involve normal or increased LAL activity, whereas later stages of atherosclerosis may have an acquired dysfunction in LAL hydrolytic activity leading to lysosomal cholesterol sequestration. Thus, LAL may represent one possible mechanism contributing to the accelerated atherosclerosis and increased cardiovascular risk reported in patients with NAFLD (Sookoian and Pirola, 2008, Pastori et al., 2015b). However, further research on this issue is needed.

Limitations of the Study

A major limitation of this study is that it is a cross-sectional study, and we therefore cannot establish a cause–effect relationship between the reduction of LAL activity and the presence of NAFLD/NASH. The association between LAL activity and NAFLD/NASH should be confirmed by a prospective study to evaluate the predictive value of LAL on the progression of fatty liver disease. A further limitation is that this is a single centre study performed in consecutive patients with US evidence of fatty liver referred to a metabolic clinic.

Conclusions

In conclusion, our findings suggest a strong association between impaired LAL activity and fatty liver disease. Our findings need to be confirmed in a larger sample of NASH patients, to establish if LAL measurement may represent a non-invasive marker of NASH. A better knowledge of the role of LAL may provide new insights in the pathogenesis and progression of NAFLD.

Declaration of Interests

None.

Research in Context

Lysosomal Acid Lipase (LAL) is a hydrolase with a key role in intra-cellular cholesterol trafficking. Genetically-determined deficiency of LAL is associated with fatty liver.

We hypothesized whether a reduction of LAL activity may be found with adult NAFLD. We measured the activity of LAL in a cohort of adult NAFLD patients. We found a significant decrease of LAL activity in NAFLD patients compared to healthy subjects, with a further reduction in a subgroup of biopsy-proven NASH. Low LAL activity was directly associated with ALT values and metabolic syndrome, and inversely with statin use.

Author Contributions

FB and DP equally contributed to the data analysis and interpretation and the paper writing; MDB, LP and SDS contributed to patient recruitment, data collection and interpretation; GL performed liver biopsies; FP and GT performed laboratory determinations; FV and FA did the study design and wrote the manuscript.