Abstract
Metabolic syndrome and type 2 diabetes mellitus constitute a major problem to global health, and their incidence is increasing at an alarming rate. Non-alcoholic fatty liver disease, which affects up to 90% of obese people and nearly 70% of the overweight, is commonly associated with MetS characteristics such as obesity, insulin resistance, hypertension and dyslipidemia. In the present study, we demonstrate that hepatic lipase (HL)-inactivation in mice fed with a high-fat, high-cholesterol diet produced dyslipidemia including hypercholesterolemia, hypertriglyceridemia and increased non-esterified fatty acid levels. These changes were accompanied by glucose intolerance, pancreatic and hepatic inflammation and steatosis. In addition, compared with WT mice, HL−/− mice exhibited enhanced circulating MCP1 levels, monocytosis and higher percentage of CD4+Th17+ cells. Consistent with increased inflammation, livers from HL−/− mice had augmented activation of the stress SAPK/JNK- and p38-pathways compared with the activation levels of the kinases in livers from WT mice. Analysis of HL−/− and WT mice fed regular chow diet showed dyslipidemia and glucose intolerance in HL−/− mice without any other changes in inflammation or hepatic steatosis. Altogether, these results indicate that dyslipidemia induced by HL-deficiency in combination with a high-fat, high-cholesterol diet promotes hepatic steatosis and inflammation in mice which are, at least in part, mediated by the activation of the stress SAPK/JNK- and p38-pathways. Future studies are warranted to asses the viability of therapeutic strategies based on the modulation of these kinases to reduce hepatic steatosis associated to lipase dysfunction.
Introduction
Patients with Metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM) have a shorter lifespan compared with the general population (Zambon et al. 2009). They constitute a major problem to global health, and their incidence is increasing at an alarming rate due to population aging and to sedentary lifestyle patterns (Wild et al. 2004). Metabolic alterations that define MetS include insulin resistance (IR), abdominal obesity, glucose intolerance, hypertension and dyslipidemia.
Hypertriglyceridemia and high levels of non-esterified fatty acids (NEFA) in the MetS promote abnormal accumulation of lipids within the liver, in a form of steatosis or non-alcoholic fatty liver disease (NAFLD) (Bugianesi et al. 2005). Thus, NAFLD affects up to 90% of obese people and nearly 70% of the overweight subjects, and it is thought to be the hepatic event in the MetS. In addition, NAFLD is commonly associated with MetS risk factors such as obesity, IR, hypertension and dyslipidemia. Albeit the precise mechanisms linking characteristics of the MetS and NAFLD are not completely known, a complex relationship exists between the two (Marchesini & Marzocchi 2007, Mitsutake et al. 2011). In this sense, an excess of glucose and triglycerides, which are key components in the MetS, is produced by fatty liver (Anstee et al. 2013, Yki-Jarvinen 2014). On the other hand, IR development, which alters carbohydrate and lipid metabolism, increases triglyceride accumulation in the liver (Samuel et al. 2010, Samuel & Shulman 2012). Moreover, excess fat in adipocytes, which occurs in overweight and obesity, aggravates fatty liver disease by increasing the release of pro-inflammatory mediators such as MCP1, IL-6 and TNFα (Sahini & Borlak 2014). Of note, one of the main contributions to steatosis in NAFLD is an excess of circulating free fatty acids (FFA) and triglycerides which are also characteristics of the MetS and IR.
Hepatic lipase (HL) is a key enzyme in lipid metabolism and biology that hydrolyses triglycerides and phospholipids in lipoproteins, thus facilitating their clearance and metabolism (Santamarina-Fojo et al. 2004, Teslovich et al. 2010). HL deficiency in the mice produces mild dyslipidemia including increased total cholesterol, phospholipids and HDL-cholesterol (Homanics et al. 1995). Upon acute fat loading (Homanics et al. 1995) or in combination with other genetic deficiencies such as apolipoprotein E deficiency or LDL-receptor deficiency (Mezdour et al. 1997, Freeman et al. 2007), HL inactivation in the mice also produces hypertriglyceridemia and atheroma lesions. Interestingly, HL deficiency did not change body weight or food intake in the mice (Escola-Gil et al. 2013). In humans, genetic studies have linked HL gene polymorphisms to progression of abdominal obesity and T2DM (Todorova et al. 2004). Reduced abdominal obesity and weight loss in subjects are associated with reduced HL activity and prevention of T2DM (Bergeron et al. 2001, Todorova et al. 2004). Other studies have shown that HL gene is associated with dyslipidemia characterised by high levels of atherogenic LDL, low HDL and high triglycerides (Teslovich et al. 2010). The role of HL in cardiovascular disease progression in humans is controversial and HL increased levels are beneficial in patients with hypercholesterolemia (Dugi et al. 2001) but detrimental in subjects with central obesity and IR (Teran-Garcia et al. 2005, Zhang et al. 2006, Brunzell et al. 2011). Therefore, the effect of HL in disease progression is highly dependent on the underlying lipoprotein and lipid phenotype (Brunzell et al. 2011).
Despite the preceding association studies, the effect of HL inactivation in the glucose metabolism homeostasis and in the development of fatty liver disease has not been fully investigated. To this end, HL-deficient (HL−/−) and WT mice were fed a high-fat, high-cholesterol (HFHC) diet for four months or fed regular chow diet (RCD) and glucose metabolism, hepatic steatosis, pancreatic characteristics and the associated inflammation were analysed.
Materials and methods
Mice and diets
Animal care was in accordance with institutional guidelines and the 2010/63/EU directive from the European Parliament. HL−/− (Jackson laboratories) and WT (Charles River) mice were on C57BL/6J background. After weaning, mice were maintained on a RCD (2.8% fat; Panlab, Barcelona, Spain) and at two months of age, mice were placed on a HFHC diet (10.8% fat, 0.75% cholesterol, S4892-E010, Ssniff, Germany) for 16 weeks or left on RCD for 16 more weeks.
Metabolic measurements
Plasma triglycerides, total cholesterol (WAKO, Neuss, Germany) and non-esterified fatty acids (NEFA, SIGMA) levels were measured using enzymatic procedures in overnight-fasted mice. HDL-cholesterol (HDL-C) was determined after precipitation of the apolipoprotein B-containing lipoproteins with dextran sulphate/MgCl2 (SIGMA) (Gonzalez-Navarro et al. 2010). For the glucose tolerance test (GTT), overnight-fasted mice received an intraperitoneal injection of glucose (2 g/Kg of body weight, BW, SIGMA) and plasma glucose and insulin levels were analysed at different time-points using a glucometer (Ascensia Elite, BAYER, Leverkusen, Germany) and an ultrasensitive anti-mouse insulin ELISA (MERCODIA, Uppsala, Sweden) respectively (Gonzalez-Navarro et al. 2007, 2008). Plasma glucose and insulin levels in feed state were measured between 10:00 and 12:00 h in the morning. For the insulin tolerance test (ITT), 4 h-fasted mice received an intraperitoneal injection of insulin (0.5 U/Kg of BW ACTRAPID, NovoNordisk, Bagsvaerd, Denmark) and plasma glucose levels were measured as before (Gonzalez-Navarro et al. 2013). HOMA-IR index was determined using the formula: fasting plasma glucose (mmol/l)×fasting plasma insulin (μU/l)/22.5. Liver triglyceride content was determined by tissue digestion and saponification in ethanolic potassium hydroxide followed by enzymatic measurement of glycerol content (Free Glycerol Reagent, SIGMA) (Norris et al. 2003).
Pancreatic islet isolation and insulin secretion assay
For islet isolation, mice were infused with Krebs buffer (127 mM NaCl, 5 mM KCl, 3 mM CaCl2, 1.5 mM MgCl2, 24 mM NaHCO3, 6 mM Hepes, 2 mg/ml glucose, 0.1% albumin, equilibrated with 5% CO2 in O2) and their pancreases were dissected and digested with collagenase-NB8 (1 mg/ml, Serva, Heidelberg, Germany) at 37 °C in a shaking waterbath for 20 min. Islets were handpicked under stereoscope (Vinue et al. 2015). Insulin secretion was evaluated by a glucose-stimulated insulin secretion assay at low (2.8 mmol/l) and high (16.7 mmol/l) glucose concentrations in KRBH buffer (140 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 20 mM Hepes, 2 mg/ml glucose, 0.1% albumin). Six assays (five islets each) were performed per condition. Insulin concentrations were measured by ELISA. The stimulation index was calculated as the ratio of glucose-stimulated insulin to basal insulin normalised by the insulin content.
Liver and pancreas immunostainings
Livers and pancreases were sectioned from mice sacrificed by cervical dislocation after perfusion with PBS and fixed with 4% paraformaldehyde/PBS 4 h and paraffin-embedded as described (Gonzalez-Navarro et al. 2013). β-cell mass was measured as the islet area relative to total pancreatic area (%) obtained from the analysis of 10–12 slides (separated 125 μm) per mouse stained with an anti-insulin antibody (described in the following paragraph). Lipid droplet (LD) images were obtained from hematoxylin/eosin stained sections.
The immunohistochemistry protocol consisted of peroxidase inactivation (H2O2 0.3% in distilled water), antigen retrieval with Sodium Citrate buffer 10 mM, pH 6.5, blocking (horse serum 5%, 1 h, RT), incubation with primary antibodies (rabbit polyclonal anti-insulin 1/200 dilution, sc-9168, Santa Cruz Biotechnologies; rat monoclonal anti-F4/80 1/50 dilution, MCA497G, AbD Serotec, ThermoFisher, Kidlington, UK) followed by biotinylated goat anti-rat or anti-rabbit secondary antibodies (1 h, RT, 1/500 dilution, sc-2491, sc-2041, Santa Cruz Biotechnologies), streptavidin-HRP (TS-060-HR, ThermoScientific, Cheshire, UK) and DAB substrate (SK4100, Vector Laboratories, Burlingame, CA, USA). Slides counterstained with hematoxylin were mounted with EUKITT (A10500, Deltalab, Barcelona, Spain). Images were captured with an OPTIKAM-PRO5 digital camera mounted on a stereomicroscope (OPTIKA, Barcelona, Spain) and analysed by computer-assisted morphometry (SigmaScan, Pro5).
Double immunofluorescences insulin/glucagon, insulin/CD3+ and insulin/Ki67 consisted of antigen retrieval (Sodium Citrate buffer 10 mM, pH 6.5 for insulin/glucagon and insulin/Ki67 and with Tris EDTA buffer 10 mM pH 9, for insulin/CD3+ high pressure and temperature) and blocking (horse serum 5%, 1 h, RT), incubation (overnight at 4 °C) with primary antibodies (mouse monoclonal anti-glucagon, 1/300, G2654, SIGMA and rabbit polyclonal anti-insulin 1/200 dilution, sc-9168, Santa Cruz Biotechnologies; mouse monoclonal anti-insulin 1/300, I2018 SIGMA; rabbit monoclonal anti-Ki67, Clone SP6,MAD-000310QD, VITRO) followed by incubation (1 h at RT) with a goat anti-mouse IgG Alexa Fluor 594 and an anti-rabbit IgG AlexaFluor488 (1/200, A11005 and A21206, Invitrogen) secondary antibodies. Nuclear staining was performed with DAPI (1/1000, 20 min, D1306, Invitrogen) and slides were mounted with Slow-Fade Gold reagent (S36936, Invitrogen) and analysed with an inverted fluorescent microscope (LEICA DMI 3000B).
Enzyme-Linked ImmunoSorbent Assay (ELISA)
MCP1 and TNF-α circulating levels were determined in isolated plasma from heparinised blood (10 U heparin/ml) from mice using the Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA).
Flow cytometry
Circulating monocytes were determined in 10 μl of heparinised whole blood incubated for 30 min at RT with Ly6C-PerCP (BD Pharmingen, Madrid, Spain) and CD115-APC (Biolegend, San Diego, CA, USA). For lymphocytes, 10 μl of heparinised whole blood was incubated for 30 min RT with 5 μl Brilliant Stain Buffer (563794, BD) and with Brilliant violet (BV)-rat anti-mouse CD4 (562891, BD), BV-rat anti-mouse CD8a (563068, BD), PE-hamster anti-mouse CD69 (553237, BD) and APC-hamster anti-mouse CD3e (553066, BD). Incubation with lysing solution (BD Facs Lysing solution) was done before analysis by flow cytometry (FACSVerse BD Biosciences). To detect functionally-polirised CD4+T lymphocyte, 100 μl of heparinised whole blood was stained with the mouse Th17/Treg phenotyping kit (BD Pharmingen, Madrid, Spain) to detect CD4+Foxp3+ and CD4+IL17 cells. Analysis of Ly6Clow- and Ly6Chi-subsets were determined in CD115+ populations.
Western blot analysis
Liver protein lysates were obtained in the presence of the ice-cold lysis TNG buffer (Tris–HCl 50 mM, pH7.5, NaCl 200 mM, Tween-20 1%, NP-40 0,2%) supplemented with Complete Mini cocktail, PhosSTOP (Roche, Mannheim, Germany), ß-glicerolphosphate 50 mM (SIGMA), 2 mM phenylmethylsulfonyl Fluoride (PMSF, ROCHE, Mannheim, Germany) and 200 μM Na3VO4 (SIGMA). Protein extracts (50–100 μg) were prepared in laemmli's buffer and analysed by 12% polyacrilamide gel electrophoresis and western blot (Gonzalez-Navarro et al. 2013, Martinez-Hervas et al. 2014). The primary antibodies used were: rabbit polyclonal anti-Phospho-p38 (1/200, sc-17852-R, Santa Cruz Biotechnologies), rabbit polyclonal anti-p38 (1/200, sc-535, Santa Cruz Biotechnologies), rabbit polyclonal anti-Phospho-SAPK/JNK (1/200, Thr183/Tyr185, 9251 Cell Signaling), rabbit polyclonal SAPK/JNK (1/200, 56G8, 9258 Cell Signaling) and mouse monoclonal anti-α-tubulin (1/500, sc-8035, Santa Cruz Biotechnologies). The HRP-conjugated secondary antibodies (1/500, Santa Cruz Biotechnologies) used were: anti-mouse IgG-HRP (sc-2005) and goat anti-rabbit IgG-HRP (sc-2004). The immunocomplexes were detected with an ECL Plus detection kit (ThermoFisher Scientific, Barcelona, Spain).
Gene expression analysis by quantitative real-time PCR
RNA (0.5–1 μg) from mouse liver obtained with TRIzol Reagent (Invitrogen), was retrotranscribed with the Maxima First-Strand cDNA Synthesis kit and amplified with Luminars Color-HiGreen/High ROX qPCR MasterMIX (Fermentas, Madrid, Spain) on 7900Fast System. Results were analysed with the provided software (Applied Biosystems). The mRNA levels were normalised to endogenous gene Cyclophilin expression and relativised to WT mRNA levels. The primers were designed with the primer express programme and were (Forward: Fw; Reverse: Rv): cyclophilin Fw: 5′-TGGAGAGCACCAAGACAGACA-3′ and Rv: 5′-TGCCGGAGTCGACAATGAT-3′; Tnf-α Fw: 5′-CCCACACCGTCAGCCGATTT-3′ and Rv: 5′-GTCTAAGTACTTGGGCAGATTGACC-3′; Mcp1 Fw: 5′-GCCCAGCACCAGCACCAG-3′ and Rv: 5′-GGCATCACAGTCCGAGTC-3′; Jnk1 Fw: 5′-CAACGTCTGGTATGATCCTTCAGA-3′; Rv: 5′-GCTCCCTCTCATCTAACTGCTTGT-3′; p38α Fw: 5′-GACTTTCCCTGTTGGACAGCTT-3′; Rv: 5′-CAACAGACTGACCCGCTAAGG-3′.
Statistical analysis
Data are presented as the mean±s.e.m. Differences were evaluated using a two-tail, unpaired Student's t-test and were considered statistically significant when P≤0.05 (GraphPad Prism Software Inc, La Jolla, CA, USA). Outliers identified with Grubbs' test (GraphPad Prism Software) were not considered.
Results
Metabolic characterisation of WT and HL−/− mice on a high-fat, high-cholesterol diet
Two-month-old WT and HL−/− male mice were fed with a HFHC diet for 16 weeks and were characterised. HL-deficiency increased total cholesterol (P<0.0001), HDL-cholesterol (P<0.02), triglycerides (P<0.01) and NEFA (P<0.02) (Fig. 1A). No changes were observed in BW between mouse groups (Fig. 1B). Fasting glucose levels were not different between genotypes but HL−/− mice exhibited augmented glucose levels in fed state compared with WT mice (Fig. 1C, P<0.02). Insulin levels were undistinguishable between mice in both fed and overnight-fasted states (Fig. 1D).
Plasmatic parameters in WT and HL−/− mice placed 4 months on a high-fat, high-cholesterol diet. (A) Total cholesterol, HDL-cholesterol, triglycerides and non-esterified fatty acids in mice. (B) Body weight, (BW) in both groups of mice. (C) Plasma glucose levels in mice fasted overnight (left panel) and in fed state (right panel). (D) Plasma insulin levels in mice fasted overnight (left panel) and in fed state (right panel). Statistical analysis was performed using Student's t-test.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
Carbohydrate metabolism by GTT showed glucose intolerance in HL−/− mice (Fig. 2A, top panel), as shown by the increased area under the curve (glucose curve vs time, AUCglucose right graph P<0.01), compared with WT mice. Insulin levels during the GTT revealed impaired glucose-stimulated insulin release in HL−/− mice as revealed by decreased AUCinsulin (insulin curve vs time) value, compared with that in WT mice (Fig. 2A, lower panel P<0.03). No differences between genotypes were found in the ITT, in the corresponding AUCglucose (Fig. 2B) or in the HOMA-IR index (Fig. 2C) indicating no difference in insulin sensitivity. Analysis of the in vitro glucose-stimulated insulin secretion in isolated islets from HFHC diet-fed mice, showed reduced secretion in HL−/− mice compared with that of WT mice (Fig. 2D, P<0.006). Thus, HL-deficiency in mice fed a HFHC diet produces glucose intolerance and impaired glucose-stimulated insulin-secretion.
HL deficiency produces glucose intolerance in mice placed on a high-fat, high-cholesterol diet for 4 months. (A) Plasmatic glucose (top panel) and insulin (lower panel) levels at the different time points during the GTT in WT and HL−/− mice which were used to calculate the AUCglucose and AUCinsulin (right panels). (B) Glucose levels (in percentage relative to the initial glucose levels) during the ITT at 0.5 U/KgBW in 4 h-fasted WT and HL−/− mice. The lower graph displays the AUCglucose parameter for the two groups of mice. (C) HOMA-IR index in both groups of mice. (D) In vitro insulin secretion of islets from WT and HL−/− mice. Statistical analysis was performed using Student's t-test.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
HL−/− mice fed a high-fat, high-cholesterol diet have increased hepatic triglyceride content
Bearing in mind the complex relationship between NAFLD, dyslipidemia and glucose metabolism derangement, hepatic analysis was next performed. Compared with WT mice, HL−/− mice had augmented liver triglyceride content (Fig. 3A, P<0.04) indicating increased steatosis as shown by the hematoxylin-eosin stained section analysis of both groups of mice (Fig. 3A images). Immunohistochemical analysis of the F4/80 macrophage marker also showed increased (kuppfer) macrophage infiltration in the liver of HL−/− mice compared with that in WT mice (Fig. 3B, P<0.03). Analysis of proinflammatory cytokine expression in the liver demonstrated enhanced mRNA levels of Mcp1 in HL−/− mice compared with those in WT mice (Fig. 3C, left panel, P<0.03). No differences between both groups of mice were observed in Tnfα mRNA expression (Fig. 3C, right panel).
Liver characterisation in WT and HL−/− mice. (A) Analysis of triglyceride content in liver from both groups of mice. Images of hematoxylin-eosin stained sections showing lipid droplets (LD). (B) Macrophage content (F4/80+ cells relative to hepatic area) in hepatic cross-sections of mice. Representative images are shown. (C) Quantification of the Mcp1 and Tnfα hepatic mRNA levels normalised to Cyclophilin and relativised to WT mouse mRNA. Statistical analysis was performed using Student's t-test. A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-15-0219.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
These results indicate that HL-deficiency increases fatty liver disease and hepatic inflammation in mice fed with a HFHC diet.
Increased inflammatory state in HL−/− mice
Analysis of systemic inflammation showed increased circulating MCP1 cytokine levels in HL−/− mice compared with those for WT mice (Fig. 4A, P=0.05). No changes were observed in TNFα circulating plasma levels. Leukocyte population analysis also revealed enhanced levels of CD115+ monocytes in HL-deficient mice compared with those in WT mice (Fig. 4B, P<0.03). Similar percentages of the proinflammatory Ly6Chi- and the patrolling Ly6Clow-monocyte subsets were similar in both WT and HL−/− mice (Fig. 4B, right panel). T-cell lymphocyte analysis showed increased number of total CD3+ (Fig. 4C, P<0.04) and CD4+T-lymphocyte subset (Fig. 4C, P<0.04) without changes in the percentage of the CD8+T-cells or in the activated CD69+-T-cells. Analysis of circulating regulatory (CD4+Foxp3+) T-cells showed no differences between both groups of mice but Th17 subtype CD4+T-cells were significantly increased in HL−/− mice compared with that in WT mice (Fig. 4D, P<0.008).
Analysis of inflammation in WT and HL−/− mice. (A) MCP1 and TNF-α circulating plasma levels in mice. (B) Percentage of circulating monocytes identified as CD115+ cells and percentage of Ly6Clow and Ly6Chi monocyte subsets in HL−/− and WT mice analysed by flow cytometry. (C) Percentage of circulating total and activated (CD69+) T-lymphocytes (CD3+) and CD4+, CD8+ and CD8+CD69+ T-lymphocyte subsets identified by flow cytometry. (D) Percentage of Tregs measured as double CD4+Foxp3+T-cells and CD4+Th17+-T-cells in both groups of mice. Statistical analysis was performed using Student's t-test.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
Altogether these results indicate that HL-deficiency in mice, increases inflammation and circulating inflammatory cells including monocytes, CD3+ and CD4+T-cells and the Th17 T-subset cells expressing IL17.
Pancreas characterisation in HL−/− and WT mice
To explore the differences in glucose-stimulated insulin-secretion between HL−/− and WT mice, pancreatic characterisation was next performed. Analysis of pancreatic islets by insulin immunohistochemistry demonstrated no differences in the relative area occupied by β-cells between HL−/− and WT mice (Fig. 5A). Similarly, no differences were observed in the α/β-cell area ratio in pancreatic islets of mice (Fig. 5B). β-cell proliferation analysis measured as double Ki67/insulin-positive cells, showed similar proliferative rates in HL−/− and WT mice (Fig. 5C). Thus, these results indicate that the observed differences in insulin-secretion were not due to differences in β-cell mass or β-cell maintenance.
Pancreatic islet characterisation in WT and HL−/− mice. (A) Quantification of β-cell area (in percentage relative to pancreatic area) identified by anti-insulin immunohistochemistry. (B) Islet α/β ratio in the pancreas of both groups of mice. (C) Quantification of proliferating β-cells, identified as double insulin/Ki67-positive cells relative to islet area determined by inmunofluorescence. (D) T-cell infiltration in the pancreas identified as CD3+-positive cells relative to pancreatic area in both groups of mice. (E) Quantification of area occupied by inflammatory foci (IF) in pancreatic regions next to islets in both groups of mice. Representative images of the immunohistochemistry, immunofluorescence and stainings are shown. Statistical analysis was performed using Student's t-test. A full colour version of this figure is available via http://dx.doi.org/10.1530/JOE-15-0219.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
Given the increase in circulating inflammatory cells in HL−/− mice, inflammatory infiltration was investigated in the pancreas. Analysis of CD3+-cells in pancreatic sections showed enhanced percentage of T-cells in the exocrine pancreas of HL−/− mice compared with that in WT mice (Fig. 5D, P<0.05). Moreover, analysis of inflammatory foci (IF) identified as a mass of cells invading the islets, also revealed augmented inflammation in the exocrine pancreas next to islets in HL−/− mice compared with that in WT mice (Fig. 5E, P<0.007). Thus, HL-deficiency in the mice produces pancreatic inflammation.
Activation of stress-related pathways in HL−/− and WT mice
Given the observed differences in inflammation and previous studies showing increased activation of the stress-MAPKinases associated with fatty liver disease (Sahini & Borlak 2014) the activation of these pathways was next evaluated. Protein lysate analysis of the p38 stress-pathway revealed increased (phospho)pp38 levels in the liver from HL−/− mice compared with those in WT mice (Fig. 6A, P<0.003). Similarly, analysis of the activated SAPK/JNK (pSAPK/JNK) protein levels were also higher in the liver of HL−/− mice compared with those in WT mice (Fig. 6B, P<0.01). No differences in the p38 and Sapk/Jnk mRNA levels were observed between genotypes (Fig. 6C). These results indicate increased activation of the stress kinases in HL−/− mice which is consistent with increased inflammatory state.
Stress-kinase expression in liver from WT and HL−/− mice. (A) Analysis of p38 activated phosphop38 (pp38) and p38 protein levels in livers from both genotypes. (B) Analysis of phospho SAPK/JNK and SAPK/JNK protein levels in liver from both groups of mice. For quantification phosphorylated forms were normalised to total p38 and SAPK/JNK protein levels. Representative blots of the phosphorylated and unphosphorylated proteins of the kinases are shown. Blots showing α-tubulin analysis is included as loading controls. (C) Quantification of the p38 (left panel) and Sapk/Jnk (right panel) mRNA expression levels in the mice. The mRNA expression levels were normalised to cyclophilin mRNA levels and relativised to WT mouse mRNA levels. Statistical analysis was performed using Student's t-test.
Citation: Journal of Endocrinology 227, 3; 10.1530/JOE-15-0219
HL-deficiency is not sufficient to induce hepatic steatosis in mice fed regular chow diet
Dietary cholesterol is an important risk factor for the progression of NAFLD (Wouters, et al. 2008), therefore mice fed RCD were also characterised (Fig. S1, see section on supplementary data given at the end of this article). HL-deficiency increased total cholesterol and triglycerides (Fig. S1A, P<0.05 and P<0.01, respectively) but no differences were observed in BW, glucose or insulin plasmatic levels between genotypes (Figure. S1B). Further analysis showed impaired glucose tolerance, demonstrated by increased AUCglucose, (Figure. S1C, right graph P<0.05) in HL−/− mice and no changes in glucose-stimulated insulin release, as revealed by similar AUCinsulin (Fig. S1C, lower panel) or in insulin-sensitivity during the ITT, as shown by similar AUCglucose (Fig. S1D). HL-deficiency in RCD-fed mice produces glucose intolerance but no changes in glucose-stimulated insulin-release.
Hepatic analysis in WT and HL−/− fed RCD revealed no differences in liver triglyceride content and steatosis (Fig. S2A, see section on supplementary data given at the end of this article) as also shown by the hematoxylin-eosin stained sections (images in Fig. S2A). Circulating plasmatic levels of MCP1 and activation of the p38-stress inflammatory pathway (Fig. S2B and C) were indistinguishable between mice indicating similar inflammation in RCD-fed HL−/− and WT mice. Pancreatic analysis revealed increased inflammatory foci, but not significant (Fig. S2D) in HL−/− mouse pancreas and no differences in CD3+ content between genotyopes (Fig. S2E). These results indicate that HL-deficiency has no effect on hepatic steatosis or inflammation in mice fed RCD.
Discussion
MetS and T2DM has become a major health burden to global health, and their incidence is increasing at an alarming rate (Wild et al. 2004). A complex relationship between NAFLD, hypertriglyceridemia, glucose intolerance and MetS exists (Bugianesi et al. 2005). In the present study, we demonstrate that HL-deficiency combined with a HFHC diet in the mice produces hypercholesterolemia, hypertriglyceridemia, increased NEFA levels and glucose intolerance. These metabolic alterations were accompanied by increased hepatic steatosis, hepatic macrophage infiltration and pancreatic inflammation. Moreover, compared with WT mice, systemic inflammation was enhanced in HL−/− mice which exhibited monocytosis, higher percentage of the proinflammatory CD4+Th17+-T-cells and augmented levels of MCP1. Analysis of inflammatory stress pathways revealed increased levels of the (activated) pp38- and pSAPK/JNK-kinases in HL−/− mice suggesting a possible role of these signalling-pathways in the metabolic alterations induced by HL-inactivation. Interestingly, HL−/− mice fed RCD diet exhibited glucose intolerance and dyslipidemia, but not systemic inflammation or hepatic steatosis suggesting that the dietary components play a main role in NAFLD and inflammation induced by HL-deficiency. Thus, NAFLD and inflammation seem to be secondary to metabolic alterations (glucose intolerance and dyslipidemia). Altogether, these studies indicate a protective role of HL in fatty liver disease associated with HFHC diet by restoring dyslipidemia, glucose tolerance and by decreasing inflammation.
Previous studies have shown that dyslipidemia, high FFA and hypertriglyceridemia induced by lipase-deficiencies promote hepatic steatosis and NAFLD. Thus, hepatic depletion of adipose triglyceride lipase (ATGL) in mice leads to severe liver steatosis (Ong et al. 2011). Decreased activity of ATGL in the liver of IR patients has also been associated with development of NAFLD (Kato et al. 2008). Consistent with these findings, hepatic overexpression of the hormone-sensitive lipase (HSL) and ATGL promotes fatty acid oxidation, ameliorates steatosis and improves insulin signal transduction in the mice (Reid et al. 2008, Turpin et al. 2011). On the other hand, loss of intracellular TGH/Ces3-lipase which, by contrary, reduces blood lipids and improves glucose tolerance, ameliorated hepatic lipid deposition (Wei et al. 2010). Surprisingly, a previous study reported decreased BW gain and reduced hepatic steatosis in HL-deficient mice (Chiu et al. 2010). These seemingly discrepancy between these later results and the findings of the present study, might be related to the different dietary regimens used in both studies. Chiu and colleagues used an obesogenic diet (21% of fat and 0.15% of cholesterol), which resulted in no differences in cholesterol or in FFA levels while in the present study, the diet contained a 10.8% fat and 0.75% of cholesterol which produced higher levels of triglycerides, NEFA and cholesterol. In fact, in the present study when mice were fed RCD HL−/− mice did not develop hepatic steatosis or inflammation. Thus, HL-deficiency in combination with a HFHC diet that causes dyslipidemia (hypertriglyceridemia, increased NEFA and high cholesterol) results in hepatic steatosis. Altogether, these studies suggest that elevated NEFA generated from the high triglyceride levels induced by lipase-deficiencies and the presence of dietary cholesterol are key factors for developing hepatic steatosis.
Of note, HL−/− mice developed hypercholesterolemia, consisting mostly of high HDL-cholesterol levels, which would rather appear as a protective mechanism of disease progression. In fact, therapies targeted to raise HDL-cholesterol are effective in treating dyslipidemia and disease progression (Rayner et al. 2010, Waksman et al. 2010). Nevertheless, consistent with our findings, recent studies have shown that chronic increased HDL-cholesterol levels produced by some of these therapies, such as long-term therapeutic silencing of miR-33 in mice challenged with a high-fat diet, produced moderate hypertriglyceridemia and hepatic steatosis (Goedeke et al. 2014). Altogether, these data suggest that modulation of lipases that develops with ‘toxic’ dyslipidemia (hypercholesterolemia, hypertriglyceridemia and high FFA) promote hepatic steatosis and NAFLD.
NAFLD is frequently associated with hepatic IR and MetS (Sabio et al. 2008, Yang et al. 2009, Gruben et al. 2014). Thus, while IR development increases triglyceride accumulation in the liver (Samuel et al. 2010, Samuel & Shulman 2012), fatty liver produces an excess of glucose and triglycerides, which are key components in IR and MetS (Anstee et al. 2013, Yki-Jarvinen 2014). Consistently, other studies have shown that decreased hepatic steatosis, induced by lipase (ATGL) overproduction is accompanied by improved insulin sensitivity and insulin-signalling (Turpin et al. 2011). In the study presented here, hepatic steatosis in HL−/− mice was accompanied by higher glucose levels in fed state and glucose intolerance. However, HOMA-IR index and sensitivity to insulin in HL−/− mice were similar to those in WT mice indicating no altered insulin sensitivity. Thus, the delayed glucose clearance observed in HL−/− mice could be caused by defective insulin secretion by the β-cells. In fact, insulin secretion during the GTT and in isolated islets revealed an impaired capacity of glucose-stimulated insulin release in HL−deficient mice. Analysis of the pancreases in HFHC-diet fed mice did not revealed differences in β-cell mass but HL−/− mice exhibited inflamed pancreas (with increased T-cells and inflammatory foci) which could mean pancreatic damage affecting pancreas functionality. In support of this hypothesis, high concentrations of FFA, which are present in the plasma of the lipoprotein lipase (LPL)-deficient mice and in patients with hypertriglyceridemia, lead to pancreatic acinar cell damage and are risk factors for acute pancreatitis (Yang et al. 2009). Interestingly, a case-report study showed association between decreased activities of LPL and HL, hypertriglyceridemia and acute pancreatitis (Fujita et al. 2010). Altogether, these studies suggest that hypertriglyceridemia and increased FFA might affect glucose homeostasis by modulating insulin sensitivity but lipase-deficiencies also might impair pancreatic function by promoting tissue inflammation.
Low grade inflammation is also a characteristic of advanced hepatic steatosis or nonalcoholic steatohepatitis (NASH) (Sabio et al. 2008, Yang et al. 2009, Gruben et al. 2014). Fatty liver in HL−/− mice was accompanied by augmented hepatic inflammation (macrophage content and Mcp1 mRNA levels) and by increased systemic inflammation including higher circulating MCP1 levels, monocytosis and higher percentage of T-cells. In addition, CD4+Th17 T-cells, which have been associated to chronic inflammation (Ramesh et al. 2014), were also significantly augmented in HL−/− mice compared with those in WT mice. Therefore, HL-deficiency in the mice fed a HFHC diet, which provides dietary cholesterol, produced advanced NAFLD or NASH. HL−/− and WT mice fed RCD did not develop systemic inflammation or hepatic steatosis indicating a role of the HFHC diet in the development of fatty liver. Consistent with our findings, dietary cholesterol has been shown to be sufficient to cause hepatic inflammation in steatosis in hyperlipidemic mouse models (Wouters et al. 2008). Altogether, these results indicate that the dyslipidemia in fatty liver disease that develops with high cholesterol levels also enhances the risk of developing inflammatory steatosis or NASH.
Several characteristics of the NAFLD progression, such as hepatocyte ballooning, lead to activation of the stress-MAPKinase signalling and previous studies have linked activation of the inflammatory stress-MAPKinases with progression of fatty liver disease (Sahini & Borlak 2014). In this sense, the activation of JNK as a main mechanism for the development of steatohepatitis has been largely described (Schattenberg et al. 2006, Tuncman et al. 2006, Singh et al. 2009). Others have shown a relevant role of p38-activation in fatty liver disease (Menghini et al. 2012, Nio et al. 2012, Song, et al. 2014). In addition, NEFA liberated by HSL activate both p38 and JNK, and p38 mediates proinflammatory cytokine expression in adipose tissue (Mottillo, et al. 2010). On the other hand, lipid-droplet formation in monocytes is mediated by p38 and SAPK/JNK activation (Guijas et al. 2012). In the line of these investigations, when mice were fed a HFHC diet, activation of the p38 and SAPK/JNK-signalling pathways was increased in the liver of HL−/− mice compared with that in WT mice. Thus, our investigations suggest that, in HFHC diet, activation of p38 and SAPK/JNK stress-pathways seems to mediate progression of hepatic inflammation, and steatosis associated with the glucose metabolism impairment and dyslipidemia (hypercholesterolemia, hypertriglyceridemia and increased NEFA) induced by HL-deficiency.
In summary, the present study demonstrates that the dyslipidemia consisting of hypercholesterolemia, hypertrigliceridemia and increased NEFA induced by HL-deficiency in combination with a HFHC diet, produces hepatic steatosis, hepatic and pancreatic inflammation and glucose intolerance and suggest a protective role of HL in the development these metabolic alterations. These changes were accompanied by an increase in systemic inflammation with enhanced percentage of monocytes and CD4+Th17-T-cells and MCP1 circulating levels. These events seem to be mediated, at least in part, by the activation of the stress p38- and JNK-signalling kinases in the liver suggesting that modulation of these kinases might prevent fatty liver disease-induced by dyslipidemia and HFHC diet. Future studies are warranted to assess the viability of therapeutic strategies based on the modulation of these kinases to reduce hepatic steatosis/NAFLD, tissue (hepatic and pancreatic) inflammation and glucose intolerance associated to lipase dysfunction.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-15-0219.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by grants from the Carlos III Health Institute (FIS: PI-CP10/00555 and PI13/00834 to HG-N) from the Spanish Ministry of Economy and Competitiveness (SAF2011-23777 to M J S) and from the European Regional Development Fund (FEDER) and research grants from Generalitat Valenciana (GVACOMP2014-006 and PROMETEO II/2013/014). H G-N is an investigator from the ‘Miguel Servet’ program (CP10/00555). I A-B and A V received salary support from Proyecto Paula. This work was supported by the CIBERDEM, a Carlos III Health Institute initiative.
Author Contribution statement
I A-B and A H-C participated in the study design, the acquisition and interpretation of data, and in writing the manuscript. A V performed metabolic studies and characterisation of pancreatic islets. S M-H participated in the design and revised the manuscript. L P and M J S participated in the stress-signalling experiments and inflammatory analysis. D J B participated in the study design and revised the manuscript. H G-N conceived and designed the study, supervised the acquisition of data, interpreted the results and wrote the manuscript.
Acknowledgements
We thank Dr A Díaz for animal care and G Herrera for flow cytometry.
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(I Andrés-Blasco and A Herrero-Cervera contributed equally to this work)
