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Molecular hydrogen attenuates fatty acid uptake and lipid accumulation through downregulating CD36 expression in HepG2 cells

Abstract

Background:

There is accumulating evidence that obesity is closely associated with an impaired free fatty acid metabolism as well as with insulin resistance and inflammation. Excessive fatty acid uptake mediated by fatty acid translocase CD36 plays an important role in hepatic steatosis. Molecular hydrogen has been shown to attenuate oxidative stress and improve lipid, glucose and energy metabolism in patients and animal models of hepatic steatosis and atherosclerosis, but the underlying molecular mechanisms remain largely unknown.

Methods:

Human hepatoma HepG2 cells were exposed to palmitate-BSA complex after treatment with or without hydrogen for 24 h. The fatty acid uptake was measured by using spectrofluorometry and the lipid content was detected by Oil Red O staining. JNK phosphorylation and CD36 expression were analyzed by Western blot and real-time PCR analyses.

Results:

Pretreatment with hydrogen reduced fatty acid uptake and lipid accumulation after palmitate overload in HepG2 cells, which was associated with inhibition of JNK activation. Hydrogen treatment did not alter CD36 mRNA expression but reduced CD36 protein expression.

Conclusion:

Hydrogen inhibits fatty acid uptake and lipid accumulation through the downregulation of CD36 at the protein level in hepatic cultured cells, providing insights into the molecular mechanism underlying the hydrogen effects in vivo on lipid metabolism disorders.

Background

Obesity and its associated disorders such as type 2 diabetes, coronary heart diseases and non-alcoholic fatty liver disease (NAFLD) are currently global health problems. There is accumulating evidence that obesity is closely associated with impaired free fatty acid (FFA) metabolism as well as insulin resistance and inflammation [1]. Excessive release of FFA from visceral fat adipocytes leads to the production of inflammatory and proatherogenic proteins through activation of the NFκB and c-Jun NH2-terminal kinase (JNK) pathways in skeletal muscle, liver, and endothelial cells, and promotes atherosclerotic vascular disease (ASVD) and NAFLD.

Fatty acid translocase CD36 mediates uptake of FFA from circulation and intracellular transport of long- chain fatty acids in diverse cell types such as monocytes, platelets, macrophages, microvascular endothelial cells, adipocytes, muscle cells, enterocytes, and hepatocytes [2]. Mice deficient in CD36 exhibit defective uptake and utilization of fatty acids. Excessive fatty acid uptake mediated by CD36 plays an important role in hepatic steatosis [3]. The expression level of CD36 is very low in normal liver tissues [4], but is drastically increased in the liver tissues of high-fat diet (HFD)-induced fatty liver mice and those of human NAFLD. Conversely, forced expression of CD36 in liver causes hepatic steatosis in the absence of HFD [5]. There is extensive evidence showing that CD36 plays significant roles in hepatic steatosis, suggesting that CD36 can be a potential drug target against NAFLD.

Since the first report in 2007, which demonstrated the effect of molecular hydrogen on brain infarction [6], hydrogen has been shown to protect against a variety of diseases including oxidative stress-related diseases, inflammation and allergy in in vivo and in vitro models as well as in humans [7]. In the metabolic diseases, hydrogen attenuates oxidative stress and improves lipid, glucose and energy metabolism in patients and animal models of hepatic steatosis and atherosclerosis, but the underlying molecular mechanisms remain largely unknown [8-11]. Although the hydrogen effects have been ascribed to a selective scavenging of hydroxyl radicals, we previously reported that hydrogen attenuates type I allergy via inhibiting intracellular signaling pathways, providing the first evidence that hydrogen modulates signaling pathways [12]. We also demonstrated that hydrogen suppresses LPS/IFNγ-induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream signaling molecules, p38, JNK and NFκB, resulting in inhibition of iNOS expression and NO production in macrophages [13]. Based on these findings, we proposed a hypothesis that hydrogen may act as a modulator of signaling pathways, thereby exhibiting protective effects against various diseases. Consistent with our hypothesis, it has been recently reported that hydrogen inhibits signaling pathways in animal models of acute liver injury [14] and amyloid-beta-induced Alzheimer’s disease [15].

In the present study, in order to understand the underlying mechanisms of hydrogen effects on lipid metabolism disorders and atherosclerosis, we examined if hydrogen could attenuate fatty acid intake and lipid accumulation caused by palmitate overload in human hepatoma HepG2 cells. We then investigated whether hydrogen could modulate signaling pathways after palmitate overload as well as CD36 expression after hydrogen treatment in this cell culture model of hepatic steatosis.

Materials and methods

Cell culture and hydrogen treatment

Human hepatoma HepG2 cells were purchased from RIKEN BioResource Center (Tsukuba, Japan) and cultured in DMEM containing 10% heat-inactivated FBS in a humidified atmosphere of 5% CO2 at 37°C. Prior to hydrogen treatment, cells were starved in serum-free DMEM for 24 h. Hydrogen treatment was performed as described previously [12]. Briefly, cells were cultured in DMEM containing 0.67% (w/v) fatty acid-free BSA (Roche, Penzberg, Germany) under a humidified condition of 75% H2, 20% O2 and 5% CO2, or 95% air and 5% CO2 in a small aluminum bag. After treatment with or without hydrogen for 24 h, cells were treated with 0.67% fatty acid-free BSA or with 0.3 and 1.0 mM sodium palmitate (Sigma, St. Louis, MO, USA)-BSA complex (containing 0.67% fatty acid-free BSA) for 24 h to analyze the lipid content. Cells were also treated with fatty acid-free BSA or with 0.3 mM sodium palmitate-BSA complex for 120 min to analyze the protein phosphorylation.

Cell viability assay

After treatment with or without hydrogen for 24 h, cell viability was determined calorimetrically using the Cell Counting kit (WST-1 assay: Wako, Osaka, Japan) according to the manufacturer’s protocol.

Measurement of fatty acid uptake and lipid content

Fatty acid uptake assay was performed as described by Liao et al. [16] with slight modification. After treatment with or without hydrogen for 24 h, cells were washed twice with Hank’s balanced salt solution (HBSS: Gibco, Langley, OK, USA) and incubated in HBSS containing 0.1% fatty acid-free BSA and 0.5 μg/ml BODIPY FL C16 (Molecular Probes, Eugene, OR, USA) for 15 min at 37°C. After washing twice with ice-cold HBSS containing 0.2% BSA, cells were detached with 10 mM EDTA/PBS and subjected to the measurement of fluorescence using the MT-600 F fluorescence microplate reader (Corona Electric, Hitachinaka, Japan). The relative BODIPY FL C16 uptake was expressed as fluorescence intensity in cells relative to the total amount of protein. To quantify the lipid content, cells were stained with Oil Red O for 10 min and then dye was extracted and measured as described previously [17].

CT-B binding assay

After treatment with or without hydrogen for 24 h, cells were washed twice with HBSS and incubated in HBSS containing 0.1% fatty acid-free BSA and 0.5 μg/ml Alexa594-conjugated cholera toxin B subunit (CT-B; Molecular Probes) for 1 h at 37°C. After washing twice with ice-cold HBSS containing 0.2% BSA, cells were subjected to the measurement of fluorescence using the fluorescence microplate reader.

Real-time RT-PCR analysis

Total RNA was extracted from cells by Isogen II (Wako) followed by DNase I treatment. cDNA was synthesized using the PrimeScript RT reagent kit (Takara, Ohtsu, Japan) and quantitative real-time PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus: Takara) and the real-time thermal cycler Dice (Takara). Primer sets were as follows: GAPDH, 5’-CCACATCGCTCAGA CACCAT-3’ and 5’-GCAACAATATCCACTTTACCAG AGTTAA -3’ [18]; CD36, 5’-TGGAACAGAGGCTGAC

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