Molecular hydrogen attenuates fatty acid uptake and lipid accumulation through downregulating CD36 expression in HepG2 cells
© Iio et al; licensee BioMed Central Ltd. 2013
Received: 25 December 2012
Accepted: 25 February 2013
Published: 1 March 2013
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.
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.
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.
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.
KeywordsMolecular hydrogen HepG2 cells Fatty acid JNK Phosphorylation CD36 Hepatic steatosis
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 . 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 . Mice deficient of CD36 exhibit defective uptake and utilization of fatty acids. Excessive fatty acid uptake mediated by CD36 plays an important role in hepatic steatosis . The expression level of CD36 is very low in normal liver tissues , 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 . 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 , 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 . 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 . 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 . 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  and amyloid-beta-induced Alzheimer’s disease .
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 . 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.  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 .
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’-CCACATCGCTCAGACACCAT-3’ and 5’-GCAACAATATCCACTTTACCAGAGTTAA -3’ ; CD36, 5’-TGGAACAGAGGCTGACAACTT-3’ and 5’-TTGATTTTGATAGATATGGGATGC-3’. The expression level of CD36 gene was determined using the comparative Ct method and normalized to that of GAPDH.
Western blot analysis
Whole cell extracts were prepared by using RIPA buffer containing the protein inhibitor cocktail (Roche) and the phosphatase inhibitor cocktail (Sigma). Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto PVDF membranes. Membranes were incubated with a primary antibody, anti-SAPK/JNK, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-GAPDH (Cell Signaling Technology, Beverly, MA, USA) or anti-CD36 (GenTex, Irvine, CA, USA), followed by incubation with a horseradish peroxidase-conjugated anti-rabbit secondary antibody. Protein bands were detected using the ECL Plus (GE Healthcare, Little Chalfont, UK) and the chemiluminescence imager LAS 4000 (Fujifilm, Tokyo, Japan).
Results were expressed as mean ± SD of three independent experiments unless otherwise noted. Data were analyzed using Student’s t-test. A value of p < 0.05 was considered significant.
Results and discussion
Hydrogen does not affect viability of HepG2 cells
Hydrogen reduces fatty acid uptake and lipid accumulation
In hepatocytes, FFA is converted to TG, which is used for production of very low-density lipoprotein (VLDL), a class of lipoproteins. VLDL transports TG from the liver and intestine to adipose and muscle tissues, but excess TG is stored in lipid droplets . The cellular lipid content in part depends on FFA uptake through transmembrane transport. In order to examine if hydrogen could influence FFA uptake, cells were treated with a BODIPY-labeled fluorescent fatty acid analog, BODIPY FL C16, for 15 min after treatment with or without hydrogen for 24 h. After washing, intracellular fluorescence was measured. As shown in Figure 2B, the fluorescent-labeled palmitate uptake was significantly reduced by hydrogen treatment. Taken together, these results suggest that hydrogen inhibits fatty acid uptake and lipid accumulation after palmitate overload in HepG2 cells.
Hydrogen inhibits palmitate-induced phosphorylation of JNK
Hydrogen downregulates protein expression of CD36
Binding of CT-B to ganglioside GM1 is a marker to identify lipid rafts, which are membrane microdomains enriched in cholesterol and sphingolipid . There are several evidences for the correlation of the level of FFA uptake with the expression level of FFA transporter proteins and with the integrity of lipid rafts . In order to examine if hydrogen affected the lipid raft integrity in HepG2 cells, we measured the intensity of fluorescent-labeled CT-B binding after hydrogen treatment using a fluorescence microplate reader. As shown in Figure 3B, hydrogen treatment did not affect the lipid raft integrity. These findings suggested the possibility that inhibition of FFA uptake by hydrogen might be due to the altered expression of FFA transporter proteins such as CD36.
Hydrogen downregulates CD36 protein expression and thereby inhibits palmitate-induced phosphorylation of JNK
In the present study, hydrogen treatment results in downregulation of CD36 protein expression in HepG2 cells, which inhibits fatty acid uptake when cells are subjected to palmitate overload. Therefore, inhibition of palmitate-induced phosphorylation of JNK is likely to be the consequence of the reduced uptake of fatty acid. Our results suggest that downregulation of CD36 expression by hydrogen pretreatment may be the primary mechanism against hepatic steatosis in this in vitro model.
Finally, it is worth to note that we investigated the molecular mechanisms of the hydrogen effects on lipid metabolism using the cell culture system we previously developed. To keep the hydrogen concentration high in the medium, cells are cultured under the condition of 75% H2, 20% O2, and 5% CO2. A recent report, however, demonstrated that oral intake of water containing a relatively lower concentration of hydrogen was effective in an animal model of Parkinson’s disease . Furthermore, the increase in hydrogen concentration in the body after taking hydrogen water should be transient. In order to precisely recapitulate the hydrogen effects in vivo, a novel cell culture system needs to be developed in which concentration and timing of hydrogen treatment can be readily changed.
Hydrogen downregulates the protein expression of CD36, and inhibits fatty acid uptake and lipid accumulation in HepG2 cells. As the consequence, hydrogen may modulate signal transduction such as the JNK pathway. Our results provide insights into the molecular mechanism underlying the hydrogen effects on lipid metabolism disorders such as hepatic steatosis and NAFLD.
Non-alcoholic fatty liver disease
Free fatty acid
c-Jun NH2-terminal kinase
Atherosclerotic vascular disease
Apoptosis signal-regulating kinase 1
Hank’s balanced salt solution
Cholera toxin B subunit
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Very low-density lipoprotein
Mitogen-activated protein kinases
This work was supported in part by Grant-in-aid for Scientific Research (22300244 to Masafumi Ito) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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