Neutral ceramidase‐enriched exosomes prevent palmitic acid‐induced insulin resistance in H4IIEC3 hepatocytes

Oversupply of free fatty acids such as palmitic acid (PA) from the portal vein may cause liver insulin resistance. Production of reactive oxygen species plays a pivotal role in PA‐induced insulin resistance in H4IIEC3 hepatocytes. Recently, we found that exosomes secreted from INS‐1 cells that were transfected with neutral ceramidase (NCDase) plasmids had raised NCDase activity; these NCDase‐enriched exosomes could inhibit PA‐induced INS‐1 cell apoptosis. Here, we showed that PA reduced insulin‐stimulated tyrosine phosphorylation of insulin receptor substrate 2 and decreased insulin‐stimulated uptake of the fluorescent glucose analog 2‐NBDG, confirming that insulin resistance occurred in PA‐treated H4IIEC3 cells. Moreover, NCDase‐enriched exosomes from INS‐1 cells rescued PA‐induced H4IIEC3 insulin resistance and blocked PA‐induced reactive oxygen species production in which ceramide was involved.

Oversupply of free fatty acids such as palmitic acid (PA) from the portal vein may cause liver insulin resistance. Production of reactive oxygen species plays a pivotal role in PA-induced insulin resistance in H4IIEC3 hepatocytes. Recently, we found that exosomes secreted from INS-1 cells that were transfected with neutral ceramidase (NCDase) plasmids had raised NCDase activity; these NCDase-enriched exosomes could inhibit PAinduced INS-1 cell apoptosis. Here, we showed that PA reduced insulinstimulated tyrosine phosphorylation of insulin receptor substrate 2 and decreased insulin-stimulated uptake of the fluorescent glucose analog 2-NBDG, confirming that insulin resistance occurred in PA-treated H4IIEC3 cells. Moreover, NCDase-enriched exosomes from INS-1 cells rescued PA-induced H4IIEC3 insulin resistance and blocked PA-induced reactive oxygen species production in which ceramide was involved. Impaired hepatic insulin sensitivity leads to more glucose production in the liver, resulting in hyperglycemia [1] and development of type 2 diabetes [2]. Too much dietary lipid, especially free fatty acids (FFAs), passing through the portal vein and being deposited in the liver might cause the liver's insulin resistance [2]; an increase in FFAs in blood decreased insulin sensitivity in some target tissues [3]. A report in mice confirmed that oxidative stress induced by lipid caused insulin resistance in liver [4], and reactive oxygen species (ROS) production plays a pivotal role in palmitic acid (PA)-induced insulin resistance in rat H4IIEC3 hepatocytes [2].
In the sphingolipid metabolic pathway, neutral ceramidase (NCDase) is one of the most important enzymes; the catalytic action of NCDase leads to ceramide hydrolysis and sphingosine generation. It is well known that ceramide is a key molecule in the sphingolipid pathway, and is involved in many physiological and pathological processes such as apoptosis, differentiation and insulin resistance. Therefore, as an important ceramide hydrolyzing enzyme, NCDase per se is also involved in the aforementioned processes. It has been reported that exosomes function to transport a variety of substances [5], and can be internalized into cells by membrane fusion or endocytosis [6], as a result of which their contents would be transferred to the recipient cells. Our previous study confirmed that NCDase-containing exosomes isolated from the culture medium from INS-1 cells exposed to a low concentration of cytokines inhibited apoptosis induced by a high concentration of cytokines [7]. Very recently, we found that exosome secreted from INS-1 cells transfected with NCDase plasmids had raised NCDase activity, and these NCDaseenriched exosomes (NCDase-Exos) could inhibit PAinduced INS-1 cell apoptosis (S. Tang, F. Luo, Y. Feng, X. Wei, H. Miao, Y. Lu, Y. Tang, D. Ding, J. Jin and Q. Zhu, unpublished data). The action of NCDase-Exos on PA-induced insulin resistance is investigated in the present study. We hypothesized that NCDase-Exos from INS-1 cells could prevent PA-induced H4IIEC3 insulin resistance by decreasing ROS production.

Cells and materials
All cells were incubated in a humidified atmosphere at 37°C containing 5% CO 2 .

Preparation of exosomes and treatment
The conditioned medium from INS-1 cells transfected with pEGFP-C3-NCDase or the control vector pEGFP-C3 was prepared and then used for exosome isolation as in our previous report [7]. In brief, after conditioned medium was ultracentrifugated at 110 000 g and 4°C for 70 min, the pellets were resuspended in 19 PBS for a second ultracentrifugation. The protein concentration in exosomes was measured and 10 lg of exosomes was added to H4IIEC3 hepatocytes seeded in six-well plates.

NCDase activity assay
The enzymatic activity of NCDase in exosome was determined by measuring the amount of sphingosine generated from ceramide as in our previous report [9].

Western blot
H4IIEC3 hepatocytes with confluence of 80-90% were treated with PA for 16 h. After 15 min stimulation by insulin at a concentration of 1 ngÁmL À1 , the cells were collected and lysed. After centrifugation, 10 lg of sample from the supernatant was loaded for detection of t-IRS2. For detection of p-IRS2, 400 lg of protein from the supernatant was immunoprecipitated at 4°C for 2 h with the antibody against p-IRS2 and protein G beads before loading. The membrane was incubated with primary antibody, then with second antibody, and finally detected and exposed to X-ray film, as described previously [2].

2-NBDG uptake
2-NBDG, a fluorescent D-glucose analog, was used to determine glucose uptake by H4IIEC3 hepatocytes. Cells were treated with PA for 16 h, then stimulated with 100 nM insulin for 15 min, and finally incubated with 50 mM 2-NDBG for 20 min. After that, cells were washed with 19 PBS three times and fluorescence densities were measured (excitation wavelength: 485 nm; emission wavelength: 535 nm) and normalized per milligram of total protein. The nonspecific uptake of 2-NBDG was excluded as in a previous report [10].

ROS production
H2DCFDA, a fluorescent probe, was used to determine ROS production in H4IIEC3 hepatocytes according to a previous report [2]. H4IIEC3 hepatocytes with a 70-80% confluence were exposed to PA for 16 h, then treated with 10 lM H 2 DCFDA for 30 min and the fluorescence was measured.

PA preparation and final concentration
Palmitic acid was prepared as in our previous report [11] and was free of bovine serum albumin. It was dissolved at 70°C in a solution of 0.1 M NaOH and 70% ethanol to produce PA stock solution (50 mM); the final concentration of PA was 0.25 mM.

Statistical analysis
The data from three independent experiments are expressed as the mean AE SD and were analyzed by Student's t test or ANOVA. A value of P < 0.05 was designated as statistically significant.

Results
Very recently, we found that exosomes secreted from INS-1 cells that were transfected with NCDase plasmids had raised NCDase activity, and these NCDaseenriched exosomes could inhibit PA-induced INS-1 cell apoptosis. Further work confirmed that a change in the ceramide/sphingosine-1-phosphate (S1P) ratio might be the mechanism (S. Tang, F. Luo, Y. Feng, X. Wei, H. Miao, Y. Lu, Y. Tang, D. Ding, J. Jin and Q. Zhu, unpublished data). Therefore, we wanted to know whether or not these NCDase-enriched exosomes could prevent PA-induced H4IIEC3 insulin resistance. Firstly, INS-1 cells were transfected with pEGFP-C3-NCDase; the exosomes isolated from the culture medium of these cells are termed NCDase-Exos. Similarly, exosomes from INS-1 cells transfected with the control vector pEGFP-C3 are termed Con-Exos. As shown in Fig. 1, the NCDase-Exos had higher NCDase activity compared with the Con-Exos.

NCDase-Exos could reduce PA-induced ROS production
Because reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in H4IIEC3 hepatocytes [2], we wanted to investigate the effect of NCDase-Exos on ROS production in H4IIEC3 hepatocytes exposed to PA. As expected, PA led to ROS production in H4IIEC3 hepatocytes (Fig. 4), and NCDase-Exos could partially block PA-induced ROS production.

Ceramide plays a key role in the effect of NCDase-Exos on the reduction of PA-mediated ROS production
Because ceramide is a cause of ROS production [12] and is involved in palmitate-induced insulin resistance [13], we investigated the role of ceramide in the effect of NCDase-Exos on ROS reduction in H4IIEC3 hepatocytes exposed to PA. As expected, PA treatment led to a ceramide increase in H4IIEC3 hepatocytes ( Fig. 5), while PA had no effect on the levels of sphingosine and S1P under our conditions (data not shown), and NCDase-Exos could partly block this ceramide elevation. Interestingly, adding C6-ceramide could ameliorate the effect of NCDase-Exos on ROS production of H4IIEC3 hepatocytes under PA treatment (Fig. 6).

Discussion
Exosomes, a distinct kind of nanoparticle with a size from 40 to 100 nm, can be internalized into cells [6] and function in transporting different substances [5], including functional RNAs, lipids, proteins (or enzymes) and even viruses. They therefore act in intercellular transmission [14]. Moreover, the altered characteristics of exosome in many diseases indicate that exosomes are beneficial in the areas of diagnosis and therapy, so that the idea emerges that exosomes might be used as a vehicle to deliver drugs [15]. Thus we hypothesized that exosomes, secretory vesicles, possibly act as carriers to transport enzymes like NCDase to target cells or organs. NCDase, an important enzyme in the sphingolipid metabolic pathway, is responsible for hydrolyzing ceramide to produce sphingosine. Some researchers, including ourselves, confirmed that NCDase protects cells from ceramide's cytotoxicity induced by some cytokines such as interferon c, interleukin 1b and tumor necrosis factor a via up-regulation of NCDase mRNA and protein [9,16,17]. Lately we found that a low concentration of a cytokine mixture led to the release of exosomes containing NCDase, and these exosomes could reduce a high concentration of cytokine mixture-induced cell death [7]. Ceramide is implicated in insulin resistance mediated by FFAs such as PA [18,19], and ROS generation induced by ceramide was confirmed in many studies  H4IIEC3 hepatocytes with a 70-80% confluence exposed to PA alone or PA along with exosomes for 16 h were collected and ceramide in these cells was measured as in our previous report [25]. Data from three determinations are expressed as fold of control (means AE SD). # vs. *: P < 0.05; # vs. +: P < 0.05; * vs. +: P > 0.05; + vs. **: P < 0.05.  Fig. 6. Exogenous C6-ceramide could ameliorate the effect of NCDase-Exos on ROS production of H4IIEC3 hepatocytes under PA treatment. H4IIEC3 hepatocytes with a 70-80% confluence were exposed to PA alone or PA along with exosomes for 10 h, then 40 lM C6-ceramide (Cer) was added and cells were incubated for a further 6 h; finally, H4IIEC3 hepatocytes were treated with 10 lM H 2 DCFDA for 30 min and the fluorescence was measured. Data from three determinations are expressed as fold of control (means AE SD). # vs. *: P < 0.05; * vs. +: P < 0.05; + vs. **: P < 0.05; # vs. ++: P < 0.05. [20][21][22]. An elegant study by Nakamura et al. revealed that ROS has a pivotal role in PA-induced H4IIEC3 hepatocyte insulin resistance [2]. The authors could not determine whether ceramide is necessary for ROS production and subsequent insulin resistance, although an increment in the level of intracellular ceramide was observed in H4IIEC3 cells under PA treatment. Therefore, there was a lack of direct evidence about the signaling of ceramide-ROS-insulin resistance in PA-treated H4IIEC3 cells. In our study, firstly we confirmed in H4IIEC3 cells that PA decreased phosphorylated tyrosine in IRS2 indicating that PA induced insulin resistance, which is in agreement with Nakamura et al.'s report [2]. Interestingly, the NCDase-Exos could partially prevent PA-mediated decrease of insulin sensitivity, which is confirmed by the data from 2-NBDG uptake stimulated by insulin. NCDase-Exos could also ameliorate PA-mediated ROS production and PA-induced ceramide increase. Exogenous ceramide addition could attenuate the effect of NCDase-Exos on ROS production in PA-treated H4IIEC3 hepatocytes, exogenous ceramide alone leading to ROS production in this cell, confirming that ceramide is the upstream signal of ROS production. The findings in this report are summarized in schematic form in Fig. 7. It illustrates the signaling of ceramide-ROS-insulin resistance in PA-treated H4IIEC3 hepatocytes, and that NCDase-Exos can inhibit this signaling cascade and rescue PA-mediated H4IIEC3 insulin resistance. As we know, ceramide has an important role in the pathogenesis of types 1 and 2 diabetes [23], and ceramide is the key intermediate linking some of the steps to insulin resistance [24]. NCDase, a key ceramide-degrading enzyme, may prove efficacious as a therapeutic to combat insulin resistance, especially as exosome-packaged NCDase allows easy transportation of NCDase without degradation. A good question is why NCDase in the exosomes is the 'active' agent and not some other downstream targets of NCDase action, such as ceramide, sphingosine, or S1P. Because no differences of ceramide, sphingosine, or S1P in NCDase-Exos and in Con-Exos were observed (data not shown), these molecules were excluded as the 'active' agents in exosomes. As we know, downstream targets of NCDase action include many genes, lipids and metabolites, all of which are also present in exosomes, and therefore the possibility also exists of other targets besides NCDase involvement. However, the limit of this research is that we cannot answer how NCDase-Exos enter the cells, and how NCDase is released and where it works; further studies are needed to answer these questions.