Protection of palmitic acid-mediated lipotoxicity by arachidonic acid via channeling of palmitic acid into triglycerides in C2C12

Abstract

Background: Excessive saturated fatty acids have been considered to be one of major contributing factors for the dysfunction of skeletal muscle cells as well as pancreatic beta cells, leading to the pathogenesis of type 2 diabetes.

Results: PA induced cell death in a dose dependent manner up to 1.5 mM, but AA protected substantially lipotoxicity caused by PA at even low concentration of 62 μM, at which monounsaturated fatty acids including palmitoleic acid (POA) and oleic acid (OA) did not protect as much as AA did. Induction of cell death by PA was resulted from mitochondrial membrane potential loss, and AA effectively blocked the progression of apoptosis. Furthermore, AA rescued significantly PA-impaired glucose uptake and -signal transduction of Akt in response to insulin.Based on the observations that polyunsaturated AA generated competently cellular droplets at low concentration within the cytosol of myotubes compared with other monounsaturated fatty acids, and AA-driven lipid droplets were also enhanced in the presence of PA, we hypothesized that incorporation of harmful PA into inert triglyceride (TG) may be responsible for the protective effects of AA against PA-induced lipotoxicity. To address this assumption, C2C12 myotubes were incubated with fluorescent probed-PA analogue 4, 4-difluoro-5, 7-dimethyl-4-boro-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16) in the presence of AA and their subsequent lipid profiles were analyzed. The analyses of lipids on thin layer chromatography (TLC) showed that fluorescent PA analogue was rapidly channeled into AA-driven TG droplets.

Conclusion: Taken together, it is proposed that AA diverts PA into inert TG, therefore reducing the availability of harmful PA into intracellular target molecules.

Figure 1

Lipotoxicity of saturated fatty PA as a function of concentration (A) and the effect of unsaturated fatty acids on PA-mediated lipotoxicity (B). Cells grown at 2*10⁴ were treated with increasing concentrations of PA for 24 h. To examine the protective effects of unsaturated fatty acids on lipotoxicity of PA, cells were coincubated with 0. 5 mM PA and 62.5 μM unsaturated fatty acids indicated for 24 h. LDH release into media was assayed for the evaluation of lipotoxicity mediated by supplementation of fatty acids, and relative LDH release for each group was calculated from absorbance values being set sample completely lysed in Triton X-100 and culture medium left untreated to 100% and 0%, respectively.

Figure 2

The changes in DNA damage (A) and mitochondrial membrane potential (B) when C2C12 cells were treated with either PA alone or PA and unsaturated fatty acids. Cells at a density of 5*10⁵ were exposed to PA in combination with unsaturated fatty acids for 24 h. The genomic DNAs from fatty acids-treated myotubes were isolated and resolved on 1.5% agarose electrophoresis. For the measurement of mitochondrial membrane potential, myotubes were overloaded with 0.5 mM PA and unsaturated fatty acids at a concentration of 62.5 μM for 24 hr. The fatty acids-treated cells were rinsed with phosphate-buffered saline (PBS), incubated with culture media containing 5 μM JC-1 for 30 min and subsequently washed twice with PBS. Fluorescent ratios were calculated from fluorescence measured at 485/545 and 530/590 nm (excitation/emission), respectively. Each bar represents the mean of 4 separate experiments ± S.E.M. *p < 0.05 versus PA-treated group.

Figure 3

The dose dependent impairment of insulin-stimulated glucose uptake in C2C12 treated with PA (A) and improvement of glucose uptake in response to insulin by AA (B). Differentiated myotubes C2C12 cells were exposed to increasing concentrations of PA or 0.5 mM PA along with either AA or ETYA at the concentration of 62.5 μM for 24 h. Subsequently, cells were rinsed and subjected to glucose uptake assay in response to 100 nM insulin in a buffer containing 10 μM 2-deoxyglucose. Glucose uptake was expressed in an increase of DPM/well in response to insulin stimulation versus basal glucose uptake. *p <0.05 versus basal glucose uptake.

Figure 4

Insensitization of insulin signal pathways when supplemented with different concentrations of PA (A) and the restoration of PA-abrogated insulin signal pathway in the presence of AA (B). To optimize insensitization condition of insulin signal transduction by PA, PA in 2-fold dilution was added to myotubes culture and kept for 24 h under CO₂ incubator. In addition, to test the preventive effect of AA against insulin signal transduction impaired by PA, cells were treated with either 0.5 mM PA plus two doses of AA¹ and AA² (31 and 62.5 μM) or 0.5 mM PA plus 62.5 μM ETYA for 24 h, respectively. Fatty acids-exposed cells were rinsed with PBS, stimulated with 100 nM insulin for 30 min and then lysed in lysis buffer containing 0.5% Triton X-100, 1 mM EDTA and protease inhibitors. The prepared cell lysates were subjected to 10% SDS-PAGE and blotting onto PVDF membrane for immunoblotting against pAkt an Akt using specific antibodies.

Figure 5

Augmentation of PA-driven lipid droplets by AA loads in C2C12 cells. The driving potency of each fatty acids to produce lipid droplets during C2C12 culture was measured by Nile red staining of cells grown in media containing various doses of fatty acids-BSA complex (A). Also, images of lipid droplets within cytosol were taken when cells were overloaded with 0. 5 mM PA alone (left), 0.5 mM PA plus 62.5 μM AA (middle) or 0.5 mM PA plus 62.5 μM ETYA (right) (B). To quantitate stimulated incorporation of PA into lipid droplets by unsaturated fatty acids, PA in combination with unsaturated fatty acids at 62.5 μM were loaded into C2C12 cell culture and allowed to incubate for 24 h. Relative fluorescence of neutral droplets were measured following Nile-red staining and fixation of them (C). *p < 0.05 versus PA-treated group.

Figure 6

The incorporation of PA into AA-driven neutral lipid TG. Microscopic images showing localization of fluorescent PA analogue BODIPY FL C16 within lipid droplets of C2C12 cells exposed to AA (A) and lipid analyses extracted from C2C12 cells on TLC (B). To examine the trafficking of PA into AA-driven TG, the myotubes were supplemented with either 7.5 μM of fluorescent PA analogue 7.5 μM BODIPY FL C16 alone or 7.5 μM of BODIPY FL C16 plus 62.5 μM AA or 62.5 μM ETYA for 24 h. Cells were washed with PBS, fixed with 10% formaldehyde solution and then observed for the incorporation of PA analogue into AA-driven TG using fluorescence microscopy equipped with a B filter set. For lipid analysis, total lipids were extracted from cells treated under the same condition as in above. Lipids on TLC were developed and visualized as described in Methods. Lane 1 and 2 were, respectively, loaded with lipids extracted from cells left treated with BODIPY FL C16 or BODIPY FL C16 plus AA.

Figure 7

Schematic summary demonstrating the protective effects of AA against PA-mediated lipotoxicity.