| Skeletal muscle plays a key role in maintaining FA homeostasis. The balance of fatty acid uptake, transportation and oxidation is well maintained in healthy skeletal muscle. However, the accumulation of FA derivatives and metabolites would compromise muscle function, and result in insulin resistant which is a key characteristic of type2diabetes. CD36is an integral membrane protein and plays an important role in transporting long chain fatty acids (LCFAs) into skeletal muscle cells. Most findings of CD36focus on its role in facilitating fatty acid uptake. However, the role that CD36plays in regulating FA oxidation remains unclear. Adenosine monophosphate-activated protein kinase (AMPK) is a known major regulator of FA metabolism in skeletal muscle. The role of CD36in regulating FA oxidation by triggering AMPK signaling activation remains unclear.Objective:The current research was designed to study the role of CD36in regulating FA uptake and FA oxidation in muscle, as well as its underlying mechanism. The role of CD36in activating AMPK at basal level was observed firstly, which would provide a new view in CD36-mediated muscle FA oxidation. Secondly, due to the importance of PA in regulating the metabolic homeostasis of skeletal muscle as well as whole body, the mechanism of CD36in regulating AMPK activation with the addition of PA was further investigated, which might be helpful for understanding the occurrence and development of metabolic diseases. Finally, in order to reveal the impact of oxidation level of FAs on CD36contents, an animal model displaying different muscle FA oxidation levels was built to support the possible role of CD36in participating FA oxidation.Methods:1. The role and corresponding mechanism of CD36in regulating muscle fatty acid oxidation in basal conditions(1) The effect of CD36depletion on AMPK activation. 1) Cell culture study with C2C12:Differentiated C2C12cells were depleted of CD36by RNAi-mediated knockdown (KD). The effect of CD36deficiency on AMPK phosphorylation levels was detected by Western Blot;2) Animal study:To investigate the contribution of CD36to AMPK activation regulation in vivo, AMPK phosphorylation levels in skeletal muscle (gastrocnemius) and cardiac muscle of CD36-/-and WT mice were detected by Western Blot.(2) The mechanism of CD36depletion on AMPK activation.1) The effect of CD36depletion on LKB1phosphorylation level was detected by IP in CHO-Vector and CHO-CD36cells;2) The effect of CD36depletion on LKB1relocation to nuclear was observed by IF in CHO-Vector and CHO-CD36cells.(3) The effect of CD36depletion on palmitic acid oxidation in myotubes.1) Differentiated C2C12cells were depleted of CD36by RNAi-mediated knockdown (KD). The effect of CD36deficiency on PA oxidation was determined by high-resolution respirometry (Oxygraph-2k);2) Differentiated C2C12cells were depleted of CD36by RNAi-mediated knockdown (KD) and then incubated with PA. The effect of CD36deficiency on PA-induced AMPK and ACC phosphorylation levels was detected by Western Blot.2. The role of CD36in regulating muscle fatty acid metabolism with the stimulation of PA.(1) The effect of PA on AMPK activation:Differentiated C2C12cells were stimulated by PA, and AMPK phosphorylation levels were detected by Western Blot.(2) CHO-CD36cells were stimulated by300μM PA for15mins. The direct association of CD36with Fyn, LKB1and AMPK was determined by co-IP; The effect of PA on LKB1relocation to nuclear was observed by IF.(3) PA-induced CD36translocation to plasma membrane via AMPK/TBC1D1signaling pathway:The differentiated C2C12cells were stimulated by300μM PA for 0,5,15and30mins, respectively. The PA-facilitated CD36translocation to plasma membrane was examined by In-Cell Western; The effect of PA on AMPK phosphorylation levels were detected by Western Blot; The effect of PA on AMPK-dependent phosphorylation of TBC1D1was examined by TBC1D1immunoprecipitation followed by probing with phospho-(Ser/Thr) antibody specific for AMPK phosphorylation motifs.3. The effect of fatty acid oxidation level in groups HD, E and CR on muscle CD36contents.40male C57mice were randomly assigned into four groups:control (N), high fat diet (HD), voluntary exercise (E) and caloric restriction (CR) with10mice in each group. The intervention peroid lasted for8weeks and the body weight of the four groups was recorded weekly.(1) An animal model with different muscle FA oxidation levels was built: AMPK/ACC phosphorylation levels in skeletal muscle of groups HD, E and CR were measured by Western Blot.(2) Groups HD, E and CR were used to determine the effect of muscle fatty acid oxidation level on CD36content. CD36expression level in skeletal muscle was measured by Western Blot.Results:(1) CD36depletion in C2C12myotubes with2independent siCD36oligonucleotides resulted in a robust increase of AMPK phosphorylation at T172(P<0.05, P<0.01). Moreover, CD36deficiency in CD36-/-mouse resulted in a significant increase in AMPK phosphorylation at T172in both skeletal muscle (P<0.01) and cardiac muscle (P<0.05).(2) Compared with CHO-CD36cells, CHO-Vector cells showed a significant decrease in LKB1phosphorylation, and LKB1was predominantly found in the cytoplasma (P<0.05). This was also accompanied with a higher basal level of AMPK phosphorylation (P<0.01).(3) The addition of PA to C2C12cells treated with irrelevant siRNA significantly increased AMPK T172-phosphorylation at15min of stimulation (P<0.001). The basal level of pAMPK in CD36-depleted cells was higher and did not increase further in response to PA stimulation (P>0.05).(4) PA could obviously inhibit LKB1relocation into nucleus in CHO-CD36cells.(5) The addition of PA markedly weakened the direct association of CD36with Fyn, however significantly enhanced the direct association of CD36with LKB1and AMPK.(6) AMPK and TBC1D1phosphorylation levels were increased with PA stimulation in a time-dependent manner, which was associated with the increase in CD36relocation to the plasma membrane (P<0.05).(7) Compared with group N, the expression level of CD36in the skeletal muscle of HD mice was significantly decreased (P<0.01), the expression of CD36in the skeletal muscle of CR group was significantly increased (P<0.01), and the expression of CD36in the skeletal muscle of E group was not changed (P>0.05); When compared to group HD, the total CD36content in skeletal muscle of E and CR groups was with a higher level (P<0.01).Conclusions:(1) CD36depletion induced an increase in muscle AMPK activation in vitro and in vivo. In the absence of CD36, Fyn-dependent LKB1phosphorylation decreased results in LKB1nuclear relocation inhibited, thus advancing its access to cytosolic AMPK and resulting in increased LKB1phosphorylation of AMPK.(2) In basal condition, the deficiency of CD36induced an increase in AMPK and ACC phosphorylation levels, as well as FA oxidation levels. However, the depletion of CD36could not induce PA-stimulated AMPK phosphorylation levels further increased, suggesting the existence of CD36played a key role in regulating FA oxidation via AMPK/ACC signaling pathway.(3) PA binding to CD36induced Fyn dissociating from the CD36-LKB1-AMPK complex, allowing LKB1to remain in the cytosol and to phosphorylate AMPK. Besides that, the addition of PA could enhance the direct association of CD36with LKB1and AMPK, which could further increase LKB1phosphorylation of AMPK.(4) PA induced CD36translocation to plasma membrane via AMPK/TBC1D1signaling pathway, but not to mitochondrial membrane.(5) Compared with HD group, CD36content was significantly higher in E and CR groups in which muscle FA oxidation was also higher, indicating that total CD36content was probably related to the adaption of fatty acid oxidation in skeletal muscle. |
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