| Abstract: | Gamma 次亞麻油酸（gamma linolenic acid，GLA，18:3）為omega-6 多元不飽和脂肪酸，人體可經由delta-6 去飽和酶代謝亞麻油酸（linoleic acid，LA，18:2n-6）生成GLA ，不需要額外補充。然而，研究許多生理及病理因素可導致delta-6 去飽和酶酵素活性降低，造成體內無法自行合成GLA ，需由飲食中添加補充，故GLA 被認為是條件必需脂肪酸（conditional essential fatty acid）。目前在台灣常見市售含有GLA 植物油脂膠囊為月間草油（primrose oil 含7-10 % GLA）、黑莓油（blackcurrant oil 含15-20 % GLA）、及琉璃苣油（borage oil 含18-26 % GLA）等。由於研究已證實GLA 具有抗發炎的功效，而發炎反應在異化作用造成的肌肉耗損與高游離脂肪酸造成的肌肉胰島素阻抗扮演重要角色，故本論文主要探討GLA 對改善骨骼肌肉流失與胰島素阻抗之功效及其相關機制。相較於沙拉油，補充富含GLA 的琉璃苣油可顯著減少因脂多醣體（lipopolysaccharide）和路易氏肺癌細胞（Lewis Lung carcinoma cells）誘發C57BL/6J 小鼠的發炎反應及骨骼肌肉耗損，也可抑制肌肉組織中mitogen-activated protein kinases (MAPKs) 和nuclear factor-κB (NF-κB) 的活化；值得注意的是，補充琉璃苣油或給予GLA 處理可減少泛素-蛋白酶路徑（ubiquitin-proteasome pathway，UPP）及自噬作用-溶酶體路徑（autophagy-lysosome pathway，ALP）相關atrogenes 表現包括：muscle ring finger protein 1 （MuRF1）、 atrogin-1/muscle atrophy F-box （MAFbx）、ubiquitin （Ub）和microtubule-associated protein 1 light chain 3B （LC3B），改善脂多醣體和路易氏肺癌細胞誘發C57BL/6J 小鼠骨骼肌肉組織及C2C12 肌肉細胞肌凝蛋白重鏈（myosin heavy chain）表現量降低；GLA 處理亦可減少脂多醣體和路易氏肺癌細胞液誘發C2C12 肌肉細胞MAPKs 活化與NF-κB 轉錄活性。在棕櫚酸（palmitic acid，PA，16:0）誘發小鼠C2C12 肌肉細胞胰島素阻抗之細胞模式，共同處理棕櫚酸與次亞麻油酸（linolenic acid，LNA，18:3，n-3）、LA 或GLA 等18 碳多元不飽和脂肪酸（polyunsaturated fatty acids）可顯著降低棕櫚酸誘發的發炎反應及胰島素阻抗，減少促發炎細胞激素：介白素-6（interleukin-6）和腫瘤壞死因子-α（tumor necrosis factor-α）表現，回復胰島素誘發的AKTser473 與AS160thr642 蛋白質磷酸化，增加葡萄糖轉運子4 號（glucose transporter type 4）轉移至細胞膜，提高肌肉細胞的葡萄糖攝入；18 碳多元不飽和脂肪酸也可顯著抑制棕櫚酸誘發MAPKs 活化與NF-κB 轉錄活性。補充富含GLA 的琉璃苣油可顯著減少高脂飲食合併鏈脲佐菌素（high fat diet/streptozotocin）誘發第一型糖尿病小鼠肌肉組織中促發炎細胞激素表現及MAPKs 和NF-κB 活化。利用持續活化型IKKβ 質體暫時轉染入C2C12 肌肉細胞，使NF-κB 持續活化，可逆轉GLA 負向調控NF-κB 路徑改善肌肉發炎反應、肌肉耗損與胰島素阻抗等現象。綜合以上研究，GLA 可透過抑制MAPKs/NF-κB 訊息傳遞路徑，減少骨骼肌肉促發炎細胞激素及蛋白質降解相關分子表現與提升胰島素敏感度，減少由異化作用造成的肌肉耗損及高游離脂肪酸造成的肌肉胰島素阻抗。此研究成果有助於提供未來發展GLA 用於減少肌肉耗損及胰島素阻抗的相關保健食品的參考依據。
Gamma linolenic acid (GLA, 18:3), an n-6 polyunsaturated unsaturated fatty acid, can be produced by desaturated linoleic acid (LA, 18:3 n-6), catalyzed by delta-6 desaturase. Although delta-6 desaturase activity is downregulated in several physiologic/pathophysiologic states and dietary conditions, supplementation with GLA could ameliorate the signs and symptoms of inflammation-related diseases. Hence, GLA is considered a conditionally essential fatty acid. In Taiwan, the commercial GLA gel capsule formulation contained borage oil (18–26 % GLA), blackcurrant oil (15–20 % GLA), and evening primrose oil (7–10 % GLA). In recent years, accumulating evidence has shown that GLA has anti-inflammatory activity. Inflammation-induced catabolism is involved in muscle atrophy, and high free fatty acids are involved in muscle insulin resistance. Herein, we investigated the effects of GLA on improvement of conditions of muscle mass loss and insulin resistance in skeletal muscle. Compared with soybean oil, supplementation with borage oil (rich in GLA) significantly decreased mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation, as well as reduced inflammation and muscle mass loss in skeletal muscles of lipopolysaccharide (LPS)-challenged or Lewis lung carcinoma (LLC)-bearing C57BL/6J mice. It is also worth noting that supplementation with borage oil or treatment with GLA significantly reduced muscle mass loss and reversed myosin heavy chain protein levels through decreasing ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP) activation, as evidenced by reductions in LPS- and LLC-induced expression of muscle ring finger protein 1 (MuRF1), atrogin-1/muscle atrophy F-box (MAFbx), ubiquitin (Ub), and microtubule-associated protein 1 light chain 3B (LC3B) in C57BL/6J mice and C2C12 myotubes. Pre-incubation of GLA also significantly repressed LPS- and LLC-conditioned medium (LCM)-induced MAPK and NF-κB activation in C2C12 myotubes. Exposure of C2C12 myotubes to palmitic acid (PA, 16:0) led to activation of MAPKs and NF-κB to enhance expression of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and these changes were inhibited by linolenic acid (LNA, 18:3, n-3), LA, or GLA, 18-carbon polyunsaturated fatty acids (18-carbon PUFAs). Moreover, 18-carbon PUFAs, especially GLA, reversed PA-inhibited phosphorylation of AKTSer473 and AS160Thr642, and subsequent upregulation of glucose transporter type 4 (GLUT4), which translocated into the membrane to enhance glucose uptake in C2C12 myotubes. Supplementation with GLA-rich borage oil also dramatically reduced pro-inflammatory cytokine expression as well as MAPK and NF-κB activation in the skeletal muscles of high fat diet/streptozotocin mice. Furthermore, GLA does not reverse LPS-, LCM- or PA-induced inflammation, muscle mass loss or insulin resistance in muscle cells transfected with a constitutively active mutant IKKβ plasmid, which indicated the importance of the inhibition of NF-κB activation by GLA. Overall, GLA significantly reduced inflammation-induced catabolism and high free fatty acid-induced expression of pro-inflammatory cytokines and downregulated activation of MAPKs and NF-κB, leading to decreasing UPP and ALP activation and increasing insulin sensitivity in skeletal muscles. These data could provide information for the development of functional foods containing GLA to reduce muscle mass loss and skeletal muscle insulin resistance. |
