探討高脂飲食引起的脂肪組織胰島素阻抗與細胞自噬間之關聯性

Abstract

第二型糖尿病（Type 2 diabetes, T2D）是一種由周邊組織胰島素阻抗（insulin resistance）造成血糖濃度異常升高的代謝疾病，而肥胖（obesity）是目前公認此疾病之主要危險因子。過多的能量攝取容易造成脂肪組織胰島素阻抗，使得細胞內脂解作用增加而大量釋出游離脂肪酸，進而影響其他組織代謝並誘發 T2D。細胞自噬（autophagy）是細胞內重要的降解系統之一，參與許多代謝調節及壓力反應。近期許多研究指出肥胖或代謝疾病患者，其脂肪組織的自噬狀態會改變，然而自噬在肥胖所誘導的胰島素阻抗之角色，仍未完全明瞭。 本研究首先以高脂飼糧誘導的肥胖小鼠模式，瞭解其脂肪自噬之活性，並同時藉由注射胰島素以評估胰島素阻抗發生之時間點。試驗結果顯示，持續 16 週的高脂飼糧會引發 C57BL/6 小鼠的脂肪組織有自噬後期抑制、胰島素阻抗、內質網壓力（ER stress）以及細胞凋亡（apoptosis）的現象，同時也發現 Rubicon 的表現量增加，可能為阻斷自噬後期的因素之一。為了進一步確認自噬後期的抑制是否會誘發胰島素阻抗，我們直接對小鼠的附睪脂肪組織注射自噬後期的抑制藥物氯喹（chloroquine, CQ）。結果發現注射 CQ 後 10 小時，脂肪組織的胰島素訊息傳遞路徑並未受影響；但注射後 24 小時，脂肪組織的胰島素下游標的 Akt 及 GSK3β 的磷酸化程度卻下降，顯示自噬作用的抑制與胰島素阻抗的發生具相關性。另外，我們測定了胰島素訊息的負調節因子PHLPP1 及 PTEN 的蛋白質表現量，然結果顯示注射 CQ 的組別，兩種蛋白質皆無顯著增加，意味著自噬抑制阻斷的胰島素下游訊息傳遞，與 PHLPP1 及 PTEN 的作用路徑無關。 此外，我們也利用分化的 3T3-L1 脂肪細胞株進行體外試驗，經由棕櫚酸（palmitic acid）及油酸（oleic acid）共處理 48 小時後，細胞內油滴大量堆積並引起自噬流（autophagic flux）的降低，表示自噬作用是受到抑制的。隨後以 CQ 處理 24 小時，亦觀察到胰島素阻抗、內質網壓力及細胞凋亡的情形。然而為了摒除如細胞凋亡等機制之影響，我們將 CQ 的處理濃度降低，卻並未造成細胞的胰島素訊息傳遞之異常。不過有趣的是，若處理時間延長至 48 小時，此時脂肪細胞便會發生胰島素阻抗，並伴隨著內質網壓力與細胞凋亡的現象。由此可知，自噬後期的抑制本身可能並非直接誘發脂肪細胞的胰島素阻抗，而是經由併發的內質網壓力或細胞凋亡間接所致。 綜上所述，長期的高脂飲食除了促使脂肪組織發生胰島素阻抗外，還導致Rubicon 的表達增加進而抑制自噬後期，並伴隨著內質網壓力及細胞凋亡等現象。其中自噬後期的抑制與胰島素敏感性下降具有相關性，然而前者並非經由磷酸酶的大量表現影響胰島素訊息的傳遞，而是藉由內質網壓力或細胞凋亡間接引起脂肪組織的胰島素阻抗。

Type 2 diabetes is a metabolic disease with abnormally elevated blood glucose level due to insulin resistance in peripheral tissues, and obesity is recognized as a risk factor for this disease. Excessive calorie intake may lead to insulin resistance and increase free fatty acid releasing from adipose tissue. This situation may alter the metabolic functions of other peripheral tissues to accelerate the development of type 2 diabetes. Autophagy is an intracellular degradation system, which is important for maintaining metabolic homeostasis. Recent studies have demonstrated that the autophagic flux is changed in the adipose tissue of patients with obesity or metabolic disease. However, the role of autophagy in obesity-induced insulin resistance is less clear. In this study, we first confirmed the state of autophagy and insulin signaling in adipose tissue from high-fat diet (HFD)-fed C57BL/6 mice. The results showed late-stage autophagy inhibition, insulin resistance, endoplasmic reticulum (ER) stress and apoptosis in the adipose from mice fed with 16-week HFD. Importantly, the increased level of Rubicon was one of the factors blocking the late-stage autophagy. To clarify whether inhibiting autophagy mediates HFD-induced insulin resistance, mouse epididymal adipose was locally injected with chloroquine (CQ), a late-stage autophagy inhibitor. We found that autophagy inhibition prominently caused insulin resistance in adipose tissue. Additionally, results demonstrated that the cascade of phosphorylation events in insulin signaling pathway blocked by CQ are independent of PHLPP1 and PTEN. Furthermore, the differentiated 3T3-L1 adipocyte was used for in vitro experiment. After co-treatment with palmitic acid and oleic acid for 48 hours, a large amount of lipid droplets accumulated and caused a reduction in autophagic flux, indicating that autophagy was inhibited. Subsequently, treatment with CQ (40 μM) for 20 hours also observed insulin resistance, ER stress and apoptosis. However, when the concentration of CQ was reduced to prevent apoptosis, no significant abnormality in the insulin signaling was observed in the first 24 hours. Interestingly, if the treatment period was extended to 48 hours, the insulin resistance occurred in the 3T3-L1, accompanied by ER stress and apoptosis. It implied that late-stage autophagy inhibition didn’t directly lead to insulin resistance, but indirectly by other pathways. In summary, long-term high-fat diet promotes insulin resistance, late-stage autophagy inhibition, ER stress and apoptosis in adipose tissue. Among these conditions, inhibition of late-stage autophagy is indeed related to a decrease in insulin sensitivity. However, autophagy suppression does not affect the insulin signaling transduction via the large amount of phosphatase expression, but causes insulin resistance indirectly through ER stress or apoptosis.