肥胖是脂肪組織的功能異常，並且和脂肪組織的慢性發炎、免疫細胞浸潤以及纖維化息息相關。在肝臟和肺臟的研究已經指出纖維化和組織硬度的增加相關。然而在肥胖中脂肪組織纖維化對於硬度的改變及後續生理代謝的影響仍未知。賴胺醯氧化酶( Lysyl oxidase, LOX) 是一個對於細胞外基質( Extracellular matrix, ECM) 的鍵結以及組織軟硬度影響很大的酵素。基質的軟硬度已被證實會影響細胞的行為。在我們實驗室先前的研究發現硬的基質會降低脂肪細胞的脂肪激素釋放、胰島素敏感性及脂解作用，並增加脂肪細胞的免疫反應。然而，LOX所調控的組織軟硬度是否影響脂肪組的功能仍未知。我們假設發炎所引發的巨噬細胞的LOX釋放會導致脂肪組織硬化，進而造成脂肪組織功能異常。我們的結果顯示，在所有脂肪組織的細胞種類裡，巨噬細胞會在發炎誘導因子( LPS) 的刺激之下增加LOX的表現。LPS也在巨噬細胞中增加活化LOX所需要的酵素蛋白表現。LPS刺激後的巨噬細胞會硬化他們所處在膠原蛋白膠，但這樣的硬化現象會因為添加LOX的抑制劑( BAPN) 而被緩解。接著，我們想要透過膠原蛋白膠的細胞培養系統，來了解LOX所調控的ECM硬化對於脂肪細胞功能的影響。我們發現培養在LPS刺激後的巨噬細胞所調控的環境下的脂肪細胞，其脂肪細胞激素的釋放以及對於胰島素的敏感性會下降，而這樣的現象會因為BAPN的添加的緩解。免疫螢光染色的結果顯示在肥胖小鼠的脂肪組織中，LOX會大量表現在巨噬細胞較多的區域。我們也發現從肥胖小鼠腹腔取得的巨噬細胞會有較高的LOX及其活化所需酵素的蛋白表現，且這些巨噬細胞較能使膠原蛋白膠硬化。而從肥胖患者的血液所取出的周邊血液單核球細胞顯示，在減重手術後LOX的表現量會下降。
接著，我們執行了一系列的實驗: 體內LOX抑制、體內去除巨噬細胞以及在骨髓細胞中調降LOX表現量，來瞭解巨噬細胞LOX對整體代謝的影響。體內LOX抑制降低了LOX在脂肪組織的活性，並緩解肥胖小鼠的代謝異常。去除巨噬細胞降低了脂肪組織LOX表現量，並減少了脂肪組織的硬度，及增強了整體代謝情形。在骨髓細胞中去除LOX表現，有效地在脂肪組織中降低了pro-LOX蛋白表現及LOX酵素活性，並伴隨著較低的脂肪組織硬度及較佳的代謝狀態。Pioglitazone是臨床用於增加胰島素敏感性的藥物，已被證實能有效降低脂肪組織的發炎情況；我們的結果顯示，Pioglitazone降低脂肪組織中LOX的mRNA 表現，以及脂肪組織硬度。這些結果代表Pioglitazone可能部分地，利用調控脂肪組織的硬度來改善整體的代謝。整體而言，我們的結果顯示，被發炎所刺激的巨噬細胞LOX會透過增加脂肪組織硬度來惡化全身性的代謝。

Obesity, the dysfunction of adipose tissue, is associated with chronic inflammation, local infiltration of macrophage, and fibrosis in adipose tissue. Studies in liver and lung have shown that fibrosis is associated with increased tissue stiffness. However, the change of adipose tissue stiffness and its consequent influence on metabolism in obesity is still unclear. Lysyl oxidase (LOX) is an enzyme crucial for extracellular matrix (ECM) crosslinking and tissue stiffening. Substrate stiffness has been proved to affect cell behaviors. Our previous results demonstrated that stiff substrate decreased adipokine secretion, insulin sensitivity and lipolysis, and increased inflammation in adipocytes. Nevertheless, how LOX-mediated tissue stiffening is involved in adipose tissue dysfunction is still unknown. Thus, we hypothesize that inflammation-induced macrophage LOX leads to adipose tissue stiffening resulting in adipocyte dysfunction. Our results showed that, among several cell types in adipose tissue, macrophage responded to an inflammation inducer—LPS with an upregulation in LOX. LPS also upregulated LOX secretion, processing proteins, and activity in macrophages. LPS-stimulated macrophages stiffened the collagen gel they were cultivated on, but the stiffening effect was abrogated by a LOX inhibitor – BAPN. We further elucidated the effect of LOX-mediated ECM stiffening on adipocyte function by a collagen gel system. We found that adipokine secretion and insulin sensitivity were diminished in adipocytes seeded on LPS-treated macrophage-processed gel, but were restored in adipocytes seeded on the gel co-treated with BAPN. Immunofluorescent staining revealed that LOX was highly expressed in macrophage-enriched region in ob/ob adipose tissue. Consistent with the insight that obesity is characterized by chronic inflammation, we found that obesity also upregulated LOX and its processing proteins in peritoneal macrophages, resulting in stiffening of the collagen gel. Peripheral blood mononuclear cells isolated from obese human subjects after weight loss surgery also demonstrated reduction in LOX. To examine the importance of macrophage LOX in systemic metabolism, we performed several experiments, including LOX inhibition, macrophage depletion and LOX knockdown in bone marrow cells, in vivo. LOX inhibition in vivo reduced adipose tissue LOX enzymatic activity and ameliorated metabolic impairments in ob/ob mice. Macrophage depletion by clodronate liposome abrogated LOX and reduced adipose tissue stiffness, associated with improvements in metabolic profile. LOX knockdown in hematopoietic cells with shLOX lentiviral infection in bone marrow cells decreased pro-LOX protein level and LOX enzymatic activity in adipose tissue. Mice reconstituted with LOX knockdowned cells exhibited lower adipose tissue stiffness and improved metabolic profiles. Lastly, a clinically used insulin sensitizer pioglitazone that has been shown to lower adipose tissue inflammation was used. Pioglitazone downregulated LOX mRNA level and decreased tissue stiffness in the gonadal adipose tissue. These results suggest that pioglitazone improves metabolism, at least in part, by decreasing adipose tissue stiffness. Taken together, our results suggest that increased macrophage LOX by an inflammatory stimulation deteriorates adipocyte function in obesity via increasing adipose tissue stiffness.

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