Norwich Medical School, University of East Anglia, Norwich, UK.
School of Biological Sciences, University of East Anglia, Norwich, UK.
Lancet. 2015 Feb 26;385 Suppl 1:S35. doi: 10.1016/S0140-6736(15)60350-6.
Metabolically unhealthy obesity is associated with adipose tissue inflammation and increased risk of type 2 diabetes. Dysfunctional adipose tissue remodelling has been implicated in development of metabolically unhealthy obesity, but the pathogenesis remains poorly characterised. We hypothesised that in health, tissue inhibitor of metalloproteinases 3 (TIMP3) modulates adipose tissue remodelling by regulating extracellular matrix turnover and shedding of the adipogenic regulator DLK1, but that in adipose tissue inflammation it might drive development of metabolically unhealthy obesity.
Primary pre-adipocyte and in-vitro-differentiated adipocyte cultures were established from abdominal subcutaneous adipose tissue donated by healthy women undergoing breast reconstruction. Cells were seeded onto collagen I, and subsequently treated with differentiation medium or tumour necrosis factor (TNF) alpha (50 ng/mL). Adenoviral transduction allowed TIMP3 overexpression. Media and lysates were collected for quantitative RT-PCR, and immunoblot and hydroxyproline release assays. Statistical analysis was performed with t testing or ANOVA.
Induction of differentiation in human pre-adipocytes reduced TIMP3 mRNA levels by 75% (n=3, p<0·0001). Hydroxyproline release by differentiating pre-adipocytes was 2·3 times greater than that by control-treated cells (mean 5·66 μg/mL [SD 0·77] vs 2·45 [0·36], p<0·0001) indicating greater collagen I degradation. TNF alpha reduced TIMP3 mRNA levels by 66% in in-vitro-differentiated adipocytes (n=3, p<0·0001); reduced TIMP3 expression was confirmed by western blot. Shedding of soluble DLK1 (sDLK1) by pre-adipocytes was increased by TNF alpha and by overexpression of adenovirally delivered TIMP3 compared with control conditions, as confirmed by immunoblot (n=3). Addition of recombinant human sDLK1 (500 pM) to pre-adipocyte cultures reduced adipogenesis, as assessed by oil red O staining (n=2).
We have shown that TIMP3 is downregulated in adipogenesis, and by inflammatory signals in adipocytes. Furthermore, TIMP3 modulates sDLK1 shedding and collagen I degradation. TIMP3 is known to inhibit ADAM17 (DLK1 sheddase) and MMP14 (implicated in extracellular matrix turnover). TIMP3 might therefore integrate inflammatory signals with adipose remodelling. Subversion of remodelling pathways by chronic adipose inflammation might lead to maladaptive adipose expansion and metabolically unhealthy obesity.
British Heart Foundation, Diabetes Research and Wellness Foundation Open Funding 2011.
代谢不健康的肥胖与脂肪组织炎症和 2 型糖尿病风险增加有关。功能失调的脂肪组织重塑与代谢不健康肥胖的发展有关，但发病机制仍知之甚少。我们假设在健康状态下，组织金属蛋白酶抑制剂 3（TIMP3）通过调节细胞外基质的转化和脂肪生成调节因子 DLK1 的脱落来调节脂肪组织重塑，但在脂肪组织炎症中，它可能会导致代谢不健康肥胖的发展。
从接受乳房重建的健康女性腹部皮下脂肪组织中建立原代前脂肪细胞和体外分化的脂肪细胞培养物。细胞接种在胶原蛋白 I 上，然后用分化培养基或肿瘤坏死因子（TNF）α（50ng/ml）处理。腺病毒转导允许 TIMP3 过表达。收集培养基和裂解物进行定量 RT-PCR、免疫印迹和羟脯氨酸释放测定。用 t 检验或 ANOVA 进行统计分析。
诱导人前脂肪细胞分化使 TIMP3 mRNA 水平降低 75%（n=3，p<0.0001）。分化前脂肪细胞的羟脯氨酸释放量比对照处理的细胞高 2.3 倍（平均 5.66μg/ml[SD0.77] vs 2.45[0.36]，p<0.0001），表明胶原 I 降解增加。TNFα 使体外分化的脂肪细胞中的 TIMP3 mRNA 水平降低 66%（n=3，p<0.0001）；Western blot 证实 TIMP3 表达减少。与对照条件相比，TNFα 和过表达腺病毒递送的 TIMP3 增加了前脂肪细胞中可溶性 DLK1（sDLK1）的脱落，这一点通过免疫印迹得到了证实（n=3）。向原代脂肪细胞培养物中添加重组人 sDLK1（500pM）可通过油红 O 染色评估脂肪生成减少（n=2）。
我们已经表明，TIMP3 在脂肪生成过程中以及在脂肪细胞中被炎症信号下调。此外，TIMP3 调节 sDLK1 的脱落和胶原 I 的降解。TIMP3 已知可抑制 ADAM17（DLK1 脱落酶）和 MMP14（参与细胞外基质转化）。因此，TIMP3 可能将炎症信号与脂肪重塑整合在一起。慢性脂肪炎症对重塑途径的颠覆可能导致适应性脂肪扩张和代谢不健康肥胖。
英国心脏基金会，糖尿病研究和健康基金会 2011 年开放基金。
