Fernández-Galilea Marta, Tapia Pablo, Cautivo Kelly, Morselli Eugenia, Cortés Víctor A
Department of Nutrition, Diabetes and Metabolism, Pontificia Universidad Católica de Chile, Santiago, Chile.
Department of Physiology, Pontificia Universidad Católica de Chile, Santiago, Chile.
Biochem Biophys Res Commun. 2015 Nov 6;467(1):39-45. doi: 10.1016/j.bbrc.2015.09.128. Epub 2015 Sep 28.
Mutations in 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) result in lipodystrophy, insulin resistance and diabetes. Autophagy is required for normal adipogenesis and adipose tissue development. The aim of this study was to determine whether impaired autophagy or excessive cell death underlie the adipogenic inability of Agpat2(-/-) mice preadipocytes.
Preadipocytes were isolated from interscapular brown adipose tissue (BAT) of Agpat2(-/-) and Agpat2(+/+) newborn mice and cultured/differentiated in vitro. Intracellular lipids were quantified by oil red O staining. Cell death was assessed by lactate dehydrogenase (LDH) activity. Apoptosis and autophagy regulatory factors were determined at the mRNA and protein level with Real-time PCR, immunoblot and immunofluorescence.
Adipogenically induced Agpat2(-/-) preadipocytes had fewer lipid-loaded cells and lower levels of adipocyte markers than wild type preadipocytes. Before adipogenic differentiation, autophagy-related proteins (ATGs) ATG3, ATG5-ATG12 complex, ATG7 and LC3II were increased but autophagic flux was reduced, as suggested by increased p62 levels, in Agpat2(-/-) preadipocytes. Adipogenic induction increased LDH levels in the culture media in Agpat2(-/-) preadipocytes but no differences were observed in the activation of Caspase 3 or in markers of autophagic flux.
AGPAT2 is required for in vitro adipogenesis of mouse preadipocytes. Autophagy defects or apoptosis are not involved in the adipogenic failure of Agpat2(-/-) preadipocytes.
1-酰基甘油-3-磷酸O-酰基转移酶2（AGPAT2）突变会导致脂肪营养不良、胰岛素抵抗和糖尿病。自噬是正常脂肪生成和脂肪组织发育所必需的。本研究的目的是确定自噬受损或过度细胞死亡是否是Agpat2基因敲除小鼠前脂肪细胞脂肪生成能力缺失的原因。
从Agpat2基因敲除和野生型新生小鼠的肩胛间棕色脂肪组织（BAT）中分离前脂肪细胞，并进行体外培养/分化。通过油红O染色对细胞内脂质进行定量。通过乳酸脱氢酶（LDH）活性评估细胞死亡情况。利用实时PCR、免疫印迹和免疫荧光在mRNA和蛋白质水平测定凋亡和自噬调节因子。
与野生型前脂肪细胞相比，经脂肪生成诱导的Agpat2基因敲除前脂肪细胞中脂质负载细胞较少，脂肪细胞标志物水平较低。在脂肪生成分化之前，Agpat2基因敲除前脂肪细胞中自噬相关蛋白（ATG）ATG3、ATG5-ATG12复合物（此处原文可能有误，推测为ATG5-ATG12复合体）、ATG7和LC3II增加，但自噬通量降低，这可通过p62水平升高得到提示。脂肪生成诱导使Agpat2基因敲除前脂肪细胞培养基中的LDH水平升高，但在半胱天冬酶3的激活或自噬通量标志物方面未观察到差异。
AGPAT2是小鼠前脂肪细胞体外脂肪生成所必需的。自噬缺陷或凋亡与Agpat2基因敲除前脂肪细胞的脂肪生成失败无关。
