Nck2, an unexpected regulator of adipogenesis

Abstract

The regulation of adipose tissue expansion by adipocyte hypertrophy and/or hyperplasia is the topic of extensive investigations given the potential differential contribution of the 2 processes to the development of numerous chronic diseases associated with obesity. We recently discovered that the loss-of-function of the Src homology domain-containing protein Nck2 in mice promotes adiposity accompanied with adipocyte hypertrophy and impaired function, and enhanced adipocyte differentiation in vitro. Moreover, in severely-obese human's adipose tissue, we found that Nck2 expression is markedly downregulated. In this commentary, our goal is to expand upon additional findings providing further evidence for a unique Nck2-dependent mechanism regulating adipogenesis. We propose that Nck2 should be further investigated as a regulator of the reliance of white adipose tissue on hyperplasia versus hypertrophy during adipose tissue expansion, and hence, as a potential novel molecular target in obesity.

Keywords: 3T3-L1 and SGBS preadipocytes; Adipogenesis; PERK activation and signaling; PPARγ regulation; Src homology adaptor protein Nck2; adipocyte differentiation.

Figure 1.

Expression of Nck2 in human SGBS (Simpson-Golabi-Behmel syndrome) cells during adipocyte differentiation. Nck2 levels were evaluated in SGBS cells at different time points during differentiation by western blot using a specific antibody that recognized human Nck2 (OriGene TA321585) and total cell lysate normalized for protein content. HSP90 was used as loading control. Upper panel represents western blot from a typical experiment, while the bottom panel represents quantitation of Nck2 expression normalized over HSP90 compared with day 0 from 3 independent experiments. Data are the mean ± SEM. *statistical significance at least p ≤ 0.05 compared with Day 0 using unpaired Student t-test.

Figure 2.

Overexpression of Nck2 in murine 3T3-L1 preadipocytes. A) Equivalent amount of proteins from total cell lysates of 3T3-L1 preadipocytes stably transfected with pcDNA3.1 or pcDNA3.1 encoding Flag-Nck2 were probed with anti-Flag antibody to detect Flag-Nck2 or RasGap antibody as loading control. B) Phase-contrast images (10X) of 3T3-L1 preadipocytes control or overexpressing Nck2 at day 0, 6 and 20 of differentiation. C) Cebpa and Cebpb mRNA levels determined using qPCR in control and Nck2 overexpressing 3T3-L1 cells at day 15 of differentiation. Shown is the mean ± SEM of 2 experiments performed in triplicate. D) Equivalent amount of proteins from total cell lysates of control and Nck2 overexpressing 3T3-L1 cells at day 15 of differentiation were subjected to western blot analyses using indicated antibodies. Shown is a typical experiment performed in duplicate. E) Pparg mRNA levels determined using qPCR in indicated 3T3-L1 cells at 15 d of differentiation. Shown is the mean ± SEM of 4 independent experiments. **, statistical significant at p ≤ 0.01 using unpaired Student t-test.

Figure 3.

PPARγ cellular distribution in induced control and Nck2 overexpressing 3T3-L1 cells. A) DIC and fluorescent images of control and Nck2 overexpressing 3T3-L1 cells induced for differentiation under the same set up were used to investigate PPARγ nuclear localization. DIC: light microscopy images at 40X; Blue: nuclear staining with Dapi; Red: PPARγ immunofluorescence using a specific PPARγ antibody and a secondary antibody-coupled to Alexa Fluor® 594; Merge: superposition of Dapi and PPARγ signals. B) Model of adipogenesis regulation by Nck2. In response to adipogenic cues, loss-of-function of Nck2 promotes adipogenesis by enhancing PPARγ nuclear translocation, while Nck2 gain-of-function promotes PPARγ expression, but directly or indirectly, represses PPAR γ nuclear translocation.