Inhibition of lipolysis in the novel transgenic quail model overexpressing G0/G1 switch gene 2 in the adipose tissue during feed restriction

Abstract

In addition to the issue of obesity in humans, the production of low-fat meat from domestic animals is important in the agricultural industry to satisfy consumer demand. Understanding the regulation of lipolysis in adipose tissue could advance our knowledge to potentially solve both issues. Although the G0/G1 switch gene 2 (G0S2) was recently identified as an inhibitor of adipose triglyceride lipase (ATGL) in vitro, its role in vivo has not been fully clarified. This study was conducted to investigate the role of G0S2 gene in vivo by using two independent transgenic quail lines during different energy conditions. Unexpectedly, G0S2 overexpression had a negligible effect on plasma NEFA concentration, fat cell size and fat pad weight under ad libitum feeding condition when adipose lipolytic activity is minimal. A two-week feed restriction in non-transgenic quail expectedly caused increased plasma NEFA concentration and dramatically reduced fat cell size and fat pad weight. Contrary, G0S2 overexpression under a feed restriction resulted in a significantly less elevation of plasma NEFA concentration and smaller reductions in fat pad weights and fat cell size compared to non-transgenic quail, demonstrating inhibition of lipolysis and resistance to loss of fat by G0S2. Excessive G0S2 inhibits lipolysis in vivo during active lipolytic conditions, such as food restriction and fasting, suggesting G0S2 as a potential target for treatment of obesity. In addition, transgenic quail are novel models for studying lipid metabolism and mechanisms of obesity.

Figure 1. Lentiviral vector construct and its expression in adipose tissues of transgenic quail.

(A) Diagram of lentiviral vector used for producing transgenic quail. The vector contains 2 kb promoter of chicken fatty acid binding protein and chicken G0S2 coding DNA sequences. The primers for detecting the transgene are presented as ^f1 for forward primer (LTG0S2-F) and ^r1 and ^r2 for reverse primers (LT-R and WPRE-R, respectively). (B) and (C) The expression level of G0S2 protein in adipose tissues of adult transgenic (Tg) and non-transgenic (Non-Tg) quail. Values are represented as mean ± SEM (n = 3). * indicates significance level of P<0.05.

Figure 2. Analysis of growth and organ weights of transgenic and non-transgenic quail fed ad libitum.

(A and B) Growth curve of FG1 and FG3 lines of transgenic and non-transgenic quail fed ad libitum. Body weights of male transgenic and non-transgenic quail were measured every week from hatch to 8 weeks of age. (C and D) Organ weights of FG1 quail fed ad libitum. Organ weights were measured and calculated to the percentage of total body weight. There was no difference between transgenic and non-transgenic quail in the tissue and organ weights (n = 5). SF: subcutaneous fat, AF: abdominal fat, LPM: left pectoralis muscle, H; heart, Li: liver, Lu: lung, and K: kidney. (E and F) Organ weights of FG3 quail fed ad libitum. FG3 did not show a difference in organ weight between transgenic (n = 6) and non-transgenic (n = 8) quail under the ad libitum feeding. Values are represented as mean ± SEM.

Figure 3. Analysis of body and organ weights of transgenic and non-transgenic quail that underwent feed restriction.

(A) Changes of body weights after feed restriction for two weeks. Non-transgenic quail lost more body weight than transgenic quail in both lines. (B and C) Weights of fat pads after feed restriction. Both transgenic quail of FG1 and FG3 had significantly greater fat pads than non-transgenic quail after feed restriction. FR2W: feed restriction for 2 weeks. (D and E) Organ weights after feed restriction. There was no difference in non-adipose organ weights between transgenic and non-transgenic quail after feed restriction. n = 6 for each. Values are represented as mean ± SEM. *, **, and *** indicate significance levels of P<0.05, P<0.01, and P<0.001, respectively.

Figure 4. Comparison of fat cell size under the different feeding conditions.

(A and B) Histology of fat tissues from transgenic and non-transgenic quail of FG1 and FG3. Fat cell size was almost the same between transgenic and non-transgenic quail in both lines fed ad libitum, while it shrank to different levels between them after feed restriction. All scale bars: 50 µm. (C and D) Numerical data of average fat cell size in different feeding conditions. The average fat cell size of non-transgenic quail was significantly smaller than that of transgenic quail only after feed restriction (n = 3). AL: ad libitum and FR2W: feed restriction for 2 weeks. Values are represented as mean ± SEM. * and *** indicate significance levels of P<0.05 and P<0.001, respectively.

Figure 5. Blood NEFA levels and the expression of G0S2 and ATGL in fat tissues in different feeding conditions.

(A) and (B) The levels of NEFA in serum were significantly lower in transgenic quail than in non-transgenic quail under the short-term fasting and feed restriction conditions (n≥5). AL: ad libitum, F4H: fasting for 4 hours, and FR2W: feed restriction for 2 weeks. (C) and (D) The expression of G0S2, ATGL, and FABP4 in fat tissues depending on feeding conditions, ad libitum (AL) vs. fasting for 12 hours (F12H). G0S2 was continuously higher in transgenic quail than non-transgenic quail. The expression of ATGL was higher in fasting condition in both transgenic and non-transgenic quail. The ratio of ATGL to G0S2 was highest in non-transgenic quail under the fasting condition. The expression of FABP4 was not significantly variable regardless of feeding condition. Values are represented as mean ± SEM. *, **, and *** indicate significance levels of P<0.05, 0.01, and 0.001, respectively. NS means non-significant.