Association of growth hormone deficiency with an increased number of preadipocytes in subcutaneous fat

Figures

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  Figure 1. Differentiation of stromal vascular fraction (SVF) of subcutaneous fat from lit/+ and lit/lit mice to adipocytes. Stromal vascular fractions were isolated from inguinal subcutaneous fat pads of 13-week-old mice and were induced to differentiate to adipocytes in culture using a standard protocol (see text for details). This cell culture experiment was repeated 4 times, each time using SVF from a pair of lit/lit and lit/+ male mice. (A) Representative images of cells. Microscopic pictures were taken on day 0 (d0), d2, d4, and d8 of differentiation at 10x magnification. Cells on d8 of differentiation were stained with Oil Red O. (B) Expression levels of pparg, cebpa, plin1, and lipe mRNAs in differentiating SVF cells. Relative abundance of mRNA was quantified by RT-qPCR. Cebpa, C/EBPα; pparg, PPARγ; plin1, perilipin-1; lipe, lipase e, hormone sensitive type, also known as hormone sensitive lipase. Data are expressed as means ± SE (n = 4 mice). mRNA expression was significantly different between days for both lit/lit and lit/+ (P < 0.01). *P < 0.05, **P < 0.01, and §P < 0.1 lit/lit vs. lit/+.

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  Figure 2. Effect of growth hormone (GH) on differentiation of stromal vascular fraction (SVF) from lit/lit mice to adipocytes. Stromal vascular fractions were isolated from inguinal subcutaneous fat pads of 14-week-old male lit/lit mice. Cells were induced to differentiate to adipocytes in the presence or absence of 100 ng/ml of recombinant bovine GH. This cell culture experiment was repeated 3 times, each time using SVF from a different mouse. (A) Representative pictures of cells. Microscopic pictures were taken on day 0 (d0), d2, d4, and d8 of adipocyte differentiation at 10x magnification. Cells on d8 of differentiation were stained with Oil red O. Note that there was no apparent difference in the formation of adipocytes between lit/lit and lit/lit+GH SVF. (B) Expression levels of pparg, cebpa, lipe and plin1 mRNAs in differentiating SVF cells. Data are expressed as means ± SE (n = 3 mice). The expression of these genes in both groups increased (P < 0.03) during adipocyte differentiation; however, the expression of these genes was not different (P > 0.8) between the two groups on the same days of differentiation.

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  Figure 3. Fluorescence-activated cell sorting (FACS) of subcutaneous adipose SVF from lit/+ and lit/lit mice. The SVFs were isolated from 3 16-week-old male lit/+ and 3 16-week-old male lit/lit mice. (A) Representative FACS staining profiles and gating (white lines) of subcutaneous adipose SVF from a lit/lit mouse. The propidium iodide (PI)-negative singlets, i.e., live singlets, were sorted first for lack of Ter119 and CD31 and then lack of CD45 on cell surface. Ter119−, CD31−, and CD45− cells (i.e., Lin− cells) were then sorted on the basis of expression of CD34 and CD29. CD34+ and CD29+ cells were further sorted for expression of Sca-1 and CD24. (B) The percentages of total live single SVF cells expressing the indicated markers. Results are shown as mean ± SE (n = 3 mice). *P = 0.06, †P = 0.14-0.18, and §P = 0.73 vs. lit/+.

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  Figure 4. Quantification of mRNA levels of preadipocyte markers in subcutaneous adipose SVF cells. Stromal vascular fractions were isolated from inguinal subcutaneous fat pads of 16-week-old male lit/+ and lit/lit mice. mRNA expression was quantified by RT-qPCR. Data are expressed as means ± SE (n = 3 and 4 mice for lit/+ and lit/lit, respectively). *P < 0.05, †P = 0.07, and §P = 0.23 vs. lit/+. Dlk1, itgb1, ly6a, and pparg are also known as Pref-1, CD29, Sca-1, and PPARγ, respectively.

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  Figure 5. Model of how growth hormone (GH) inhibits subcutaneous adipogenesis in mice. It is hypothesized that GH inhibits the formation of adipocytes in mice by inhibiting the recruitment of mesenchymal stem cells (MSCs) to the subcutaneous fat depot, or the commitment of the multipotent MSCs to preadipocytes in the subcutaneous fat depot, or both. In this model, GH does not affect preadipocyte proliferation or terminal differentiation of preadipocytes into adipocytes.