Breeding pairs of C57BL/6J-Ghrhr^lit mice, a lit/lit female and a lit/+ male, were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were housed on an automatically timed 12 h light/dark cycle at 23°C with free access to standard rodent chow and water. Male lit/+ littermates were used as controls for lit/lit mice because wild-type littermates were not available from the breeding program and because lit/+ mice did not differ from wild-type mice in body weight (38). Mice were euthanized by CO₂ inhalation and cervical dislocation. Euthanasia was performed between 9:00 a.m. and 11:00 a.m. Mice were euthanized for collection of the inguinal fat pads. The inguinal fat pads in this study referred to the subcutaneous fat pads between the lumbar spine and the groin. All animal-related procedures were approved by the Virginia Tech Institutional Animal Care and Use Committee.

The stromal vascular fraction (SFV) of adipose tissue was isolated as previously described with minor modifications (40). Fat pads were minced and digested using 1 mg/ml of collagenase D (Roche, Indianapolis, IN, USA) in HEPES buffer (100 mM HEPES pH 7.4, 120 mM NaCl, 50 mM KCl, 0.5 mM glucose, 1.5% bovine serum albumin, and 1 mM CaCl₂) at 37 °C with shaking at 115 rpm for 1 h. After centrifugation at 500 ×g for 10 min at room temperature, the pelleted SVF was washed twice with growth medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM)/F12 (Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum or FBS (Atlanta Biologicals, Lawrenceville, GA, USA), 2.5 mM of L-glutamine, and 1% of antibiotics-antimycotics or ABAM (Mediatech, Manassas, VA, USA).

The SVF cells were initially cultured in growth medium in a CO2 incubator at 37 °C. Four or 5 days later, the SVF cells from the same mouse were collected and reseeded in 6- or 24-well plates at a density of 25,000/cm². When cells reached 100% confluence, they were induced to differentiate into adipocytes using the previously described method (40). This method included initially culturing the cells in the DMEM/F12 medium supplemented with 5% FBS, 17 nM insulin (Sigma-Aldrich), 0.1 μM dexamethasone (Sigma-Aldrich), 250 μM 3-isobutyo-1-methylxanthine (IBMX) (Sigma-Aldrich), and 60 μM indomethacin (MP Biomedical, Solon, OH, USA) for two days, then in DMEM/F12 medium supplemented with 17 nM insulin and 10% FBS for another two days, and lastly in DMEM/F12 supplemented with 10% FBS for four days. Each cell culture experiment was repeated 3 or 4 times, each time using cells isolated from different lit/lit or lit/+ mice.

Cells were washed with phosphate buffered saline (PBS) twice and fixed with 10% phosphate buffered formalin (Fisher Scientific, Pittsburg, PA, USA) for 1 h. Cells were washed first with water and then 60% isopropanol (Fisher Scientific). Cells were stained with 2.1 mg/ml Oil Red O solution prepared in 60% isopropanol for 1 h and then washed with water as previously described (41).

Total RNA from cultured adipocytes was isolated using TRI reagent (Invitrogen, Grand island, NY, USA) according to the manufacture’s instruction. Total RNA (1 μg/reaction) was reverse-transcribed into cDNA using ImProm-II Reverse Transcription system (Promega, Madison, Wisconsin, USA). cDNA (20 ng/reaction) was quantified by PCR using gene-specific primers ( Table 1 ) and Fast SYBRGreen Master Mix on an Applied Biosystems 7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). Specificity of primers was validated by gel electrophoresis and DNA sequencing of PCR products, and efficiency of primers was validated by amplifying a serial dilution of cDNA. Each sample was quantified in duplicate. The PCR data were analyzed using the 2(-ΔΔCt) method (42). The mouse hmbs gene was chosen as the reference gene because, based on the Ct values, its mRNA expression was stable across the samples.

Preadipocytes from the mouse subcutaneous SVF were sorted and counted as previously described (43). To describe in brief, freshly isolated SVF cells were resuspended in ice-cold DMEM with 2% FBS and then incubated with the following antibodies at 1:100 dilution on ice for 15 min: APC-Cy7 rat anti-mouse CD45 (Cat# 557659, RRID : AB_396774), PE-Cy7 rat anti-mouse CD31 (Cat# 561410, RRID : AB_10612003), PerCP-Cy5.5 rat anti-mouse TER-119/erythroid cells (Cat# 560512, RRID : AB_10561844), PE hamster anti-rat CD29 (Cat# 562154, RRID : AB_10897843), BV421 rat anti-mouse CD34 (Cat# 562608, RRID : AB_11154576), PE-CF594 rat anti-mouse Ly-6A/E (also known as Sca-1) (Cat# 562730, RRID : AB_2737751), and APC rat anti-mouse CD24 (Cat# 562349, RRID : AB_11151896), all from BD Biosciences (San Jose, CA, USA). Following the incubation, cells were washed with Hank’s Balanced Salt Solution (HBSS) and then resuspended in HBSS supplemented with 0.5 g/ml propidium iodide (BD Biosciences). Cells were subsequently sorted on a BD FACSARIA Fusion Flow Cytometer equipped with BD FACSDiva Software (BD Biosciences). Cells were sorted first for singlets, followed by exclusion of propidium iodide-stained cells, i.e., dead cells. Live single cells were then sorted on the basis of cell-surface markers CD45, CD31, and TER-119. Cells negative for these markers (i.e., lineage negative or LIN−) were then sorted based on their expression of CD29, CD34, Sca-1, and CD24, the cell surface markers for preadipocytes (43).

Data from multiple groups were analyzed by ANOVA followed by Tukey’s test. Data from two groups were analyzed by t-test. These analyses were performed using the General Linear Model in JMP (SAS Institute Inc., Cary, NC, USA). The experimental units for all experiments in this study were mice. All data are expressed as the mean ± standard error (SE).

The subcutaneous fat SVFs isolated from 13-week-old male lit/+ and lit/lit mice were induced to differentiate into adipocytes in culture. During the 8-day course of differentiation induction, the SVF cells from both the lit/lit and lit/+ mice underwent similar morphological changes: they both changed from the fibroblast-like shape to spherical shape, and they gradually accumulated lipid droplets ( Figure 1A ). However, it was apparent that a much greater portion of the SVF cells from the lit/lit mice became lipid droplets-containing adipocytes than that from the lit/+ mice at days 2 and 4 of differentiation ( Figure 1A ), and these differences were confirmed by Oil Red O staining of cells at day 8 of differentiation ( Figure 1A ).

To further assess the adipogenic potential of the subcutaneous fat SVF cells from the lit/lit and lit/+ mice, mRNAs of four adipocyte marker genes were quantified. These marker genes were peroxisome proliferator activated receptor γ (pparg), CCAAT/enhancer-binding protein α (cebpa), perlipin-1 (plin1), and hormone sensitive lipase (lipe). Genes pparg and cebpa are two master transcriptional regulators of adipogenesis (27); genes plin1 and lipe play an important role in lipid metabolism in adipocytes (44). At days 2, 4, and 8 of differentiation, almost all of these marker genes were expressed at much higher levels in the lit/lit than lit/+ SVF cells (P < 0.05, Figure 1B ). These differences further indicated that the subcutaneous fat SVF cells from the lit/lit mice had much greater potential than those from the lit/+ mice at the same age to differentiate into adipocytes.

The aforementioned data that the subcutaneous fat SVF cells from the GH-deficient lit/lit mice had greater adipogenic potential than those from the GH-normal lit/+ mice suggested that GH inhibited subcutaneous fat adipogenesis in mice. To further determine the effect of GH on adipogenesis, we differentiated the subcutaneous fat SVF from the lit/lit mice into adipocytes in the presence of 100 ng/ml bovine GH, which was previously shown to be effective in stimulating liver IGF-I gene expression or inhibiting adipose tissue growth in mice (45, 46). Surprisingly, based on the formation of lipid droplets-containing adipocytes ( Figure 2A ) and the expression levels of adipocyte marker genes ( Figure 2B ), GH addition had no significant effect on the differentiation of the lit/lit SVF cells into adipocytes in culture.

The SVF consists of several other types of cells in addition to preadipocytes (47). The aforementioned results that the SVF cells from the GH-deficient lit/lit mice had greater potential to become adipocytes than those from the GH-normal lit/+ mice yet that GH had no effect on the differentiation of the lit/lit SVF cells into adipocytes in vitro suggested that the lit/lit subcutaneous fat SVF might contain more preadipocytes, the adipocyte precursor cells, than the lit/+ subcutaneous fat SVF. To test this possibility, we first used FACS to compare the percentage of preadipocytes between the lit/lit and lit/+ SVFs. As shown in Figure 3A , we sorted and counted preadipocytes in the SVF based on their expression of surface markers CD29, CD34, Sca-1, and CD24 (43). The SVF from the lit/lit mice contained more Ter119–, CD31–, and CD45– cells, i.e., Lin– cells, than the SVF from the lit/+ mice (P < 0.05, Figure 3B ). The SVF from the lit/lit mice had the tendency (P < 0.18, n = 3) to contain more Lin–, CD34+, CD29+, Sca-1+, and CD24+ cells, i.e., preadipocytes, than the SVF from the lit/+ mice ( Figure 3B ). CD24− preadipocytes are considered more committed toward becoming adipocytes than CD24+ preadipocytes (43, 48). The percentage of Lin–, CD29+, CD34+, Sca-1+, and CD24– cells, i.e., the more committed preadipocytes, was not different between the lit/lit and lit/+ SVF ( Figure 3B ).

We also quantified the expression levels of cd29, cd24, itgb1, ly6a, dlk1, and pparg mRNAs in the subcutaneous fat SVF cells from the lit/lit and lit/+ mice. Itgb1, ly6a, dlk1, and pparg are genes encoding CD29, Sca-1, Pref-1, and PPARγ, respectively. Besides CD29, CD34, Sca-1, and CD24, which are cell surface proteins (43), Pref-1, a transmembrane protein and PPARγ, a nuclear protein, are also considered markers of preadipocytes (49, 50). In particular, Pref-1 is considered an excellent preadipocyte marker because it is highly expressed in preadipocytes but silenced in adipocytes (51). As shown in Figure 4 , cd29, itgb1, ly6a, dlk1, and pparg mRNAs were all expressed at higher levels in the lit/lit than in the lit/+ SVF cells (P < 0.05); cd24 mRNA expression had a tendency to be higher in the lit/lit than the lit/+ SVF cells (P < 0.1, n = 4). These gene expression data further indicated that the subcutaneous fat SVF from the GH-deficient lit/lit mouse contained more preadipocytes than that from the GH-normal lit/+ mouse.