Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

ETHICS STATEMENT: Animals are housed in standardized conditions following the guidelines of the German Animal Welfare Act. Animals are fed a standard diet and water ad libitum and kept with a 12 hr day/night cycle. All experiments with animals are authorized by the local ethics committee.

1. In Vivo Analysis of Adipocyte Homeostasis in Adult Males

1. To quantify hypoxia in vivo, first determine the body weight of the mice, then inject 60 mg/kg body weight of solid pimonidazole hydrochloride intra-peritoneally (for example: inject 1.5 mg into a 25 g mouse). Pimonidazole is an effective hypoxic marker, which forms adducts with thiol groups in proteins, peptides and amino acids and is detected by a specific antibody.
2. Sacrifice mice and remove the perigonadal fat pads (Figure 1).
   1. 45 min after injection, sacrifice mice by CO₂ asphyxiation and subsequent cervical dislocation.
   2. Pin down the limbs of mice (as illustrated in Figure 1) and open the peritoneal cavity. Remove the left and the right perigonadal (epididymal) fat pad inside the peritoneal cavity.
      Note: Fat pad are bound to the epididymis by the peritoneal leaflets as shown in Figure 1 (perigonadal fat pads are indicated by arrows).
   3. Take care to remove the gonadal tissues from the fat pad. Determine fat pad weights to calculate the ratio: fat pad weight (g) per body weight (g).

Figure 1: Location of the perigonadal fat pads in the peritoneal cavity of mice. Picture of perigonadal fat localization in adult mice after sacrifice. Perigonadal fat pads are indicated by the arrows. Please click here to view a larger version of this figure.

3. To compile a quantitative gene expression profile, use one perigonadal fat pad to isolate RNA. Note: Until the tissue is processed, store tissue samples in RNA stabilization solution at -80 °C or in liquid nitrogen.
   1. To homogenize the fat pad, add the fat pad to 1 ml single-phase solution of guanidine isothiocyanate and phenol. Use tubes containing ceramic beads (1.4 mm) to crush the tissue in a homogenizer at 6,500 rpm (2 times 20 sec, with 30 sec pause).
   2. Isolate the RNA as follows (single-step method by Chomczynski and Sacchi ¹⁷).
      1. To separate the phases, transfer homogenized fat pad to microcentrifuge tube, add 0.2 ml volume chloroform, shake for 15 sec, incubate for 5 min at room temperature and centrifuge 12,000 x g for 5 min. Transfer the upper aqueous phase (around 400 µl), which contains the RNA, into a new microcentrifuge tube. Do not include the DNA-containing interphase or the protein-containing phenol phase.
      2. To precipitate the RNA, add 1 volume of isopropanol, mix and incubate for 15 min at 4 °C (it is also possible to incubate at -20 °C overnight). Centrifuge at 12,000 x g for 10 min. Remove isopropanol supernatant carefully. Wash twice with 75% ethanol with centrifuge steps of 12,000 x g for 10 min.
      3. After drying the precipitated RNA for around 10 min at room temperature, dissolve it in 50 µl H₂O (RNase-free). To facilitate this, incubate for 2 min at 65 °C.
         Note: Keep RNA in 75% ethanol at -80 °C or in liquid nitrogen for long-term storage.
      4. Quantify the RNA preparations by A_260/280 (optimal quotient is between 1.8 and 2.2) and calculate the concentration by A₂₆₀:
         
   3. To avoid DNA contamination, digest 1 µg of the RNA preparation with 1 U DNase I for 30 min at 37 °C in a volume of 10 µl. Inactivate at 65 °C for 10 min.
      Note: This step is optional.
   4. Use 10 µl of RNA preparation containing 1 µg RNA for the reverse transcriptase reaction to generate single-stranded cDNA, suitable for quantitative PCR application. The components and their amounts are listed in Table 1.
   5. Use a PCR Master Mix for a quantitative real-time PCR reaction. To determine metabolic changes in the whole fat pad, use specific primers for genes involved in AT homeostasis (Table 2) and the PCR conditions listed in Table 3.
   6. Real-time PCR data analysis.
      1. Define the baseline (Figure 2), normally cycle 1 to 15, where there is no change in fluorescence signals. The real-time PCR software normalizes specific fluorescence signals to the baseline fluorescence and to the internal reference dye, ROX, resulting in the magnitude of the specific signals by the primers, delta Rn (ΔRn).
      2. Set the threshold within the exponential phase of the amplification curve. The intersection defines the threshold cycle (Ct). Based on the CT value, calculate the relative expression (ΔCt) and the fold change (ΔΔCt):
         
         

Table 1: Components with respective volume for the reverse transcriptase reaction to generate single-stranded cDNA.

Table 2: List of genes with sequence of the respective primers used for analyzing adipocyte homeostasis.

Table 3: Real-time PCR conditions.

Figure 2: Characteristics of real-time PCR amplification curve. Please click here to view a larger version of this figure.

4. To perform histological analysis of the adipocyte homeostasis, use the second perigonadal fat pad. Do not desiccate the tissue!
   1. Fix the fat pad in 3.7% PBS-buffered formaldehyde overnight, embed in paraffin (follow the instructions as described elsewhere ¹⁸) and cut the embedded tissue into 2-5 µm thick sections (maximum 5 µm).
   2. Determine the number of adipocytes per field and adipocyte size in a bright-field microscope after hematoxylin and eosin (H&E) staining:
      1. Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol. Finally, wash the section in distilled H₂O for 5 min.
      2. Stain with hematoxylin, diluted 1:5 with distilled H₂O, for 10 min at room temperature and wash in H₂O for 5 min. Stain with eosin solution (20 ml 5% eosin Y/210 ml distilled H₂O/25 µl glacial acetic acid) for 30 sec and wash again with H₂O for 5 min.
      3. Dehydrate sections using 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min. Mount the sections with anhydrous mounting agent.
      4. Evaluate the sections under a bright-field microscope. A representative example of how to analyze adipocytes with ImageJ 1.48v ¹⁹ is shown in Fig. 3: number of cells per area (µm²), cell area (x 10³ µm²), cell size (µm).
         1. Open the picture of the section with ImageJ 1.48v. If the parameters of the image are indicated in pixels instead of a unit of length, adjust the scale.
         2. Select *Straight line* in the toolbar and adjust the line to a known distance by reference to the scale bar. Go to Analyze -> Set Scale. The distance of the line is shown in pixels; add the known distance and the unit of length, e.g., µm. Confirm with OK. The size of the picture and the analysis of the parameters are indicated in the unit of length given.
         3. To determine the parameters of the adipocytes, adjust the threshold. Go to Image -> Adjust -> Threshold to open the Threshold window. Select the following settings: Thresholding method: Default; Threshold color: B&W; Color space: HSB. The picture is now shown in black and white. Adjust the Brightness to clear white adipocytes and to close black intercellular spaces, as in Fig. 3b.
         4. Count the number of adipocyte per µm² (Figure 3e) with the *multi-point* selection in the toolbar and mark each cell for counting.
         5. To determine the adipocyte size, select *Straight line* again in the toolbar and draw the diameter of an adipocyte (Figure 3c). Go to Analyze -> Measure and a new window will appear, with the length of the diameter in µm.
         6. To determine the adipocyte area, select *Wand (tracing) tool* and click inside the adipocyte. The inner wall of the adipocyte is selected in red (Figure 3d). Go to Analyze -> Measure and a new window will appear, with the area of this adipocyte in µm².
   3. For immunohistochemistry, prepare the section for antibody and TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining as follows:
      1. Deparaffinize sections by washing 3 times for 5 min in xylene and rehydrate the section 2 times for 2 min in 100% ethanol and 2 times for 2 min in 96% ethanol. Finally, wash the section in distilled H₂O.
      2. For antigen retrieval, digest the tissue section for 30 min at 37 °C with Proteinase K working solution (20 µg/ml in 10 mM Tris/HCl, pH 7.4-8) and rinse with PBS.
   4. Perform antibody staining in wet chambers:
      1. To block the endogenous peroxidase, use 3% hydrogen peroxide in PBS for 10 min, with subsequently washing 2 times for 5 min in PBS. To block the unspecific binding of antibodies, use 10% serum in PBS. Use the serum of the host of the secondary antibody, in this case goat.
      2. For the staining, use the antibodies for apoptosis, proliferation and hypoxia detection (Table 4). Dilute antibodies in PBS/10% goat serum. Incubate at 4 °C overnight. Wash the sections 3 times for 5 min in PBS.
      3. To enhance the signal use a biotinylated secondary antibody, with the dilution as listed in Table 5. For hypoxia detection, use HRP conjugated rabbit anti-FITC as the secondary antibody. Incubate for 1 hr at room temperature. Wash the sections 2 times 5 min with PBS.
         Note: For the hypoxia staining with FITC-MAb1, skip step 1.4.4.4) and continue with step 1.4.4.5).
      4. For each slide, pre-incubate 50 µl avidin solution with 50 µl biotinylated peroxidase H for 30 min at room temperature (this method is also referred to as avidin/biotin ABC complex formulation) and then add to the sections for another 45 min. Wash 2 times for 5 min with PBS.
      5. Incubate the sections in peroxidase substrate solution until the staining become more intense (between 5 to 10 min). Wash for 5 min with distilled H₂O.
      6. For counterstaining, stain with hematoxylin diluted 1:5 with distilled H₂O for 10 min at room temperature and wash in H₂O for 5 min.
      7. Dehydrate sections in 96% ethanol and 100% ethanol 2 times each for 2 min, and xylene 3 times for 5 min. Seal the sections with coverslips and anhydrous mounting agent. Evaluate the sections under a bright-field microscope.
   5. Determine apoptosis by TUNEL assay and follow manufacturer's instructions:
      1. Add 50 µl enzyme solution into 450 µl label solution. Then apply 50 µl TUNEL reaction mixture on histologic sections and incubate for 60 min at +37 °C in a humidified atmosphere in the dark. Wash the sections 3 times with PBS for 5 min and mount with fluorescence mounting medium including DAPI (4',6-diamidino-2-phenylindole) for counterstaining.
      2. Evaluate the section under a fluorescence microscope with 100 fold magnification. For measurement of Fluorescein use an excitation wavelength of 488 nm and detect between 515-565 nm (green laser); DAPI excites at about 360 nm and emits at about 460 nm when bound to DNA (blue laser).
      3. Quantify the overlays of DAPI and Fluorescein:
         

Figure 3: Analyzing adipocyte characteristics in fat pad sections. Section pictures of the perigonadal fat pad of male mice treated with a high-fat diet (HFD) or normal diet (ND) with a bright-field microscope (a); the threshold is adjusted into black and white (b) and the adipocyte size (length; c), area (d) and adipocyte cell number per mm² (e) are quantified with ImageJ 1.48v. Please click here to view a larger version of this figure.

Table 4: Antibodies with respective dilution used for the immunohistological staining of AT sections.

Table 5: Secondary antibodies with dilution used for immunohistological staining.

2. In Vitro Analysis of Adipocyte Homeostasis Influenced by Hypoxia

1. Sacrifice mice and remove the subcutaneous adipose tissue.
   1. Sacrifice the mice by CO₂ asphyxiation and subsequent cervical dislocation.
   2. Pin down the limbs of mice as illustrated in Figure 4. Detach the skin from the upper leg, loin and flank and pin it down with needles as in Figure 4. Then remove the subcutaneous adipose tissue, which is located posterior at the base of the hind legs, surrounding the inguinal lymph nodes (as shown in Figure 4, left: subcutaneous fat pads indicated by the arrows; right: inguinal lymph node indicated by the arrow).

Figure 4: Location of the subcutaneous fat pads. Picture of subcutaneous fat pad; the left arrows indicate the subcutaneous fat pad and the right arrow indicates the inguinal lymph node. Please click here to view a larger version of this figure.

2. Isolate adipose-derived stem cells (ADSC) as previously described ²⁰.
3. Seed ADSC 4,000 cells/cm² in Dulbecco's modified Eagle's medium-Ham's F-12 supplemented with 10% normal calf serum, 1% penicillin/streptomycin, 0.5% amphotericin B, 16 µM biotin, 18 µM pantothenic acid and 100 µM ascorbic acid and grow culture to confluence around 70 to 80%, which is reached after 4 to 6 days of culture.
4. Induce Adipogenic Differentiation.
   1. Remove the adherent ADSC from the surface by trypsin treatment.
      1. Remove medium, wash with PBS and add 0.025% trypsin solution (preheated to 37 °C) for 2 min (until cells detach from the surface). Immediately add medium and wash the cells.
   2. Count the cells using a Neubauer chamber.
      1. Put the glass cover on the central area of the Neubauer chamber. Dilute the cell suspension 1:10 and load the chamber with 10 µl diluted cell suspension. Count the cells in 4 squares located at the corners, each composed of 16 smaller squares. Calculate the cell number per ml:
         
   3. Seed ADSC (as described in point 2.2) in 12-well culture plates and grow culture to confluence around 70 to 80% (reached after 4 to 6 days).
   4. Induce adipogenic differentiation by adding 5 µg/ml insulin, 1 µM dexamethasone and 5 µM 3-isobutyl-1-methylxanthine (IBMX) to the cultures. Renew the medium every 2 days. Cells will be fully differentiated after 7 days.
5. To analyze the adipogenic differentiation, stain with Oil Red O, which stains triglycerides of mature adipocytes.
   Note: Work must be performed under a fume hood!
   1. Remove the medium, wash the adipocytes gently with PBS and fix the cells for 60 min with 2 ml 10% formalin.
   2. To prepare Oil Red O staining solution, mix 3 parts of the Red Oil O stock solution (300 mg Red Oil O powder dissolved in 100 ml 99% isopropanol) with 2 parts distilled H₂O and incubate for 10 min at room temperature. Filter the Oil Red O working solution through a 0.2 µm injection filter.
      Note: The working solution is stable for 2 hr.
   3. For the Oil Red O staining, remove the formalin, wash adipocytes with H₂O, incubate with 2 ml 60% isopropanol for 5 min, remove the isopropanol and add 2 ml Oil Red O working solution for 5 min. Rinse the cells with tap water until the water is clear. Counterstain with hematoxylin as in point 1.4.4.5).
   4. Evaluate the plates under a phase contrast microscope with 100 fold magnification. The lipids of the adipocytes will appear red and the nuclei will appear blue.
6. Optional step: Silence the gene of interest by transfection with shRNA.
   1. Change the medium and add serum-free medium.
   2. For the transfection of adipocytes, use lipofection. Follow the manufacturer's instructions and use 1 µg shRNA per 12-well tissue plates. After addition of the lipid-DNA-complex, incubate adipocytes for 48 hr at 37 °C.
7. To analyze adipocytes subjected to hypoxia, use a hypoxic work station or hypoxic incubator to maintain the cells under hypoxic conditions. Depending of the study, hypoxia could be 0.5, 1 or 2% oxygen.
   1. Analyze apoptosis by FITC-labeled Annexin V and subsequent flow cytometry analyses.
      1. Gather adipocytes with an extra soft cell scraper, wash with PBS and add 1 x 10⁶ cells to 100 µl Annexin V-binding buffer (10 mM Hepes/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl₂). Add the amount of Annexin V-FITC recommended by the manufacturer and incubate at room temperature for 15 min.
      2. For flow cytometry measurement, add 200 µl Annexin V-binding buffer and 1 µM of nuclear counterstain. Determine the fluorescence in the green and violet channel. Annexin V⁺/nuclear counterstain⁺ cells are defined as secondary necrotic and Annexin V⁺/nuclear counterstain^– cells are defined as apoptotic cells (Figure 5).
   2. Quantitatively analyze adipocyte RNA level to determine the homeostasis under hypoxic conditions.
      1. Add 1 ml single-phase solution of guanidine isothiocyanate and phenol to each well and isolate the RNA [using single-step method by Chomczynski and Sacchi ¹⁷ (step 1.3.2)] and proceed as in steps 1.3.2.1) to 1.3.5.2).

Figure 5: Adipocyte apoptosis analysis by flow cytometry. Dot plot presentations of the FACS for Annexin V-FITC and TO-PRO-3 staining of adipocytes. Please click here to view a larger version of this figure.