Supplemental Figure 1: Location of oxygen probe insertion and AT biopsy
Abdominal subcutaneous adipose tissue oxygenation [AT pO2] was measured supine, on the left side of the abdomen, at 1/3 of the distance between the umbilicus and the superior iliac crest and with the skin uncovered with ambient room temperature of approximately 25oC. The AT biopsy was performed on the right side of the lateral abdomen.
Supplemental Figure 2: Measurement of AT oxygenation
Oxygen was measured by 2 complementary methods: the indirect method [A] and the direct method [B].
- The indirect method: after intradermal anesthesia at the insertion and exit sites a gas permeable silicone tubing [10 cm long, 1.2 mm outer diameter, 0.8 mm inner diameter] with a 20 Ga needle at one end and a Luer lock at the other end was channeled in the AT approximately 1 cm under the skin for a distance of approximately 6 cm. The silastic tube was flushed with sterile Ringer solution via the Leur lock. After confirming calibration against room air, an oxygen probe and a temperature were introduced in the subcutaneous portion of the silastic tube, on the inferior and superior ends, respectively and connected to an electronic unit.
- The direct insertion method developed by our laboratory. After cleaning the skin with povidone-iodine solution and removal of the dried iodine with sterile saline on gauze, a skin wheal was raised with 1% lidocaine. A combined oxygen and temperature probe was inserted through a 3.2 cm long 14 Ga IV catheter to a depth of 1 cm.
Supplemental Figure 3: Trace of oxygen and temperature measurements in AT
[A] AT pO2 [blue dots] and AT temperature [green dots] were measured every 20 seconds for 1.4 hours. The last 10 minutes represent the steady state.
[B] We evaluated the effect of temperature on the measurement of pO2 using the LICOX device and probes. After calibrating the oxygen probe, we inserted the probe in a water bath ( while room air was pumped in) and varied the temperature beyond the range of temperatures observed in subcutaneous adipose tissue in this investigation [35°C to 29°C]. The pO2 varied less than 1 mmHg, showing that the pO2 value displayed by LICOX is controlled for changes in temperature [as described in the LICOX manual].
Supplemental Figure 4: AT staining for nuclei, capillaries and plasmallema
In a preliminary experiment, the AT sections were stained with UEA lectin (orange) to label capillaries, GS lectin (green) to label the adipocyte plasmalemma and DAPI (blue) to label nuclei. UEA lectin stains only capillaries.
Supplemental Table: Oligonucleotide sequences for primer/probe sets used in Taqman analysisCommon Gene Name / Accession number /
Reverse primerCYCB / XM_042251 / GGAGATGGCACAGGAGGAAA / CATCTACGGTGAGCGCTTCCCCG / CGTAGTGCTTCAGTTTGAAGTTCTCA
CD68 / NM_001040059 / GCTTCTCTCATTCCCCTATGGA / CAGCTTTGGATTCATGCAGGACCTCC / ATGTAGCTCAGGTAGACAACCTTCTG
MAC-2 / NM_004244 / TGCAGAAAACCCCACAAAAAG / CACAACAGGTCGCTCATCCCGTCA / CAAGGATCCCGACTGCAATAA
MIP-1 / NM_002983 / ACAGAATTTCATAGCTGACTACTTTGAGA / AGTGCTCCAAGCCCGGTGTCATCTTC / GCCGGCTTCGCTTGGT
MCP1 / NM002982 / GATCTCAGTGCAGAGGCTCG / AGCTATAGAAGAATCACCAGCAGCAAGTGTCCC / AATGGTCTTGAAGATCACAGCTTCT
VEGF / NM_001025366.1 / AGCCTTGCCTTGCTGCTCT / ACCTCCACCATGCCAAGTGGTCCC / TCCTTCTGCCATGGGTGC
COL6 / NM_004369.2 / CTCTCAGGCTTTCCTACAACTCATG / CAGCAGCAAGCAGCACAAGACTCTGCT / GTGTTGTTTGATCCATCAATAAGGAA
PPARγ1 / NM_005037 / GTCAAACGAGAGTCAGCCTTTAACG / AGAGATGCCATTCTGGCCCACCAACTT / CCACGGAGCTGATCCCAA
PDK1 / NM_002613 / CGGGAACGCACACAAGTG / TGCAGGAAGATCCAG / TGGTATCGCTGCCTCCAAAC
ANG1 / NM_001146 / CCTCTCTCAGACTGCAGAGCAG / CTAATAAACTGGGTCTCAACATCTGTCAGCTTTCTG / CCTTGTGTTTTCCTTCCATTTCTAAG
CYCB: Cyclophilin β ; CD68, CD68 antigen; MAC-2, macrophage-associated antigen; MIP-1, macrophage inflammatory protein 1, alpha subunit; MCP-1, Macrophage chemoattractant protein 1; VEGF, vascular endothelial growth factor; COL6, collagen VI alpha 3 subunit, PPAR1, peroxisome proliferative activated receptor, gamma 1; PDK1: pyruvate dehydrogenase kinase; Ang1: Angiopoetin 1. For all assays performed using Taqman primers and probe, Cyclophilin β was used as the internal control.