Subcutaneous Fat, Abdominal

A genome-wide SNP genotype dataset will be generated for all participants using the H3Africa SNP array. It is enriched for common variation in multiple African populations. Data generated by these studies will be used both for genome-wide exploratory research to identify novel genetic associations, as well as hypothesis-driven research, including replication studies. The genetic association studies will focus initially on the body composition and anthropometric variables, particularly the levels of visceral and abdominal subcutaneous fat, and the blood and urine biomarkers as risk factors or determinants of cardiometabolic outcomes.

Body composition analyses for abdominal fat and muscles were performed from the reconstructed water and fat images, using the commercially available service AMRA^® Profiler (Advanced MR Analytics AB, Linköping, Sweden). The methods used in AMRA^® Profiler have been thoroughly described in earlier publications [10 (link), 11 (link), 15 (link), 33 ] but briefly the analysis consisted of the following steps: (1) image calibration to fat referenced images, (2) labels of fat and muscle compartments registered to the acquired volumes, (3) quality control of labels performed by trained analysis engineers at Advanced MR Analytics (Linköping, Sweden), and (4) quantification of fat and muscle volumes based on the calibrated images by integrating over the quality controlled labels. This process was described in detail in [11 (link)]. The included fat and muscle compartments were visceral adipose tissue (VAT), abdominal subcutaneous adipose tissue (ASAT), posterior thigh muscles, anterior thigh muscles, lower leg muscles, and abdominal muscles, detailed definitions of the anatomical regions used for compartmental fat and muscle segmentations and quality control are listed in Table 1. Finally, the individual muscles latissimus dorsi, pectoralis major, and rhomboideus were included.
Muscle fat infiltration was measured for each muscle. The MFI measurements were defined as the average PDFF of the muscle tissue, i.e. muscle tissue with an adipose tissue concentration of less than 50%. As the calibrated fat images are T1-corrected [20 (link)], and represent the adipose tissue concentration of the tissue, the MFI was calculated by scaling the adipose tissue concentration with the PDFF of adipose tissue. In this study a constant PDFF of 93.7% was assumed for adipose tissue to convert adipose tissue concentration to PDFF.
Based on water-fat images acquired with a 5° flip angle, the liver-fat was measured as the average PDFF of three 22x22x28 mm³ regions of interest (ROI) manually placed in right liver lobe, avoiding major vessels and bile ducts. The liver test and re-test scans were pooled and analysed in randomized order.

Murine 3T3-L1 cells (ATCC, Manassas, VA, USA) were cultured in a growth media containing Dulbecco’s Modified Eagles’ Medium (DMEM), 10% heat-inactivated fetal calf serum (Gibco, Grand Island, NY, USA), and 1% penicillin–streptomycin (Welgene, Daegu, Republic of Korea). The adipocyte differentiation was induced by changing the medium to DMEM supplemented with 10% fetal bovine serum (FBS) (Welgene, Daegu, Republic of Korea) added with a cocktail of hormones (MDI) that include 0.5 mM IBMX (M), 0.5 µM dexamethasone (D), and 5 µg/mL insulin (I) either with or without TAK-715 at the indicated concentrations. On day 2, the first differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 µg/mL insulin either with or without TAK-715 at the indicated doses for an additional 3 days. The cells were further fed with DMEM containing 10% FBS in the presence or absence of TAK-715 for an additional 3 days. On day 8, the preadipocytes became mature adipocytes that were rounded up and filled with lipid droplets.
The hASCs were isolated from abdominal subcutaneous adipose tissue of female patients admitted to Keimyung University Dongsan Hospital (KUDH), Daegu, Republic of Korea. The Ethics Committee of KUDH approved the study protocol (No. 2021-02-063-018), and the informed consent was obtained from the patients. The hASCs isolated were then cultured in growth media containing Dulbecco’s Modified Eagles’ Medium/ Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, and 0.25 μg/mL fungizone. The adipocyte differentiation of hASCs was induced by using adipocyte differentiation medium (Zenbio, DM-2) for 7 days, and then the medium was changed into adipocyte maintenance medium (Zenbio, AM-1) until 5 days in the absence or presence of TAK-715 at the indicated concentrations. On day 12, hASCs became mature adipocytes that rounded with large lipid droplets in the cytoplasm.

Data collection and collection tools: study participants underwent one visit every four weeks and were asked to report SMBG by fingerstick and to use the CGM (Medtronic Minimed, Northridge, CA, USA) during at least 48 hours at baseline. Glucose levels in the extracellular fluid of the abdominal subcutaneous tissue were monitored every five minutes during the CGM and participants performed an eight-point profile of SMBG (preprandial, 90 min postprandial, bedtime and 03:00 hours). During the three weeks when CGM was not done, participants performed a seven-point SMBG (preprandial, postprandial and bedtime) for at least two week days and one day in the week-end. Hemocue Glucose 201 Plus meter (Hemocue, Angelholm, Sweden) was used for the eight-point SMBG while OneTouch Ultra glucose monitoring devices (Lifescan, Milpitas, CA, USA) were used for the seven-point SMBG. At the last visit, only the seven-point SMBG was performed. Fasting BG was considered to be the pre-breakfast BG and pre- and post-prandial measurements from SMBG (Hemocue) were used to calculate mean pre- and post-prandial BG, as well as pre- and post-breakfast, lunch and dinner values. The measurement of A1C was done at the National Obesity Centre using the DCCT-aligned machine DCA 2000®+, Bayer Healthcare LLC, inc. Elkhart, IN 46514 USA. For the second measurement, frozen samples were shipped overnight on dry ice to the European Reference Laboratory for A1C at Zwolle, Netherlands. A1C values retained were the mean of 4 measurements obtained from different DCCT-aligned assays, including a high-performance liquid chromatography assay (Tosoh G7; Tosoh Bioscience, Tokyo, Japan), two immunoassays (Roche A1C and Roche Tina-quant; Roche Diagnostics), and an affinity assay (Primus Ultra-2; Primus Diagnostics, Kansas City, MO) all approved by the National Glycohemoglobin Standardization Program (NGSP) as previously reported [17 (link)].
For optimal accuracy, the initial two hours of CGM (calibration period) were excluded and CGM results were only used if at least one successful 24-h profile out of the 2-3 days of monitoring was available with no gaps >120 min and a mean absolute difference compared with the Hemocue calibration results <18% [7 (link)]. Those results were pooled with specific SMBG results to calculate the estimate of eAG concentration.
Sample size: study participants were consecutively included and a convenience sample was used for the purpose of the study.
Data analysis: statistical analyses were done using SPSS for Windows software version 22.0 (SPSS, Chicago, IL). Results are expressed as median (interquartile range) or number of observation (percentage). Test of normality was performed using Kolmogorov-Smirnov test. Continuous variables were compared by Kruskal-Wallis one-way analysis of variance or Mann-Whitney U test and categorical variables by Chi-square test with Yates´ continuity correction or Fischer´s exact test. Correlations between variables were evaluated using both Spearman's rank test using a complete case analysis. The bias obtained from the use of the global ADAG equation was calculated as the percentage of difference between the population specific eAG and the result obtained with the ADAG equation. The cutoff for significance was p value < 0.05.
Ethical consideration: the study was approved by the National Ethics Committee of Cameroon and all study participants or their legal representative provided informed consent.